CN111454355B - SOX6 nano antibody and application thereof - Google Patents
- ️Fri Mar 24 2023
具体实施方式Detailed ways
本发明提供了一种SOX6纳米抗体，所述SOX6纳米抗体具有SEQ ID No.1所示的氨基酸序列，具体如下：The present invention provides a SOX6 nanobody, the SOX6 nanobody has the amino acid sequence shown in SEQ ID No.1, specifically as follows:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP。ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP.
在本发明中，所述SOX6纳米抗体的筛选方法，优选包括以下步骤：In the present invention, the screening method of the SOX6 Nanobody preferably comprises the following steps:
1)将黑素瘤纳米抗体库进行第一轮淘洗，得到B16-SOX6-VHH1；1) Perform the first round of elutriation on the melanoma nanobody library to obtain B16-SOX6-VHH1;
所述第一轮淘洗的SOX6蛋白的包被浓度为18～22μg/ml；The coating concentration of the SOX6 protein in the first round of elutriation is 18-22 μg/ml;
2)将所述步骤1)得到的B16-SOX6-VHH1依次进行第二轮、第三轮和第四轮淘洗，得到噬菌体溶液；2) The B16-SOX6-VHH1 obtained in the step 1) is subjected to the second round, the third round and the fourth round of elutriation in order to obtain a phage solution;
所述第二轮淘洗的SOX6蛋白的包被浓度为8～12μg/ml；The coating concentration of the SOX6 protein in the second round of elutriation is 8-12 μg/ml;
所述第三轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml；The coating concentration of the SOX6 protein in the third round of elutriation is 3-8 μg/ml;
所述第四轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml；The coating concentration of the SOX6 protein in the fourth round of elutriation is 3-8 μg/ml;
3)将所述步骤2)得到的噬菌体液与TG1菌液混合、感染后进行培养，得到菌株；3) mixing the phage liquid obtained in the step 2) with the TG1 bacterial liquid, and culturing after infection to obtain bacterial strains;
4)将所述步骤3)得到的菌株与KM13辅助噬菌体混合、感染，将得到的感染物进行第一振荡培养后进行第一离心，将得到的第一沉淀经液体培养基重悬后进行第二振荡培养后，进行第二离心，将得到的第二上清液与封闭液混合、孵育后进行间接ELISA检测，检测第二上清液同SOX6蛋白的反应性，以确定所述菌株与SOX6蛋白具有反应性；4) Mix and infect the bacterial strain obtained in step 3) with the KM13 helper phage, perform the first shaking culture on the obtained infected material, and then perform the first centrifugation, resuspend the obtained first precipitate in liquid medium, and then perform the second centrifugation. 2. After shaking culture, carry out the 2nd centrifugation, the 2nd supernatant that obtains is mixed with blocking solution, after incubation, carry out indirect ELISA detection, detect the reactivity of the 2nd supernatant with SOX6 protein, to determine that described bacterial strain and SOX6 The protein is reactive;
所述第一振荡的温度为35～42℃，所述第二振荡的温度为28～32℃；The temperature of the first oscillation is 35-42°C, and the temperature of the second oscillation is 28-32°C;
所述第一离心的离心力为7500～8500g，所述第二离心的离心力为2000～2100g；The centrifugal force of the first centrifugation is 7500-8500g, and the centrifugal force of the second centrifugation is 2000-2100g;
5)将所述步骤4)与SOX6蛋白具有反应性的菌株进行质粒提取，以所述质粒为模版，用质粒引物对进行PCR扩增，得到纳米抗体VHH片段，将所述纳米抗体VHH片段与表达载体连接，得到重组质粒；5) performing plasmid extraction on the bacterial strain reactive with the SOX6 protein in step 4), using the plasmid as a template, and performing PCR amplification with a pair of plasmid primers to obtain a Nanobody VHH fragment, and combining the Nanobody VHH fragment with The expression vector is connected to obtain a recombinant plasmid;
所述质粒引物包括质粒上游引物和质粒下游引物，所述质粒上游引物具有SEQ IDNo.2所示的核苷酸序列，具体序列如下：The plasmid primers include plasmid upstream primers and plasmid downstream primers. The plasmid upstream primers have the nucleotide sequence shown in SEQ ID No.2, and the specific sequences are as follows:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA，其中，GGATCC为BamHⅠ酶切位点。GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA, wherein GGATCC is a BamHI restriction site.
所述质粒下游引物具有SEQ ID No.3所示的核苷酸序列；The downstream primer of the plasmid has the nucleotide sequence shown in SEQ ID No.3;
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC，其中，GTCGAC为SaⅠl酶切位点。TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC, wherein, GTCGAC is a SaIl restriction site.
6)将所述步骤5)得到的重组质粒、pColdⅠ转入大肠杆菌，得到纳米抗体表达菌株，对所述纳米抗体表达菌株进行IPTG诱导后，提取得到诱导后的纳米抗体表达菌株的蛋白，根据蛋白分子量及His-tag标签对所述蛋白进行Western Blotting鉴定，蛋白分子量为15kDa的蛋白为SOX6纳米抗体。6) Transform the recombinant plasmid and pColdI obtained in step 5) into Escherichia coli to obtain a Nanobody expression strain, and after the Nanobody expression strain is induced by IPTG, extract the protein of the induced Nanobody expression strain, according to Protein molecular weight and His-tag tag The protein was identified by Western Blotting, and the protein with a protein molecular weight of 15kDa was the SOX6 nanobody.
在本发明中，所述黑素瘤纳米抗体库优选为中国专利CN201910057953.4中公开的黑素瘤纳米抗体库。In the present invention, the melanoma nanobody library is preferably the melanoma nanobody library disclosed in Chinese patent CN201910057953.4.
在本发明中，所述第一轮淘洗的SOX6蛋白的包被浓度为18～22μg/ml，优选为10μg/ml。本发明对所述第一轮淘洗的方法没有特殊限定，采用本领域常规淘洗的方法即可。In the present invention, the coating concentration of the SOX6 protein washed in the first round is 18-22 μg/ml, preferably 10 μg/ml. In the present invention, the method of the first round of elutriation is not particularly limited, and conventional elutriation methods in the art can be used.
本发明将得到的B16-SOX6-VHH1依次进行第二轮、第三轮和第四轮淘洗，得到噬菌体溶液；所述第二轮淘洗的SOX6蛋白的包被浓度为8～12μg/ml；所述第三轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml；所述第四轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml。In the present invention, the obtained B16-SOX6-VHH1 is subjected to the second round, the third round and the fourth round of elutriation in order to obtain a phage solution; the coating concentration of the SOX6 protein in the second round of elutriation is 8-12 μg/ml ; The coating concentration of the SOX6 protein in the third round of elutriation is 3-8 μg/ml; the coating concentration of the SOX6 protein in the fourth round of elutriation is 3-8 μg/ml.
在本发明中，所述第二轮淘洗的SOX6蛋白的包被浓度为8～12μg/ml，优选为10μg/ml；所述第三轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml，优选为5μg/ml；所述第四轮淘洗的SOX6蛋白的包被浓度为3～8μg/ml，优选为5μg/ml。本发明对所述第二轮淘洗、第三轮淘洗和第四轮淘洗的方法没有特殊限定，采用本领域常规淘洗的方法即可。In the present invention, the coating concentration of the SOX6 protein in the second round of elutriation is 8-12 μg/ml, preferably 10 μg/ml; the coating concentration of the SOX6 protein in the third round of elutriation is 3-8 μg /ml, preferably 5 μg/ml; the coating concentration of the SOX6 protein in the fourth round of elutriation is 3-8 μg/ml, preferably 5 μg/ml. In the present invention, there is no special limitation on the methods of the second round of elutriation, the third round of elutriation and the fourth round of elutriation, and conventional elutriation methods in the field can be used.
本发明将得到的噬菌体液与TG1菌液混合、感染后进行培养，得到菌株。In the present invention, the obtained phage liquid is mixed with the TG1 bacterial liquid, infected and cultured to obtain bacterial strains.
在本发明中，所述噬菌体液与TG1菌液的体积比优选为1:4。在本发明中，所述TG1菌液的OD600值优选为0.4。在本发明中，所述培养的温度优选为25～35℃，更优选为30℃。In the present invention, the volume ratio of the phage liquid to the TG1 bacterial liquid is preferably 1:4. In the present invention, the OD 600 value of the TG1 bacterial solution is preferably 0.4. In the present invention, the culture temperature is preferably 25-35°C, more preferably 30°C.
本发明将得到的菌株与KM13辅助噬菌体混合、感染，将得到的感染物进行第一振荡培养后进行第一离心，将得到的第一沉淀经液体培养基重悬后进行第二振荡培养后，进行第二离心，将得到的第二上清液与封闭液混合、孵育后进行间接ELISA检测，检测第二上清液同SOX6蛋白的反应性，以确定所述菌株与SOX6蛋白具有反应性；所述第一振荡的温度为35～42℃，所述第二振荡的温度为28～32℃；所述第一离心的离心力为1700～1900g，所述第二离心的离心力为2000～2100g。In the present invention, the obtained bacterial strain is mixed and infected with the KM13 helper phage, the obtained infected substance is subjected to the first shaking culture and then the first centrifugation is performed, and the obtained first precipitate is resuspended in the liquid medium and then the second shaking culture is carried out. Carrying out the second centrifugation, mixing the obtained second supernatant with the blocking solution, and performing indirect ELISA detection after incubation to detect the reactivity of the second supernatant with the SOX6 protein to determine that the strain has reactivity with the SOX6 protein; The temperature of the first oscillation is 35-42°C, the temperature of the second oscillation is 28-32°C; the centrifugal force of the first centrifugation is 1700-1900g, and the centrifugal force of the second centrifugation is 2000-2100g.
在本发明中，所述菌株与KM13辅助噬菌体混合后优选以静置的方式进行感染，所述感染的时间优选为25～35min，优选为30min。In the present invention, the bacterial strain is mixed with the KM13 helper phage to infect preferably in a static manner, and the infection time is preferably 25-35 minutes, preferably 30 minutes.
在本发明中，所述第一振荡的温度优选为35～42℃，更优选为37℃；所述第二振荡的温度优选为28～32℃，更优选为30℃；所述第一离心的离心力优选为1700～1900g，更优选为所述第二离心的离心力为1800g；所述第二离心的离心力优选为2000～2100g，更优选为2020g。In the present invention, the temperature of the first oscillation is preferably 35-42°C, more preferably 37°C; the temperature of the second oscillation is preferably 28-32°C, more preferably 30°C; the first centrifugation The centrifugal force is preferably 1700-1900g, more preferably the second centrifugal force is 1800g; the second centrifugal force is preferably 2000-2100g, more preferably 2020g.
本发明将与SOX6蛋白具有反应性的菌株进行质粒提取，以所述质粒为模版，用质粒引物对进行PCR扩增，得到纳米抗体VHH片段，将所述纳米抗体VHH片段与表达载体连接，得到重组质粒；所述质粒引物包括质粒上游引物和质粒下游引物，所述质粒上游引物具有SEQ ID No.2所示的核苷酸序列；所述质粒下游引物具有SEQ ID No.3所示的核苷酸序列。In the present invention, plasmids are extracted from strains reactive with the SOX6 protein, and the plasmids are used as templates to perform PCR amplification with plasmid primer pairs to obtain nanobody VHH fragments, and the nanobody VHH fragments are connected to expression vectors to obtain Recombinant plasmid; said plasmid primers include plasmid upstream primers and plasmid downstream primers, said plasmid upstream primers have a nucleotide sequence shown in SEQ ID No.2; said plasmid downstream primers have a core sequence shown in SEQ ID No.3 nucleotide sequence.
本发明对质粒的提取没有特殊限定，采用常规提取质粒的方法即可。本发明对所述PCR扩增使用的体系和程序没有特殊限定，采用常规使用的体系和程序即可。在本发明中，所述所述质粒引物包括质粒上游引物和质粒下游引物，所述质粒上游引物优选具有SEQID No.2所示的核苷酸序列，具体如下：The present invention has no special limitation on the extraction of the plasmid, and a conventional method for extracting the plasmid can be used. In the present invention, there is no special limitation on the system and program used in the PCR amplification, and conventionally used systems and programs can be used. In the present invention, the plasmid primers include plasmid upstream primers and plasmid downstream primers, and the plasmid upstream primers preferably have the nucleotide sequence shown in SEQID No.2, specifically as follows:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA;
所述质粒下游引物具有SEQ ID No.3所示的核苷酸序列，具体序列如下：The downstream primer of the plasmid has the nucleotide sequence shown in SEQ ID No.3, and the specific sequence is as follows:
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC。TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC.
本发明对所述纳米抗体VHH片段与表达载体连接的方法没有特殊限定，采用本领域常规连接方法即可。In the present invention, the method for linking the Nanobody VHH fragment to the expression vector is not particularly limited, and conventional linking methods in the art can be used.
本发明将得到的重组质粒、pColdⅠ转入大肠杆菌，得到纳米抗体表达菌株，对所述纳米抗体表达菌株进行IPTG诱导后，提取得到诱导后的纳米抗体表达菌株的蛋白，根据蛋白分子量(15kDa左右)及His-tag标签对所述蛋白进行Western Blotting鉴定，蛋白分子量为15kDa的蛋白为SOX6纳米抗体。In the present invention, the obtained recombinant plasmid and pColdI are transferred into Escherichia coli to obtain a nanobody expression strain. After the nanobody expression strain is induced by IPTG, the protein of the induced nanobody expression strain is extracted. According to the protein molecular weight (about 15kDa ) and His-tag tags were used to identify the protein by Western Blotting, and the protein with a protein molecular weight of 15 kDa was a SOX6 nanobody.
本发明对所述重组质粒、pColdⅠ转入大肠杆菌的转入方法没有特殊限定，采用常规方法即可。本发明对所述纳米抗体表达菌株进行IPTG诱导的诱导方法除浓度外没有特殊限定，采用常规诱导方法即可。本发明对提取得到诱导后的纳米抗体表达菌株的蛋白的提取方法没有特殊限定，采用常规提取微生物中的蛋白的方法即可。本发明对所述SDS-PAGE鉴定的方法没有特殊限定，采用常规即可。In the present invention, there is no special limitation on the transfer method of the recombinant plasmid and pColdI into Escherichia coli, and conventional methods can be used. In the present invention, the induction method for IPTG induction of the nanobody expressing strain is not particularly limited except for the concentration, and conventional induction methods can be used. In the present invention, there is no special limitation on the method for extracting the protein of the induced nanobody expression strain, and a conventional method for extracting protein from microorganisms can be used. In the present invention, there is no special limitation on the identification method of SDS-PAGE, and conventional methods can be adopted.
本发明还提供了上述技术方案所述的SOX6纳米抗体在制备抑制黑色素瘤细胞迁移的药物中的应用。本发明对所述药物的剂型没有特殊限定，采用SOX6纳米抗体在医学上可接受的剂型即可。在本发明中，所述药物中以SOX6纳米抗体为唯一活性物质。本发明对所述药物中SOX6纳米抗体的含量没有特殊限定，采用药物中活性物质的常规含量即可。The present invention also provides the application of the SOX6 nanobody described in the above technical scheme in the preparation of a drug for inhibiting melanoma cell migration. In the present invention, the dosage form of the drug is not particularly limited, and a medically acceptable dosage form of the SOX6 nanobody can be used. In the present invention, the SOX6 nanobody is the only active substance in the drug. The present invention has no special limitation on the content of the SOX6 nanobody in the medicine, and the conventional content of the active substance in the medicine can be used.
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.
实施例1Example 1
将实验室保留的黑素瘤纳米抗体库(中国专利CN201910057953.4中公开的黑素瘤纳米抗体库)进行第一轮淘洗，得到B16-SOX6-VHH，分装冻存于-70℃。The melanoma nanobody library kept in the laboratory (the melanoma nanobody library disclosed in Chinese patent CN201910057953.4) was subjected to the first round of elutriation to obtain B16-SOX6-VHH, which was subpackaged and frozen at -70°C.
淘洗时用50mM碳酸钠/碳酸氢钠缓冲液作为包被缓冲液，包被浓度20μg/ml，包被体积2ml，以SOX6多肽蛋白包被免疫管。During elutriation, 50 mM sodium carbonate/sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg/ml, and the coating volume was 2 ml, and the immunotube was coated with SOX6 polypeptide protein.
淘洗方法如下：The washing method is as follows:
(1)将500μl黑素瘤纳米抗体库接种于100ml 2×YTAG培养基，37℃200rmp振荡培养1小时至OD600为0.4；(1) Inoculate 500 μl of the melanoma nanobody library in 100 ml of 2×YTAG medium, shake and culture at 200 rpm at 37°C for 1 hour until the OD 600 is 0.4;
2)加入KM13辅助噬菌体，100ml菌液加100μl KM13辅助噬菌体，37℃静置感染30分钟，而后振荡培养30分钟；2) Add KM13 helper phage, add 100μl KM13 helper phage to 100ml bacterial solution, let stand for infection at 37°C for 30 minutes, then shake and culture for 30 minutes;
3)4000×g离心10分钟，去除培养基上清，用100ml 2×YTAK培养基重悬菌体沉淀，30℃200rmp振荡培养过夜；3) Centrifuge at 4000×g for 10 minutes, remove the supernatant of the medium, resuspend the bacterial pellet with 100ml 2×YTAK medium, and cultivate overnight at 30°C and 200rmp with shaking;
4)次日上午11000×g，4℃离心过夜培养菌液10分钟，将上清转至新的离心瓶并加入20ml PEG/NaCl溶液，混匀冰浴90分钟；4) The next morning, 11000×g, centrifuge overnight culture solution at 4°C for 10 minutes, transfer the supernatant to a new centrifuge bottle and add 20ml PEG/NaCl solution, mix well and bathe in ice for 90 minutes;
5)11000×g，4℃离心30分钟，弃上清，而后再次离心2分钟，彻底吸尽上清；5) Centrifuge at 11000×g for 30 minutes at 4°C, discard the supernatant, then centrifuge again for 2 minutes, and completely suck up the supernatant;
6)使用2.6ml PBS缓冲液重悬沉淀，而后将其分装于2个1.5ml离心管中，11600×g离心10分钟；6) Use 2.6ml PBS buffer to resuspend the pellet, then divide it into two 1.5ml centrifuge tubes, and centrifuge at 11600×g for 10 minutes;
7)回收上清，命名为ZJ-B16-SOX6-VHH1，取100μl待用于滴度测定，剩余同1.6mlMPBS溶液混合，室温共孵育1h，得到混合液(MPBS溶液处理过的SOX6-VHH1)，待用。7) Recover the supernatant, name it ZJ-B16-SOX6-VHH1, take 100 μl for titer determination, mix the rest with 1.6ml MPBS solution, and incubate at room temperature for 1 hour to obtain a mixed solution (SOX6-VHH1 treated with MPBS solution) ,stand-by.
包被蛋白处理：Coating Protein Treatment:
(1)包被蛋白次日，将免疫管内的液体倒出，使用PBS缓冲液洗管3次。(1) The next day after coating the protein, pour out the liquid in the immunotube, and wash the tube 3 times with PBS buffer.
(2)在每管中加满MPBS，室温封闭2h后使用PBS缓冲液洗管3次。(2) Fill each tube with MPBS, block at room temperature for 2 hours, and wash the tube with PBS buffer 3 times.
(3)在免疫管中加入2ml上述步骤(7)得到的混合液，室温孵育2h后使用PBST溶液洗管10次，而后用PBS缓冲液洗管10次。(3) Add 2 ml of the mixture obtained in the above step (7) to the immunotube, incubate at room temperature for 2 hours, wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer solution.
(4)在每管中加入2ml 100mM TEA溶液，室温轻摇15min洗脱结合的噬菌体，而后加入2ml Tris-HCl溶液中和。(4) Add 2ml of 100mM TEA solution to each tube, shake gently at room temperature for 15min to elute the bound phage, and then add 2ml of Tris-HCl solution for neutralization.
(5)将洗脱的噬菌体(命名为XT-B16-SOX6-VHH1)转至50ml离心管，并加入16mlOD600为0.4的TG1菌液，37℃水浴30分钟，使洗脱的噬菌体感染TG1菌液。(并在免疫管内加入4ml的OD600为0.4的TG1菌液进行感染，最后合并，总共24ml的体积)(5) Transfer the eluted phage (named XT-B16-SOX6-VHH1) to a 50ml centrifuge tube, add 16ml of TG1 bacterial solution with an OD600 of 0.4, and bathe in water for 30 minutes at 37°C to infect the TG1 bacteria with the eluted phage liquid. (And add 4ml of TG1 bacterial solution with OD600 of 0.4 to the immune tube for infection, and finally merge, a total volume of 24ml)
(6)取100μl菌液待用于滴度测定，剩余菌液于4000g离心10min。(6) Take 100 μl of bacterial liquid for titer determination, and centrifuge the remaining bacterial liquid at 4000 g for 10 min.
(7)使用1ml 2×YT培养基重悬菌体沉淀，将重悬后的菌液涂布于5个2×YTAG固体培养板(150mm平板)，置于30℃孵箱培养过夜。(7) Use 1ml of 2×YT medium to resuspend the bacterial pellet, spread the resuspended bacterial solution on five 2×YTAG solid culture plates (150mm plates), and culture in a 30°C incubator overnight.
(8)次日用2×YT培养基收集平板上长出的菌落，加入60％的甘油至终浓度为15％，其即为一级文库菌，命名为B16-SOX6-VHH1，分装冻存于-70℃。(8) The next day, use 2×YT medium to collect the colonies grown on the plate, add 60% glycerol to a final concentration of 15%, which is the first-level library bacteria, named B16-SOX6-VHH1, and freeze Store at -70°C.
测定拯救噬菌体滴度：ZJ-B16-SOX6-VHH1进行梯度稀释，稀释度从10-7～10-13；每个稀释度取10μl噬菌体感染190μl OD600为0.4的TG1菌液；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算ZJ-B16-SOX6-VHH1滴度。Determination of rescue phage titer: ZJ-B16-SOX6-VHH1 was serially diluted from 10 -7 to 10 -13 ; 10 μl of phage was used for each dilution to infect 190 μl of TG1 bacterial solution with OD600 of 0.4; each dilution was taken 100 μl of bacterial solution was applied to 2×YTAG solid culture plates, and cultured in a 30°C incubator overnight; the colonies on the assay plates were counted, and the titer of ZJ-B16-SOX6-VHH1 was calculated.
测定洗脱噬菌体滴度：将用于滴度测定的菌液梯度稀释，稀释度从10-1～10-5；每个稀释度取100μl菌液涂布2×YTAG固体培养板，置于30℃培养箱培养过夜；对测定板上的菌落计数，计算XT-B16-SOX6-VHH1滴度；进而计算第一轮淘洗的输入输出比I/O。Measure the titer of the eluted phage: dilute the bacterial solution used for titer determination in a gradient from 10 -1 to 10 -5 ; take 100 μl of the bacterial solution for each dilution and spread it on a 2×YTAG solid culture plate, place it at 30 Cultivate overnight in an incubator at ℃; count the colonies on the assay plate to calculate the titer of XT-B16-SOX6-VHH1; then calculate the input-output ratio I/O of the first round of elutriation.
在一轮淘洗的基础上，依次进行二至四轮淘洗：SOX6多肽蛋白包被浓度分别为10μg/ml、5μg/ml、5μg/ml；拯救噬菌体滴度测定稀释度分别为10-7～10-12、10-8～10-11、10-8～10-11；洗脱噬菌体M13-SOX6的滴度测定稀释度分别为10-1～10-6、10-3～10-6、10-3～10-6；洗脱的噬菌体用Tris-HCl溶液(1M，pH值为7.4)中和后，取200μl噬菌体感染800μl OD600为0.4的TG1菌液(取100μl进行梯度稀释，剩余的进行保菌)，而后做10-3～10-6共4个稀释度，每个稀释度涂布3个2×YTAG固体培养板(150mm平板)，每板100μl菌液，置于30℃培养过夜；对培养板菌落计数，计算滴度，并将培养板标记为平板，置于4℃冰箱待用。On the basis of one round of elutriation, two to four rounds of elutriation were carried out sequentially: the SOX6 polypeptide protein coating concentrations were 10 μg/ml, 5 μg/ml, and 5 μg /ml; ～10 -12 , 10 -8 ～10 -11 , 10 -8 ～10 -11 ; the titer determination dilutions of eluted phage M13-SOX6 are 10 -1 ～10 -6 , 10 -3 ～10 -6 respectively , 10 -3 ~ 10 -6 ; after the eluted phages were neutralized with Tris-HCl solution (1M, pH value 7.4), take 200 μl phages and infect 800 μl OD600 of 0.4 TG1 bacterial solution (take 100 μl for gradient dilution, The rest should be preserved), and then make 4 dilutions from 10 -3 to 10 -6 , spread 3 2×YTAG solid culture plates (150mm plate) for each dilution, 100μl bacterial solution per plate, and place at 30°C Cultivate overnight; count the colonies on the culture plate, calculate the titer, mark the culture plate as a plate, and place it in a 4°C refrigerator for use.
特异性纳米抗体的筛选：Screening of specific nanobodies:
单克隆噬菌体上清的制备：从平板各挑取130个单克隆菌株共接种2块96孔深孔培养板，每孔中均含200μl 2×YTAG培养基，培养板分别标记为E-1、E-2，于30℃振荡培养。8h后，从每孔中吸取20μl菌液接种于180μl 2×YTAG培养基，于37℃振荡培养，原平板的剩余菌液中则加入60μl 60％的甘油至终浓度为15％，冻存于-80℃。转接平板振荡培养1h后，在每孔中加入20μl KM13(60μlKM13+12ml2*YTAG)辅助噬菌体，37℃静置感染30min，而后37℃振荡培养40min。1800×g离心深孔板10min，弃上清并在每孔中加入400μl 2×YTAK培养基重悬沉淀，30℃振荡培养过夜。次日，最大转速2020xg离心20分钟，从各孔中吸250μl噬菌体上清转移至新的深孔板中，并在每孔中加入250μl封闭液(含3％BSA的PBS缓冲溶液)常温共孵育1小时，待用于间接ELISA检测。Preparation of monoclonal phage supernatant: pick 130 monoclonal strains from each plate and inoculate two 96-well deep-well culture plates, each well contains 200 μl 2×YTAG medium, and the culture plates are respectively marked as E-1, E-2, cultured with shaking at 30°C. After 8 hours, draw 20 μl of bacterial solution from each well and inoculate it into 180 μl of 2×YTAG medium, culture it with shaking at 37°C, add 60 μl of 60% glycerol to the remaining bacterial solution on the original plate to a final concentration of 15%, and freeze it in -80°C. After the transfer plate was cultured with shaking for 1 hour, 20 μl KM13 (60 μl KM13+12 ml 2*YTAG) helper phage was added to each well, and the infection was allowed to stand at 37°C for 30 minutes, and then cultured with shaking at 37°C for 40 minutes. Centrifuge the deep-well plate at 1800×g for 10 minutes, discard the supernatant and add 400 μl of 2×YTAK medium to each well to resuspend the pellet, shake and culture at 30°C overnight. The next day, centrifuge at a maximum speed of 2020xg for 20 minutes, transfer 250 μl of phage supernatant from each well to a new deep-well plate, and add 250 μl of blocking solution (PBS buffer solution containing 3% BSA) to each well for co-incubation at room temperature 1 hour, to be used for indirect ELISA detection.
特异性单克隆噬菌体的鉴定：通过间接ELISA试验检测噬菌体上清同SOX6蛋白的反应性，具体方法如下：使用SOX6蛋白，包被96孔酶标板，包被浓度为2μg/ml，每孔100μl，置于4℃过夜。次日弃孔内包被液体，在每孔中加入100μl封闭液于37℃封闭1h。弃孔内封闭液，在每孔分别中加入100μl封闭液处理过的四轮筛选得到的噬菌体上清作为一抗，37℃孵育1h。用PBST洗液洗板12次。在每孔中加入100μl二抗(HRP-M13Antibody，稀释度1:10000)，37℃孵育1h。用PBST洗液洗板12次。在每孔中加入100μl显色底物，避光反应5-15min，而后在每孔中加入50μl终止液终止反应。将96孔酶标板置于读板机上读取OD450吸收值。对ELISA结果进行分析并确定阳性孔号。Identification of specific monoclonal phage: Detect the reactivity of phage supernatant with SOX6 protein by indirect ELISA test, the specific method is as follows: use SOX6 protein, coat 96-well microtiter plate, the coating concentration is 2 μg/ml, 100 μl per well , placed at 4°C overnight. The next day, the coating liquid in the wells was discarded, and 100 μl of blocking solution was added to each well for blocking at 37°C for 1 h. The blocking solution in the well was discarded, and 100 μl of the phage supernatant obtained from the four rounds of screening treated with the blocking solution was added to each well as the primary antibody, and incubated at 37°C for 1 hour. Plates were washed 12 times with PBST wash solution. Add 100 μl of secondary antibody (HRP-M13 Antibody, dilution 1:10000) to each well and incubate at 37°C for 1 hour. Plates were washed 12 times with PBST wash solution. Add 100 μl of chromogenic substrate to each well, and react in the dark for 5-15 minutes, then add 50 μl of stop solution to each well to stop the reaction. Place the 96-well ELISA plate on a plate reader to read the OD 450 absorbance value. Analyze the ELISA results and determine the number of positive wells.
通过间接ELISA方法检测130个单克隆对应的噬菌体上清同SOX6蛋白的反应性，根据间接ELISA试验的结果挑选出了20个单克隆，这些单克隆均同SOX6蛋白有较好的反应性且同BSA蛋白的反应值较弱。将20个单克隆的培养菌液送测序公司测序。The reactivity of the phage supernatant corresponding to 130 monoclonals with SOX6 protein was detected by indirect ELISA method, and 20 monoclonals were selected according to the results of indirect ELISA test, and these monoclonals all had good reactivity with SOX6 protein and the same The response value of BSA protein was weak. The culture liquid of 20 single clones was sent to a sequencing company for sequencing.
SOX6纳米抗体表达活性和亲和性：SOX6 nanobody expression activity and affinity:
原核表达重组质粒的构建：将上述测序结果正确的克隆株的甘油菌接种5ml 2×YTAG培养基培养，并利用质粒小量提取试剂盒提取质粒作为原核表达的模板质粒。之后设计用于原核表达的引物，并在引物的5’端和3'端分别引入BamHⅠ和SalI酶切位点。利用设计的引物扩增纳米抗体VHH序列，并通过上述酶切位点将其连接入pColdⅠ原核表达载体，构建纳米抗体原核表达重组质粒以进行纳米抗体的SOX6特异性鉴定。Construction of recombinant plasmids for prokaryotic expression: inoculate 5 ml of 2×YTAG medium with glycerol bacteria of the clones with correct sequencing results, and use the plasmid mini-extraction kit to extract plasmids as template plasmids for prokaryotic expression. Afterwards, primers for prokaryotic expression were designed, and BamHI and SalI restriction sites were introduced at the 5' and 3' ends of the primers, respectively. The designed primers were used to amplify the VHH sequence of the Nanobody, and then ligated it into the pColdI prokaryotic expression vector through the restriction site mentioned above to construct a recombinant plasmid for the prokaryotic expression of the Nanobody to identify the SOX6 specificity of the Nanobody.
原核表达的引物：Primers for prokaryotic expression:
(SEQ ID No.2)F:(SEQ ID No. 2)F:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA;
(SEQ ID No.3)R:(SEQ ID No.3)R:
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC。TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC.
筛选步骤如下The screening steps are as follows
将重组质粒和pColdⅠ空载转化入BL21(DE3)菌株并获得相应的纳米抗体表达菌株。而后对纳米抗体进行诱导表达，具体方法为：The recombinant plasmid and pCold I were transformed into BL21(DE3) strain with empty load, and the corresponding nanobody expression strain was obtained. Then, the expression of the nanobody is induced, and the specific method is as follows:
将转化后涂板后的菌液进行过夜培养，次日挑取培养板上的单克隆菌落过夜培养。将次日培养的菌液进行保菌。The transformed bacterial solution was cultured overnight, and the next day, the monoclonal colonies on the culture plate were picked and cultured overnight. Bacteria preservation was carried out on the cultured bacteria solution the next day.
吸取10μl甘油菌接种于5mlAmp抗性的LB培养基，30℃振荡培养过夜；Take 10 μl of glycerol bacteria and inoculate them in 5ml of Amp-resistant LB medium, shake and culture at 30°C overnight;
第二天吸取50μl菌液接种5mlAmp抗性的LB培养基，各接种2管，37℃振荡培养至OD600为0.6；On the second day, draw 50 μl of bacterial liquid to inoculate 5 ml of Amp-resistant LB medium, inoculate 2 tubes each, and culture with shaking at 37°C until the OD 600 is 0.6;
在其中1管菌液中加入IPTG诱导(终浓度0.2mM)，另1管不加IPTG做为未诱导对照，15℃振荡培养过夜；Add IPTG to one tube of bacteria solution (final concentration 0.2mM) for induction, and the other tube without IPTG as the uninduced control, shake culture overnight at 15°C;
同时做BL21(DE3)空菌株对照，空菌株对照培养使用无抗性的LB培养基。At the same time, do BL21 (DE3) empty strain control, and the empty strain control culture uses LB medium without resistance.
SOX6纳米抗体纯化：Purification of SOX6 Nanobody:
将诱导表达成功的1L SOX6纳米抗体菌液分装，11000×g，4℃离心15min，弃掉上清。使用100ml PBS重悬菌体沉淀后分装50ml离心管，每管分装25ml。将50ml离心管中的菌液分别在超声破碎仪中进行超声破碎处理。待超声结束，16500×g离心10min，收集超声上清并过滤0.02μm滤膜，以备上机(AKTApure)纯化。Aliquot 1L of the SOX6 nanobody bacterial solution that successfully induced expression, centrifuge at 11,000×g for 15 minutes at 4°C, and discard the supernatant. Use 100ml PBS to resuspend the bacterial pellet, and then divide into 50ml centrifuge tubes, each tube is divided into 25ml. The bacterial solution in the 50ml centrifuge tube was ultrasonically disrupted in a sonicator. After the sonication is over, centrifuge at 16500×g for 10 min, collect the sonicated supernatant and filter it through a 0.02 μm filter membrane for purification on a machine (AKTApure).
根据所纯化蛋白大小，设置0.5Mpa柱前压，2Mpa系统压。待SOX6纳米抗体超声样品全部上柱后，分别设置5％，10％，20％，30％，40％，50％，60％的咪唑(咪唑浓度为500mM)进行洗脱。并回收洗脱液鉴定是否为目的蛋白。According to the size of the purified protein, set the pre-column pressure of 0.5Mpa and the system pressure of 2Mpa. After all the ultrasonic samples of SOX6 Nanobody were applied to the column, 5%, 10%, 20%, 30%, 40%, 50%, and 60% imidazole (imidazole concentration was 500mM) were respectively set for elution. And recover the eluate to identify whether it is the target protein.
纳米抗体的SDS-PAGE鉴定：SDS-PAGE identification of Nanobodies:
将对纳米抗体的表达进行SDS-PAGE鉴定，具体方法为：The expression of nanobodies will be identified by SDS-PAGE, the specific method is:
吸取1ml菌液于1.5ml离心管，13000rpm离心2min；Pipette 1ml of bacterial solution into a 1.5ml centrifuge tube, centrifuge at 13000rpm for 2min;
弃上清，使用PBS缓冲液洗涤菌体沉淀2次；Discard the supernatant, and wash the cell pellet twice with PBS buffer;
用20μl PBS缓冲液重悬菌体沉淀，而后加入5μl 5×蛋白上样缓冲液，并于沸水中煮样5分钟。用10％的聚丙烯酰胺凝胶对样品进行电泳。待电泳结束后，用考马斯亮蓝染液染胶1h，而后用脱色液进行脱色。Resuspend the bacterial pellet with 20 μl PBS buffer, then add 5 μl 5× protein loading buffer, and boil the sample in boiling water for 5 minutes. The samples were electrophoresed on a 10% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue staining solution for 1 h, and then decolorized with decolorizing solution.
SOX6纳米抗体的亲和性：Affinity of SOX6 Nanobodies:
用5μg/ml得SOX6包被ELISA板；经BSA封闭后将纯化稀释后的SOX6纳米抗体作为一抗，分别梯度稀释到5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.3125μg/ml，进行ELISA鉴定。Coat the ELISA plate with SOX6 obtained at 5 μg/ml; after blocking with BSA, use the purified and diluted SOX6 Nanobody as the primary antibody, and serially dilute to 5 μg/ml, 2.5 μg/ml, 1.25 μg/ml, 0.625 μg/ml, 0.3125μg/ml for ELISA identification.
结果：result:
1.ELISA筛选结果1. ELISA screening results
通过间接ELISA方法检测130个单克隆对应的噬菌体上清同SOX6蛋白的反应性，根据间接ELISA试验的结果挑选出了10个单克隆，这些单克隆均同SOX6蛋白有较好的反应性且同BSA蛋白的反应值较弱(表1)。将10个单克隆的培养菌液送测序公司测序。The reactivity of the phage supernatant corresponding to 130 monoclonals with SOX6 protein was detected by indirect ELISA method, and 10 monoclonals were selected according to the results of indirect ELISA test, and these monoclonals had good reactivity with SOX6 protein and the same The response value of BSA protein was weak (Table 1). The culture liquid of 10 single clones was sent to a sequencing company for sequencing.
表1 SOX6单克隆ELISA筛选结果Table 1 SOX6 monoclonal ELISA screening results
经ELISA筛选后，阳性最强的两个克隆是SOX6-VHH1和SOX6-VHH2，将上述所有10个克隆进行测序，经测序并预测的氨基酸序列为：After screening by ELISA, the two most positive clones were SOX6-VHH1 and SOX6-VHH2. All the above 10 clones were sequenced, and the sequenced and predicted amino acid sequences were:
N.1.(SEQ ID No.1)(SOX6-VHH1)氨基酸序列：N.1. (SEQ ID No.1) (SOX6-VHH1) amino acid sequence:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP；ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP;
N.2.(SEQ ID No.4)(SOX6-VHH2)氨基酸序列：N.2. (SEQ ID No.4) (SOX6-VHH2) amino acid sequence:
ESGGGSVQPGGSLRLSCAASGFTFSGYYMSWVRQAPGEEPEWVTFITNDGSGVRYADSVKGRFTVSRNNVENTVYLRMDNLQPNDTARYYCVRGRLTATSPLIPDDSWGQGTQVTVSS；ESGGGSVQPGGSLRLSCAASGFTFSGYYMSWVRQAPGEEPEWVTFITNDGSGVRYADSVKGRFTVSRNNVENTVYLRMDNLQPNDTARYYCVRGRLTATSPLIPDDSWGQGTQVTVSS;
N.3.(SEQ ID No.5)(SOX6-VHH3)氨基酸序列：N.3. (SEQ ID No.5) (SOX6-VHH3) amino acid sequence:
ESGGGLVQPGGSLRLSCLASGFSFDSYAMSWYRQAPGKEREWVAHITSGGSTNYSDSVKGRFTISRDNAKNAVYLQMDNLKPEDTAVYYCNEVSTSLDDYDYWGKGTQVTVSA；ESGGGLVQPGGSLRLSCLASGFSFDSYAMSWYRQAPGKEREWVAHITSGGSTNYSDSVKGRFTISRDNAKNAVYLQMDNLKPEDTAVYYCNEVSTSLDDYDYWGKGTQVTVSA;
N.4.(SEQ ID No.6)(SOX6-VHH4)氨基酸序列：N.4. (SEQ ID No.6) (SOX6-VHH4) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVS；ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVS;
N.5.(SEQ ID No.7)(SOX6-VHH5)氨基酸序列：N.5. (SEQ ID No.7) (SOX6-VHH5) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS;
N.6.(SEQ ID No.8)(SOX6-VHH6)氨基酸序列：N.6. (SEQ ID No.8) (SOX6-VHH6) amino acid sequence:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVS；ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVS;
N.7.(SEQ ID No.9)(SOX6-VHH7)氨基酸序列：N.7. (SEQ ID No.9) (SOX6-VHH7) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFTLSSYNMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；ESGGGLVQPGGSLRLSCAAPGFTLSSYNMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS;
N.8.(SEQ ID No.10)(SOX6-VHH8)氨基酸序列：N.8. (SEQ ID No.10) (SOX6-VHH8) amino acid sequence:
ESGGGLVQPGGSLRLSCLAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；ESGGGLVQPGGSLRLSCLAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS;
N.9.(SEQ ID No.11)(SOX6-VHH9)氨基酸序列：N.9. (SEQ ID No.11) (SOX6-VHH9) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYSSWGQGTQVTVSS；ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYSSWGQGTQVTVSS;
N.10.(SEQ ID No.12)(SOX6-VHH10)氨基酸序列：N.10. (SEQ ID No.12) (SOX6-VHH10) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMDSLKPEDTALYYCAKRNRAGLDAYDYWGQGIQVTVSS。ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMDSLKPEDTALYYCAKRNRAGLDAYDYWGQGIQVTVSS.
2.SOX6纳米抗体亲和性检测：2. SOX6 Nanobody Affinity Detection:
选择上述两个最强的阳性克隆进行表达后，亲和性检测结果为：SOX6纳米抗体N.1对SOX6蛋白的灵敏度为1.25μg/ml；SOX6纳米抗体N.2对SOX6蛋白的灵敏度为2.5μg/ml。After the above two strongest positive clones were selected for expression, the affinity test results were as follows: the sensitivity of SOX6 Nanobody N.1 to SOX6 protein was 1.25 μg/ml; the sensitivity of SOX6 Nanobody N.2 to SOX6 protein was 2.5 μg/ml.
由以上实施例可知，本发明提供的SOX6纳米抗体能够特异性结合SOX6蛋白，对该蛋白的灵敏度为1.25μg/ml。It can be seen from the above examples that the SOX6 nanobody provided by the present invention can specifically bind to the SOX6 protein, and the sensitivity to this protein is 1.25 μg/ml.
3.对SOX6纳米抗体1进行表达和纯化3. Expression and purification of SOX6 Nanobody 1
SOX6纳米抗体在40％咪唑浓度成功洗脱下来，经SDS-PAGE鉴定条带单一，大小为15kDa。根据蛋白分子量及His-tag标签对所述蛋白进行Western Blotting鉴定，蛋白分子量为15kDa的蛋白为SOX6纳米抗体。实验结果如图1。The SOX6 nanobody was successfully eluted at a concentration of 40% imidazole, and the band was identified by SDS-PAGE as a single band with a size of 15kDa. According to the protein molecular weight and His-tag tag, the protein was identified by Western Blotting, and the protein with a protein molecular weight of 15kDa was the SOX6 nanobody. The experimental results are shown in Figure 1.
实施例2Example 2
细胞培养及SOX6纳米抗体功能：研究发现SOX6在黑色素瘤细胞中高表达且与细胞增殖迁移有重要联系。通过细胞划痕实验和细胞免疫组织化学来鉴定SOX6纳米抗体。用含10％FBS的DMEM高糖培养基培养B16细胞，待细胞长至6孔板80％时消化传代，将一部分细胞1:2传至新的6孔板中，另一部分传至有无毒的载玻片上，摇匀，置于37℃，5％CO2培养箱中培养过夜。Cell culture and SOX6 nanobody function: The study found that SOX6 is highly expressed in melanoma cells and has an important relationship with cell proliferation and migration. The SOX6 nanobody was identified by cell scratch assay and cell immunohistochemistry. Cultivate B16 cells with DMEM high-glucose medium containing 10% FBS, digest and subculture when the cells grow to 80% of the 6-well plate, transfer a part of the cells to a new 6-well plate at a ratio of 1:2, and pass the other part to a non-toxic Shake well and place in a 37°C, 5% CO 2 incubator overnight.
免疫细胞化学定位：待载玻片的B16细胞长满后用PBS冲洗3次，之后加入500μl4％的多聚甲醛于4℃环境中静置30min；之后使用PBS冲洗3次并加入3％的H2O2溶液，静置15min；PBS充分冲洗，加入牛血清封闭20min后加入SOX6纳米抗体置于4℃环境过夜孵育；第二天将孵育盒放于室温复温30min后，PBS冲洗3次；加入带有His标签的二抗在37℃孵育30min，PBS冲洗3次，滴加50μl DAB在暗盒孵育3-10min后于显微镜下观察。显色完成后滴加蒸馏水终止染色，弃掉蒸馏水后加入苏木精进行核染，30s后自来水冲洗返蓝。Immunocytochemical localization: After the B16 cells on the slide are overgrown, wash with PBS 3 times, then add 500 μl of 4% paraformaldehyde and let stand at 4°C for 30 minutes; then wash with PBS 3 times and add 3% H 2 O 2 solution, let it stand for 15 minutes; PBS was fully rinsed, added bovine serum to block for 20 minutes, then added SOX6 nano-antibody and incubated overnight at 4°C; the next day, put the incubation box at room temperature for 30 minutes, then rinsed with PBS 3 times; Add the secondary antibody with His tag and incubate at 37°C for 30min, wash with PBS 3 times, add 50μl DAB dropwise, incubate for 3-10min in the dark box and observe under the microscope. After the color development was completed, distilled water was added dropwise to stop the staining. After the distilled water was discarded, hematoxylin was added for nuclear staining. After 30 seconds, it was washed with tap water to return to blue.
细胞划痕实验鉴定：DMEM高糖培养基培养B16细胞在6孔板中长满一层后，用消毒的直尺和中枪头在培养孔中划1条直线，划时枪头保持竖直。PBS冲洗3次，去除掉落的细胞，重新加入无血清的DMEM高糖培养基置于培养箱中培养。每6个小时在同一位置记录一次并拍照。Cell scratch test identification: After culturing B16 cells in DMEM high-glucose medium and growing a layer in the 6-well plate, draw a straight line in the culture well with a sterilized ruler and medium tip, and keep the tip vertical when drawing . Rinse with PBS 3 times, remove the fallen cells, add serum-free DMEM high-glucose medium again and place in the incubator for culture. Record and photograph every 6 hours at the same location.
结果：result:
1.SOX6纳米抗体在B16细胞中的定位1. Localization of SOX6 nanobody in B16 cells
对照组添加的是等量的PBS，实验组添加SOX6纳米抗体的浓度为100μg/ml。根据结果发现：SOX6纳米抗体主要分布到B16细胞的细胞质中，说明该纳米抗体可以进入到细胞内发挥作用。实验结果如图2(箭头所示)。The same amount of PBS was added to the control group, and the concentration of SOX6 Nanobody was added to the experimental group at a concentration of 100 μg/ml. According to the results, it is found that the SOX6 nanobody is mainly distributed in the cytoplasm of B16 cells, indicating that the nanobody can enter the cell and play a role. The experimental results are shown in Figure 2 (arrows).
2.添加SOX6纳米抗体对B16细胞划痕实验的影响2. Effect of adding SOX6 nanobody on B16 cell scratch experiment
对照组添加的是等量的PBS，实验组添加SOX6纳米抗体的浓度为100μg/ml。根据结果发现：添加PBS的组在36小时内，划痕基本长满细胞。实验组在24小时处最明显，36小时还有20％左右未长满，结果说明SOX6纳米抗体添加B16细胞后，随着时间的增长起到了抑制B16细胞迁移的过程。实验结果如图3。The same amount of PBS was added to the control group, and the concentration of SOX6 Nanobody was added to the experimental group at a concentration of 100 μg/ml. According to the results, it was found that in the group added with PBS, the scratches were basically covered with cells within 36 hours. In the experimental group, it was most obvious at 24 hours, and about 20% were not overgrown at 36 hours. The results showed that after the SOX6 nanobody was added to B16 cells, it inhibited the migration of B16 cells as time went on. The experimental results are shown in Figure 3.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.
序列表sequence listing
<110> 山西农业大学<110> Shanxi Agricultural University
<120> 一种SOX6纳米抗体及其应用<120> A kind of SOX6 nanobody and its application
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1 5 10 151 5 10 15
Cys Leu Ala Ser Gly Phe Ser Phe Asp Ser Tyr Ala Met Ser Trp TyrCys Leu Ala Ser Gly Phe Ser Phe Asp Ser Tyr Ala Met Ser Trp Tyr
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Ser Arg Asp Asn Ala Lys Asn Ala Val Tyr Leu Gln Met Asp Asn LeuSer Arg Asp Asn Ala Lys Asn Ala Val Tyr Leu Gln Met Asp Asn Leu
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100 105 110 100 105 110
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Cys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp ValCys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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115 115
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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1 5 10 151 5 10 15
Cys Ala Ala Pro Gly Phe Thr Leu Ser Ser Tyr Asn Met Ser Trp ValCys Ala Ala Pro Gly Phe Thr Leu Ser Ser Tyr Asn Met Ser Trp Val
20 25 30 20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala AlaArg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45 35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe ThrSer Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn ThrIle Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
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Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn ArgLeu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95 85 90 95
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100 105 110 100 105 110
Val Ser SerVal Ser Ser
115 115
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<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu SerGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 151 5 10 15
Cys Leu Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp ValCys Leu Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30 20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala AlaArg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
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Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe ThrSer Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
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Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn ThrIle Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
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Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn ArgLeu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95 85 90 95
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100 105 110 100 105 110
Val Ser SerVal Ser Ser
115 115
<210> 11<210> 11
<211> 115<211> 115
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu SerGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 151 5 10 15
Cys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp ValCys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30 20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala AlaArg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45 35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe ThrSer Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn ThrIle Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 8065 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn ArgLeu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95 85 90 95
Ala Gly Leu Ser Ala Tyr Ser Ser Trp Gly Gln Gly Thr Gln Val ThrAla Gly Leu Ser Ala Tyr Ser Ser Trp Gly Gln Gly Thr Gln Val Thr
100 105 110 100 105 110
Val Ser SerVal Ser Ser
115 115
<210> 12<210> 12
<211> 115<211> 115
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu SerGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 151 5 10 15
Cys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp ValCys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30 20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala AlaArg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45 35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe ThrSer Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60 50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asp SerIle Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser
65 70 75 8065 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn ArgLeu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95 85 90 95
Ala Gly Leu Asp Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val ThrAla Gly Leu Asp Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110 100 105 110
Val Ser SerVal Ser Ser
115 115