温控表达载体的构建及HIV-1p24的表达与纯化

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

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

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.