温控表达载体的构建及HIV-1p24的表达与纯化

通过改造原核表达载体ｐ Ｂ Ｖ２２０ 和ｐ Ｅ Ｔ２８，构建出了一种新的通用型温控原核表达载体ｐ Ｖ Ｖ５，该载体携带 Ｐｒ Ｐｌ串联温控启动子以及 Ｈｉｓ Ｔａｇ 的纯化标签，利于目的蛋白的表达与纯化将 Ｈ Ｉ Ｖ１ ｇａｇ 基因的１１４８１８５７ 编码序列分别插入到ｐ Ｖ Ｖ５ｂ、ｐ Ｅ Ｔ２８ｂ 的相应位点，构建了重组表达质粒 ｐ Ｅ Ｇ１ｂ、ｐ Ｂ Ｇ７ｂ，二者在不同受体菌中，表达重组蛋白的量分别占全菌体蛋白总量的 ４２％ 和２８％ 表达产物为融合蛋白并主要以包涵体的形式存在 该蛋白 Ｎ 端与含６ 个组氨酸在内的３０ 个氨基酸残基的多肽相连，利用 Ｉ Ｍ Ａ Ｃ金属螯和层析柱，对包涵体中的重组ｐ２４ 蛋白进行纯化， 其纯度超过 ８０％ ；对上清中的可溶性ｐ２４ 蛋白进行纯化，产物最终纯度超过６７％ 纯化后的重组蛋白可与 Ｈ Ｉ Ｖ１ 型标准阳性血清发生较强的免疫学反应

Construction of Current Novel Expression Vector With High Level of Expression and Purification of NIV Capsid Protein

New kinds of novel fusion expression vector were designed and constructed for the cloning and expression of recombinant protein in E.coli including a series of plasmids named pVV5a pVV5b and pVV5c with different reading frame fitting for the cloning of various genes . With the phage promoter pR and pL as well as the 5' terminal sequence adjacent to the cloning sites that code for the peptide of 6xhis tag , these vectors provided efficient ways for high level expression of fusion target protein and for purification of the protein by immobilized metal affinity chromatography (IMAC) . To evaluate the efficiency of these vectors , HIV 1 p24 gag gene was cloned into plasmid pVV5b and another novel expression plasmid pET28b respectively . The results showed both vectors could express target products successively and the expression recombinant HIV 1 p24 gag protein take more than 25% of the cell proteins . By one step Ni NTA affinity chromatography , HIV 1 p24 gag protein was purified to near homogenity and could be recognized by sera from HIV 1 infected individuals in ELISA.