›› 2010, Vol. 37 ›› Issue (3): 65-70.

• 生物技术 • 上一篇    下一篇

猪瘟病毒四川分离株E 2基因的分子克隆、原核表达及免疫学活性分析

韩国全1，2，郭万柱1，2，林华1，王利娜3，张博1，陈弟诗1，陈杨1   

  1. (1.四川农业大学动物生物技术中心， 雅安 625014； 2.四川农业大学动物疫病与人类健康四川省重点实验室， 雅安 625014； 3.四川省成都市产品质量监督检验院， 成都 610041)
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-03-20 发布日期:2010-03-20
  • 通讯作者: 郭万柱

Cloning，Expression and Immunological Activity Analysis E 2 Gene of Classical Swine Fever Virus Isolates from Sichuan Strain

HAN Guo-quan1,2,GUO Wan-zhu1,2,LIN Hua1,WANG Li-na3, ZHANG Bo1,CHEN Di-shi1,CHEN Yang1   

  1. (1.Animal Biotechnology Center,Sichuan Agricultural University,Ya’an 625014,China; 2.Key Laboratory of Animal Disease and Human Health of Sichuan Province,Sichuan Agricultural University,Ya’an 625014,China; 3.Chengdu Product Quality Supervision and Inspection Institute of Sichuan Province,Chengdu 610041,China)
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-03-20 Published:2010-03-20
  • Contact: GUO Wan-zhu

摘要： 分子克隆猪瘟病毒(classical swine fever virus，CSFV)四川分离株E2基因，并对其进行原核表达及免疫学活性分析。PK-15细胞培养增殖CSFV四川分离株提取总RNA，运用RT-PCR扩增E 2基因，定向克隆构建原核重组表达载体，转化E.coli Rosetta-gami-TM(DE3) plysS，IPTG诱导表达，SDS-PAGE检测蛋白表达，Western blotting分析表达蛋白的免疫学活性。试验结果表明，成功克隆了E 2基因，共1119 bp，包含有编码E2蛋白的完整序列，编码373个氨基酸；成功构建了CSFV E2基因完整阅读框、主要抗原区原核表达载体pET-E2(pe)、pET-mE2(pe)；蛋白质电泳结果显示，成功表达出约45.32、28.49 ku两目的蛋白，而且表达的E2(pe)、mE2(pe)融合蛋白均能被CSFV阳性血清所识别，具有良好的免疫学反应活性。因此，本试验成功获得CSFV四川分离株E2基因，表达出具有生物学活性的E2(pe)、mE2(pe)融合蛋白。

关键词: 猪瘟病毒; 四川分离株; E 2基因; 原核表达; 免疫学活性

Abstract:

In order to investigate the cloning,expression and immunological activity analysis E2 gene of classical swine fever virus (CSFV) isolates from Sichuan. The CSFV total RNA of Sichuan strain was extracted from infected PK-15 cells. The objective E 2 gene was cloned by RT-PCR and directionally cloned into prokaryotic expression vector. The expressing plasmids were transformed into E.coli Rosetta-gami-TM(DE3) plysS. The recombinant bacteria were induced expression by IPTG. The expressed proteins were analyzed by SDS-PAGE and Western blotting. The results indicated that the objective E 2 gene was obtained and contains E2 protein complete codon DNA sequence (CDS), which have 1119 bp，encoding 373 amino acid. The recombinant expression plasmids were successfully constructed of complete CDS and N terminal major antigen gene of CSFV E2 protein,which named pET-E2(pe) and pET-mE2(pe). The recombinant bacteria could express the target fusion proteins which are about 45.32 ku and 28.49 ku by SDS-PAGE detection. The expression of E2 (pe),mE2 (pe) fusion proteins could be recognized by CSFV-positive sera,and had good immunological reaction activity. The research obtained the CSFV E2 gene of Sichuan strain. The E2 (pe)，mE2 (pe) fusion proteins were expressed and have biological activity.

Key words: classical swine fever virus; Sichuan strain; E 2 gene; prokaryotic expression; immunological activity

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