猪传染性胃肠炎病毒膜蛋白亲和肽的原核表达及其病毒亲和性分析

doi:10.7668/hbnxb.201751554

摘要/Abstract

摘要： 为了研究猪传染性胃肠炎病毒膜蛋白（M蛋白）亲和肽的病毒亲和性，建立快速有效的TGEV检测方法，设计了1条含有TGEV M蛋白亲和肽的串联基因，核苷酸序列依据大肠杆菌偏爱密码子进行优化，人工基因合成获得了含有M蛋白亲和肽基因的重组质粒pUC57-MQHT。然后，根据合成基因的序列，设计合成了1条特异性引物，以pUC57-MQHT为模板，通过PCR获得了150 bp的TGEV M蛋白亲和肽基因。亲和肽基因通过Bam HⅠ/Xho Ⅰ多克隆位点分别插入至原核表达载体pET-32a和pGEX-6p-1，获得重组质粒pET-32a-MQHT和pGEX-6p-MQHT。重组质粒经Bam HⅠ单酶切和Bam HⅠ/Xho Ⅰ双酶切及测序鉴定。重组质粒分别转化至大肠杆菌Rosetta（DE3），经IPTG诱导，获得了重组蛋白TRX-MQHT和GST-MQHT，分子质量分别为25，31 ku。切胶纯化后，重组蛋白TRX-MQHT和GST-MQHT分别经His标签单克隆抗体和GST标签单克隆抗体鉴定，鉴定结果表明，获得的纯化蛋白为目的蛋白。Western Blot分析表明，2种重组蛋白可与TGEV特异性结合。Dot-ELISA分析表明，在TGEV滴度为1×10³，5×10²，2.5×10² TCID50/mL时2种重组蛋白都可显示出阳性反应，而在1.25×10² TCID50/mL滴度时无可见斑点，具有良好的TGEV亲和性。阻断试验表明，1：100或1：200稀释的TGEV阳性血清可完全阻断TGEV H株（1×10⁵ TCID50/mL）与2种重组蛋白的结合，特异性良好。为建立TGEV诊断方法奠定了一定的理论和物质基础。

关键词: 传染性胃肠炎病毒, 膜蛋白, 亲和肽, 原核表达, 亲和性

Abstract: In order to investigate the viral affinity of Transmissible gastroenteritis virus of swine (TGEV) membrane protein (M protein) affinity peptide and establish a rapid and effective TGEV detection method, a tandem gene containing the TGEV M protein affinity peptide was designed. The nucleotide sequence was optimized according to the E. coli preference codon. A recombinant plasmid pUC57-MQHT containing M protein affinity peptide gene was obtained by artificial gene synthesis. According to the sequence of the synthetic gene, a specific primer was designed and synthesized. And then, using pUC57-MQHT as a template, TGEV M protein affinity peptide gene (about 150 bp) was obtained by PCR. The affinity peptide gene was respectively inserted into the Bam H Ⅰ/Xho Ⅰ multiple cloning sites of the prokaryotic expression vectors pET-32a and pGEX-6p-1 to obtain the recombinant plasmids pET-32a-MQHT and pGEX-6p-MQHT. The recombinant plasmids were identified by single enzyme digestion of Bam HⅠ, double enzyme digestion of Bam HⅠ/Xho Ⅰ and sequencing. The recombinant plasmids were transformed into E. coli Rosetta (DE3) and induced by IPTG to obtain the expression of recombinant proteins TRX-MQHT and GST-MQHT, respectively. The molecular weights of the two recombinant proteins were 25, 31 ku, respectively. After purification, the recombinant proteins TRX-MQHT and GST-MQHT were identified by His-tag monoclonal antibody and GST-tag monoclonal antibody, respectively. The results indicated that the purified proteins obtained were the target proteins. Western Blot analysis showed that the two recombinant proteins could specifically bind to TGEV. Dot-ELISA analysis indicated that the two recombinant proteins showed positive reaction to TGEV at 1×10³, 5×10² and 2.5×10² TCID50/mL, but no visible spot was found at 1.25×10² TCID50/mL. The results showed that the two recombinant proteins had good affinity to TGEV virions. Dot-ELISA analysis showed that the minimum binding titer of the two recombinant proteins to TGEV virions was 2.5×10² TCID50/mL. Blocking experiments showed that TGEV-positive serum diluted 1:100 or 1:200 could completely blocked the binding of the two recombinant proteins to TGEV H strain (1×10⁵ TCID50/mL). The results showed that the specificity of the two recombinant proteins was good. This study laid a certain theoretical and material basis for the establishment of TGEV diagnostic methods.

中图分类号:

S858.28

于天飞, 董慧莹, 谢鹏宇, 孙婉姝, 尹海畅, 黎明, 于志丹. 猪传染性胃肠炎病毒膜蛋白亲和肽的原核表达及其病毒亲和性分析[J]. 华北农学报, 2019, 34(5): 224-230. doi: 10.7668/hbnxb.201751554.

YU Tianfei, DONG Huiying, XIE Pengyu, SUN Wanshu, YIN Haichang, LI Ming, YU Zhidan. Prokaryotic Expression of Affinity Peptides to Membrane Protein of Transmissible gastroenteritis virus and Analysis of Viral Affinity Activity[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(5): 224-230. doi: 10.7668/hbnxb.201751554.

http://www.hbnxb.net/CN/Y2019/V34/I5/224

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