摘要
本试验旨在利用毕赤酵母系统表达狂犬病病毒(RV)糖蛋白(GP)基因,并对重组蛋白进行反应原性研究。构建重组表达质粒pPiczαA-G,线性化后电转入酵母GS115中,经平板初筛及PCR鉴定正确后命名为pPiczαA-G-GS115。重组菌经1.0%甲醇诱导表达,对目的蛋白进行双抗体夹心ELISA检测、SDS-PAGE电泳、Western blot分析。结果表明,ELISA检测诱导72 h重组蛋白表达量最高;SDS-PAGE分析显示目的蛋白相对分子量约为65 kD,与理论值相符。Western blot结果表明,重组蛋白可与RV阳性血清发生特异性反应。本试验为进一步研制狂犬病病毒ELISA抗体诊断试剂盒奠定基础。
This assay aims to express rabies virus（RV） Glycoprotein（GP） gene in Pichia pastoris,and analyze the reactogenicity of the protein.The constructed recombinant plasmid pPiczαA-G was linearizated and electrotransformated into GS115,via plate screening and PCR and was renamed pPiczαA-G-GS115.After induction by 1.0% methanol,the products were analyzed by DoubleAntibody Sandwich-ELISA,SDS-PAGE and Western blot.ELISA results indicated the recombinant protein had got the highest level of expression after induction for 72 h;SDS-PAGE analysis showed the molecular weight of GP in Pichia pastoris was about 65 kD,matching with the theoretical value.Western blot analysis indicated that the recombinant protein could specifically react with RV positive serum.These results laid foundation for further development of the RV antibody ELISA diagnostic kits.
出处
《中国兽医杂志》
CAS
北大核心
2014年第6期22-24,共3页
Chinese Journal of Veterinary Medicine
基金
国家公益性行业(农业)科研专项经费资助(201203056)
关键词
狂犬病病毒糖蛋白
毕赤酵母
反应原性
Rabies virus Glycoprotein
Pichia pastoris
Reactionogenicity
