HIV-1核心蛋白p24在大肠杆菌中的表达与纯化

探索进一步提高ＨＩＶ－１Ｐ２４ｇａｇ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，构建了一种通用型温控原核表达载体ｐＶＶ５，将ＨＩＶ－１ｇａｇ基因的１１４８－１８５７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经，纯化的表达产物用

Expression and purification of the recombinant HIV-1 capsid protein p24 in E.coli

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 way for the production of HIV 1 diagnose reagents.