抗鹅细小病毒NS1蛋白单克隆抗体可变区的原核表达

doi:10.7668/hbnxb.20192216

摘要/Abstract

摘要： 为了在大肠杆菌中表达抗鹅细小病毒（GPV） NS1蛋白单克隆抗体轻链可变区（VL）和重链可变区（VH）基因，并测定其与NS1蛋白结合活性。对抗GPV NS1蛋白单克隆抗体VL、VH基因核苷酸序列依据大肠杆菌偏爱密码子进行优化，人工合成获得了含有可变区基因的重组质粒pUC57-VL和pUC57-VH。然后用Bam H Ⅰ/Xho Ⅰ双酶切pUC57-VL、pUC57-VH ，回收340 bp的VL基因和370 bp的VH基因。目的基因通过Bam H Ⅰ/Xho Ⅰ多克隆位点分别插入至原核表达载体pET-32a，获得重组质粒pET-VL和pET-VH。重组质粒经Bam H Ⅰ单酶切和Bam H Ⅰ/Xho Ⅰ双酶切及测序鉴定。重组质粒分别转化大肠杆菌Rosetta （DE3），经IPTG诱导，获得了重组蛋白TRX-VL和TRX-VH的表达，分子量分别为30.3，31.4 ku。纯化后的重组蛋白能与His标签单克隆抗体发生特异性结合，鉴定结果表明，获得的纯化蛋白为目的蛋白。间接ELISA分析表明，0.4 μg的TRX-VH可与25 ng的GST-NS1蛋白特异性结合而0.4 μg的TRX-VL不能与各包被量的GST-NS1蛋白特异性结合，TRX-VH与GST-NS1蛋白的特异性结合可被1 ∶ 200稀释的GPV感染鹅血清完全阻断。

关键词: 鹅细小病毒, 非结构蛋白, 单克隆抗体, 抗体可变区, 原核表达

Abstract: In order to express the light chain variable domain(VL) and heavy chain variable domain(VH) genes of the monoclonal antibody against Goose parvovirus(GPV) NS1 protein in E. coli, and determine their binding activity to the NS1 protein. The nucleotide sequences of the VL and VH genes of the monoclonal antibody against GPV NS1 protein were optimized according to the preferred codons of E. coli, and recombinant plasmids pUC57-VL and pUC57-VH containing variable region genes were artificially synthesized. Then pUC57-VL and pUC57-VH were double digested with Bam H Ⅰ/Xho Ⅰ, and 340 bp of VL gene and 370 bp of VH gene were recovered. The target genes were inserted into the prokaryotic expression vector pET-32a through the Bam H Ⅰ/Xho Ⅰ multiple cloning sites, and the recombinant plasmids pET-VL and pET-VH were obtained. The recombinant plasmids were identified by Bam H Ⅰ single restriction enzyme digestion and Bam H Ⅰ/Xho Ⅰ double restriction enzyme digestion and sequencing. The recombinant plasmids were transformed into E. coli Rosetta(DE3), and induced by IPTG. The recombinant proteins TRX-VL and TRX-VH were expressed with molecular weights of 30.3, 31.4 ku, respectively. The purified recombinant proteins could specifically bind to the His tag monoclonal antibody. And the identification results showed that 0.4 μg of TRX-VH could specifically bind to 25 ng of GST-NS1 protein, but 0.4 μg of TRX-VL could not specifically bind to each coating amount of GST-NS1 protein. The specific binding of TRX-VH and GST-NS1 protein could be completely blocked by 1:200 diluted GPV infected goose serum.

Key words: Goose parvovirus, Non-structural protein, Monoclonal antibody, Antibody variable domain, Prokaryotic expression

中图分类号:

S852.65+9.2

于天飞, 谢鹏宇, 孙婉姝, 李静, 尹海畅, 黎明, 于志丹. 抗鹅细小病毒NS1蛋白单克隆抗体可变区的原核表达[J]. 华北农学报, 2021, 36(5): 211-215. doi: 10.7668/hbnxb.20192216.

YU Tianfei, XIE Pengyu, SUN Wanshu, LI Jing, YIN Haichang, LI Ming, YU Zhidan. Prokaryotic Expression of Variable Domain of Monoclonal Antibody Against Goose parvovirus NS1 Protein[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(5): 211-215. doi: 10.7668/hbnxb.20192216.

http://www.hbnxb.net/CN/Y2021/V36/I5/211

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