摘要
目的在原核表达系统中对乙型脑炎病毒包膜糖蛋白(E蛋白)的B区多肽抗原进行高效表达、纯化及血清学评价。方法利用PCR技术从乙脑减毒活疫苗中扩增编码B区的DNA片段,酶切消化后连接到表达载体pET22b上,用此连接产物转化大肠埃希菌BL21(DE3)并表达B区多肽抗原,表达产物经液相层析纯化;用乙脑病人血清对纯化后的B区多肽抗原进行评价。结果重组质粒pET22b-JEB经双酶切,其插入的外源基因片段为372 bp,与预期DNA片段大小一致。将纯化前与纯化后的蛋白作SDS-PAGE电泳,均可见一条约16kD的外源基因蛋白带,与计算的相对分子质量相符。经WB和ELISA血清学评价,表明重组B区多肽抗原具有较高的灵敏性及特异性。结论成功构建了表达载体pET22b-JEB,在原核系统中表达的B区多肽抗原具有良好的血清学检测价值。
The peptide antigen in B domain of the envelope glucoprotein （E-protein） of Japanese B encephalitis virus （JEB） was highly expressed in prokaryotic expression system, and it was purified and evaluated for its serological activities, in which the DNA fragment coding the B domain of envelope glycoprotein was amplified by PCR from the attenuated Japanese B encephalitis vaccine, enzymatic ally digested and subcloned into vector pET-22b（＋）. The recombinant B domain glycoprotein was purified by liquid chromatography and its serological activities were evaluated with sera of patients with Japanese B encephalitis infection. It was found that the size of the inserted gene fragment of the recombinant plasmid pET-22b-JEB after double enzyme digestion was 372 bps, that was just the same as that of the predicated DNA fragment value. One exogenous protein band of 16 kDa could be observed in SDS-PAGE analysis both before and after purification,that was consistent with that of the calculated relative molecular mass. As indicated by the serological evaluation with ELISA and Western blotting , higher sensitivity and specificity could be demonstrated for this recombinant B domain peptide antigen. It is evident that an expression vector pET-22b-JEB was successfully constructed and this recombinant peptide antigen expressed in E. coli has excellent potential for serum testing.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第1期53-55,82,共4页
Chinese Journal of Zoonoses
基金
广东省医学科学研究基金(No.WSTJJ20021210440102196303133230)
关键词
乙型脑炎病毒
包膜糖蛋白B区
原核表达系统
蛋白纯化
Japanese encephalitis virus
envelope B domain protein
prokaryotic expression system
protein purification
