摘要
目的构建人源单克隆抗-HBs Fab片段-IFN-α表达载体,对原核表达该融合蛋白的可行性及其效果进行实验研究。方法用聚合酶链反应(PCR)体外扩增目的基因IFN-α,单酶点插入已含人源抗-HBs Fab基因的载体,插入点位于轻链基因3′末端,利用酶切、PCR鉴定,测序筛选正确插入的阳性克隆,转化大肠埃希菌Top10,挑选单克隆表达、纯化目的蛋白,用酶联免疫吸附法(ELISA)检测融合蛋白的抗原性,用Dot-blot和WISH细胞检测其生物学活性。结果利用插入了人源抗-HBs Fab段基因及IFN-α基因的pBAD/gⅢA原核表达系统,成功表达了具生物活性的轻链与IFN-α的融合蛋白,其与HBsAg的亲合力与抗-HBs-Fab相近,而且具有干扰素的活性。融合蛋白对HepG2.2.15细胞初步实验结果亦显示出其具有良好的靶向性及致凋亡作用。结论轻链与干扰素IFN-α融合蛋白的成功表达表明,应用原核系统能够同时表达特异抗体和其介导的某些生物活性因子,不失为一种经济、有效的方法,为特异抗体及其介导融合蛋白的制备和临床应用提供了实验依据。
Objective Construct an expression vector for anti-HBs Fab fragment and IFN -αfusion protein, then transform it into E. coli Top10 in order to produce a targeting drug for hepatitis B. Methods Using PCR and molecular clone techniques, we amplify the gene fragment of IFN-α with corresponding endonuclease sites and artificial linker at 5＇,3＇ termini, then recombine it within the vector of anti-HBs Fab gene in correct endonuclease sites at 3＇ termini of the Lc gene, choosing the positive clone with PCR and transform it into E. coil Top1l0,after the expression and purification of the fusion protein, we use ELISA and Dot-blot to verify the antigenicity and binding activity to HBsAg. Results We got fusion protein consisting Lc and IFN-αIt has the IFN-α activity and HBsAg affinity which is similar to anti-HBs Fab fragment.Conclusions The successful expression of the fusion protein with both HBsAg affinity and IFN-α activity make it possible for immunobiotherapy drug targeted to HBV.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2005年第4期229-232,共4页
Chinese Journal of Infectious Diseases
