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王宏伟 金宁一 戴 炜 李体远 郭志儒 殷 震
摘 要 目的：探索进一步提高 HIV-1 p24 gag 蛋白在大肠杆菌中的表达效率，增加产物的纯化手段。方法：通过改造原核表达载体 pBV220和 pET28，构建了一种新的通用型温控原核表达载体 pVV5，将 HIV-1 gag 基因的 1 148～1 857 编码序列，插入到 pVV5b 和 pET28b 的相应位点中，构建了重组表达质粒 pEG1b 和 pEG7b，转化到大肠杆菌中进行表达，产物利用 IMAC 金属螯合层析柱进行纯化，纯化的表达产物用 HIV-1 标准阳性血清进行鉴定。结果：构建的重组表达质粒 pEG1b 和 pEG7b 在相应受体菌中表达重组蛋白的量为41%和28%，表达的外源蛋白经 IMAC 柱一步纯化后纯度超过80%，纯化后的重组蛋白可与 HIV-1 阳性血清发生 ELISA 反应。结论：构建的通用型温控原核表达载体可以高效地表达外源蛋白，在大肠杆菌中高效地表达 HIV-1 p24 可用于制备 HIV-1 感染的诊断试剂。
关键词 原核载体 人免疫缺陷病毒 核心蛋白 纯化
中国图书分类号 R373. 9
Expression and purification of the recombinant HIV-1 capsid protein p24 in E.coli
WANG Hong-Wei，JIN Ning-Yi，DAI Wei et al.
Genetic Engineering Laboratory ,University of Agriculture and Animal Sciences ， Changchun 130062
Abstract Objective:To resesarch the way of increasing the expression efficiency of HIV-1 p24 gag gene in E.coli and improving the methods for the purification of the recombinant proteins.Methods:New kinds of novel fusion expression vector plasmid pVV5 were designed and construced by the reconstructing plasmid pBV220 and pET28.Two recombinant plasmids pEG1b and pEG7b were obtained by cloning HIV-1 p24 gag gene into the fitable site of pVV5b and pET28,After trasformating and inducing ,the recombinant proteins were expressed in E.coli and purified by IMAC,the purified products were identified by standard HIV-1 positive serum.Results:The expression levels of fusion HIV-1 p24 protein reach 42% and 28% for plsamids pEG1b and pEG7b respectively and the purity of the recombinant p24 was over 80% after purified by IMAC,the purified recombinant p24 proteins could be recognized by sera from HIV-1 seropositive individuals in ELISA.Conclusion:The way of constructing current novel expression vector for high level expression and purificating target protein was reasonable and the novel expression vector pVV5 as well as the expressed recombinant HIV-1 p24 provided a cheaper and efficient wa[1] [2] 下一页
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