TNF-α纳米抗体的筛选、表达及特异性检测 <sup>*</sup>

doi:10.13523/j.cb.2002034

中国生物工程杂志

2020, Vol. 40

Issue (7): 15-21 DOI: 10.13523/j.cb.2002034

研究报告

TNF-α纳米抗体的筛选、表达及特异性检测 ^*

蔺士新¹,刘东晨²,雷云²,熊盛²,谢秋玲^1,^**(

)

1 暨南大学生命科学技术学院广州 510632
2 基因工程药物国家工程研究中心广州 510632

Screening, Expression and Specificity Detection of Anti-TNF-α Nanobody

LIN Shi-xin¹,LIU Dong-chen²,LEI Yun²,XIONG Sheng²,XIE Qiu-ling^1,^**(

)

1 College of life science and technology, Jinan university, Guangzhou 510632, China
2 National Engineering Research Center of Genetic Medicine, Jinan university, Guangzhou 510632, China

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摘要：

目的:构建肿瘤坏死因子(tumor necrosis factor,TNF)纳米抗体的噬菌体文库,筛选并表达与TNF-α具有亲和特异性的纳米抗体。方法:(1)利用TNF-α免疫羊驼,提取外周血淋巴细胞总RNA,构建噬菌体文库;多次淘洗筛选到与TNF-α有亲和力的克隆。(2)通过ExPASy分析其分子量和亲疏水性等理化性质,并将筛选得到的VHH基因在大肠杆菌E.coli DH5α中表达。(3)表达的Nb_TNF-α蛋白质经过Ni金属螯合亲和层析纯化,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测蛋白质的抗原特异性和亲和力。结果:(1)通过噬菌体文库的构建和淘洗,筛选到8个与TNF-α有亲和力的VHH基因片段。(2)通过软件预测,这8个Nb_TNF-α蛋白均为亲水性蛋白,其分子量为19.6~20.1kDa;在大肠杆菌中重组表达这8个抗体蛋白。(3)ELISA检测结果表明,有5株纳米抗体Nb_TNF-α-1、Nb_TNF-α-2、Nb_TNF-α-3、Nb_TNF-α-4和Nb_TNF-α-5能与TNF-α特异性结合。结论:成功筛选并表达了5株具有TNF-α特异性的纳米抗体,可能成为抗TNF-α的候选药物。

关键词： 纳米抗体; 肿瘤坏死因子; 噬菌体文库; 筛选

Abstract:

Objective: To construct the phage library of anti-TNF-α (vascular endothelial growth factor) andto screen and express the nanobodies which have specificity and affinity with TNF-α. Methods: (1) The llama was immunized with TNF-α, and the total RNA of peripheral blood lymphocytes was extracted to construct a phage library, the clones having affinity with TNF-α were screened by multiple panning. (2) Then their molecular weight, pI and hydrophilicity were analyzed by ExPASy. And the VHH genes were cloned into the expression vector pNCS to construct the recombinant plasmids (pNCS-Nb_TNF-α) and to express these recombinant nanobodies (Nb_TNF-α) in E.coli DH5α. (3) The recombinant nanobodies were purified by Ni metal chelate affinity chromatography, followed by detection the specificity by enzyme linked immunosorbent assay (ELISA).Results: (1) Ten VHH gene fragments having affinity with TNF-α were obtained after phage library construction and panning. (2) Based on the bioinformatics analysis, it was found that eight nanobodies were hydrophilic proteins with molecular weights of 19.6-20.1kDa. All of Nb_TNF-α were expressed in E.coli DH5α in soluble form. (3) It was showed that five recombinant nanobodies, Nb_TNF-α-1, Nb_TNF-α-2, Nb_TNF-α-3, Nb_TNF-α-4 and Nb_TNF-α-5 could specifically bind to TNF-α. Conclusion: Eight nanobodies with specificity to TNF-α were screened and expressed in E.coli successfully, and five Nb_TNF-α showed good affinity with TNF-α, which could be possible candidates for anti-TNF-α drug.

Key words: Nanobody Tumor necrosis factor Phage library Screening

收稿日期: 2020-02-22 出版日期: 2020-08-13

ZTFLH:

Q816

基金资助: * 广东省省级科技计划资助项目(2015A020211016)

通讯作者: 谢秋玲 E-mail: txql@jnu.edu.cn

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引用本文:

蔺士新,刘东晨,雷云,熊盛,谢秋玲. TNF-α纳米抗体的筛选、表达及特异性检测 ^*[J]. 中国生物工程杂志, 2020, 40(7): 15-21.

LIN Shi-xin,LIU Dong-chen,LEI Yun,XIONG Sheng,XIE Qiu-ling. Screening, Expression and Specificity Detection of Anti-TNF-α Nanobody. China Biotechnology, 2020, 40(7): 15-21.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2002034 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I7/15

表1 引物列表

图1 羊驼免疫前后血清中TNF-α抗体滴度的检测

图2 重组质粒pComb-3X-VHH酶切鉴定

图3 噬菌体文库的淘洗

图4 TNF-α纳米抗体噬菌体文库的富集

图5 TNF-α纳米抗体文库筛选结果

表2 NbTNF-α抗原蛋白的理化性质

图6 重组表达的NbTNF-α的SDS-PAGE及Western blot结果分析

图7 纯化后的NbTNF-α蛋白

图8 NbTNF-α蛋白的特异性

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