文章摘要
赵丽丽,刘敏,哈卓,刘巍巍,赵永欣,葛俊伟,乔薪瑗,李一经.传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析[J].水产学报,2010,34(4):604~610
传染性胰腺坏死病毒VP3蛋白的原核表达及抗原性分析
Prokaryotic expression of VP3 gene of infectious pancreas necrosis virus and antigenicity of expressed product
投稿时间:2009-11-04  修订日期:2009-12-28
DOI:10.3724/SP.J.1231.2010.06690
中文关键词: 传染性胰腺坏死病毒  VP3基因  原核表达  抗血清
英文关键词: Infectious Pancreas Necrosis Virus  VP3 gene  Prokaryotic Expression  antisera
基金项目:黑龙江省自然科学基金(C200837)
作者单位E-mail
赵丽丽 东北农业大学 zhaolili213@163.com 
刘敏 东北农业大学动物医学学院  
哈卓 东北农业大学动物医学学院  
刘巍巍 东北农业大学动物医学学院  
赵永欣 东北农业大学动物医学学院  
葛俊伟 东北农业大学动物医学学院  
乔薪瑗 东北农业大学动物医学学院  
李一经 东北农业大学动物医学学院  
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中文摘要:
      应用RT-PCR方法扩增了IPNV编码内衣壳VP3蛋白的基因615 bp,将VP3基因克隆至原核表达载体pET30b,并在大肠杆菌BL21中得到了表达。通过SDS-PAGE分析表明,重组菌诱导后得到了预期大小约30 ku的VP3蛋白,与理论值相符,经薄层扫描分析表明目的蛋白表达量可占菌体总蛋白的30%。用镍离子亲和层析柱纯化可溶性的VP3蛋白,并制备抗血清。Western-blotting结果显示,VP3蛋白可被兔抗IPNV阳性血清识别;间接ELISA结果显示,IPNV细胞培养物作为抗原,兔抗VP3蛋白高免血清稀释度为1∶25 600时,P/N>2,抗血清可与IPNV全病毒发生反应,以上两项结果说明,表达的VP3蛋白与天然的IPNV VP3蛋白一样具有相同的抗原性。试验利用原核表达系统成功地高效表达了IPNV VP3蛋白,融合蛋白以可溶性形式存在,并制备了高效价的抗血清。
英文摘要:
      Infectious pancreatic necrosis (IPN) virus, the etiologic agent of infectious pancreatic necrosis in salmonid fish, causes significant losses to the aquaculture industry. The gene for the viral inner capsid protein (VP3) was amplified by RT PCR method from IPNV, and cloned into pET30b vector. The expression of recombinant plasmid pET30b VP3 in E.coli BL21(DE3) was induced and detected by SDS PAGE analysis. The predicted molecular weight for unmodified r trunc VP3 was approximately 30 ku and this was found to be the case for E.coli protein. The amount of expression made up 30 percent of the bacteria protein total expression by thin layer scanning analysis. The results showed that the VP3 gene of IPNV can express successfully in E.coli BL21. The fusion protein was purified with ProBondTM resin from the suspension centrifuged and the antisera against VP3 protein was produced. The pET30b VP3 fusion protein can be recognized by the positive serum of IPNV by Western-blotting analysis. The prepared antisera reacted specifically with IPNV antigen by indirect ELISA. The antisera against VP3 protein had OD values at least twice that obtained for the negative control serum at a dilution of 1∶25 600. The results showed that the expressed VP3 protein was immunogenical and antigenical which is the same as the natural IPNV VP3 protein. In this experiment the IPNV VP3 protein was expressed successfully by using prokaryotic expression system. The expressed fusion protein was active and the antisera against VP3 protein were produced.
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