摘要
用原核表达系统表达猪圆环病毒2型ORF3基因,分析ORF3蛋白的抗原性。根据GenBank上公布的猪圆环病毒2型(PCV-2)核苷酸序列进行分析,针对ORF3基因设计并合成1对特异性引物,PCR扩增该基因,并将此片段克隆入原核表达载体pET-32a(+)上,命名为pET32a-ORF3,转化大肠杆菌Rosetta-blue(DE3)pLacⅠ感受态细胞,1.0mmol/L IPTG37℃诱导表达。结果表明,PCR扩增得到315bp的片段,重组蛋白大小约为29ku,与预期大小相符。Western blotting分析结果表明,重组蛋白能与抗His-tag的单克隆抗体反应,不与PCV-2阳性血清发生反应。猪圆环病毒2型ORF3基因能在大肠杆菌中成功表达,但与阳性血清之间没有反应原性,为进一步研究PCV-2ORF3蛋白的功能及特性奠定基础。
To express ORF3 gene of porcine circovirus 2（PCV-2）in E.coli,analyze the antigenicity of ORF3 protein,according to the amino acid sequence of PCV-2 strain in GenBank,a pair of specific primer was designed to amplify the fragment encoding ORF3 gene.This fragment was cloned into the multiple cloning sites of pET-32a（＋）vector.The recombinant plasmid was designated pET32a-ORF3 and was transformed into E.coli Rosetta-blue（DE3）pLacⅠ competent cells.Expression of recombinant protein was induced by addition of final concentration of 1.0 mmol/L IPTG under 37 ℃ condition.The length of PCR product of interest was 315 base pairs.The size of recombinant amino acid was 29 ku.The results of western blotting showed that it could be recognized by anti-His tagged mouse monoclonal antibody and could not be recognized by swine serum infected with PCV-2.The results showed that ORF3 of PCV-2 was expressed successfully in E.coli and the ORF3 protein didn＇t exhibit reaction genicity.These results were benefit to research for function and characteristics of ORF3 of PCV-2.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第6期171-174,共4页
China Animal Husbandry & Veterinary Medicine
基金
黑龙江省农垦总局课题(HNKXIV-08-07A)
黑龙江八一农垦大学博士科研启动项目(校启D2007-1)
