HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

Expression of HBcAg Gene in Methyltrophic Pichia pastoris,Purification of HBcAg Protein and Primary Research on Characterization of HBcAg Protein

为研究乙肝核心抗原蛋白 (HBcAg)在甲醇型酵母 (PichiaPastoris)中的表达和性质 ,用PCR方法将HBcAg基因 (L)克隆到P .Pastoris胞内表达载体pPIC3k中 ,并利用电击和同源重组法 ,将重组质粒pPIC3k L转化感受态的甲醇型GS1 1 5酵母菌株。经过筛选得到阳性P .Pastoris重组子。重组菌株经甲醇诱导培养 ,表达产物的Western印迹结果表明 ,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达 ,产物为一 2 1 5kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后 ,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为 1 2 5 76g ml和1 30 1 3g ml的 2个峰值处。电镜观察表明 ,该重组HBcAg蛋白能自主装配成大小不同的 2种颗粒 (即核心颗粒 ) ,大颗粒直径约 34nm左右 ,小颗粒直径约 30nm左右。同时 ,我们还观察到 ,该核心蛋白颗粒在体外可发生集聚现象。

To study expression and characterization of Hepatitis B core antigen(HBcAg) protein in methyltrophic yeast,Pichia pastoris,HBcAg gene (L gene) was cloned into the intracellular expression vector pPIC3K by PCR.The competent Pichia pastoris was transferred by the recombinant plasmid pPIC3K-L through electroporation and homologous recombination.The positive recombinant Pichia pastoris strains were screened and induced by methanol of the HBcAg expression.Western blot analysis showed that r-HBcAg protein could be expressed in the methylotrophic Pichia pastoris by the methanol induction,the product(r-HBcAg protein) was a 21^5kDa peptide.After purification by sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultralcentrifugation,analysis of ELISA and density indicated that the r-HBcAg protein(core protein) were mainly distributed at the ELISA reaction peaks corresponding to the densities of 1^2576g/ml and 1^3013g/ml respectively.Observation of r-HBcAg particles by TEM indicated that the r-HBcAg peptides could self-assemble into two kinds of different size HBcAg particles(core particles); the large particle was about 34nm in diameter and the small particle was about 30nm in diameter.Simultaneously,we found that the r-HBcAg particles which were storaged for some time could aggregate.;