单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定

目的:在大肠杆菌中表达1型单纯疱疹病毒(HSV-1)囊膜糖蛋白gD,纯化重组蛋白并对其免疫活性进行鉴定。方法:将HSV-1 gD 基因克隆入原核表达载体pET-28b,利用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒转化的大肠杆菌,探讨IPTG浓度、诱导时间、诱导温度对重组蛋白表达的影响;盐酸胍裂解变性包涵体,镍柱亲和层析法纯化gD蛋白,并对纯化后的蛋白进行透析复性;Western blot和ELISA检测gD蛋白的免疫活性。结果:酶切和测序结果表明gD基因克隆入pET-28b载体。该重组质粒转化的大肠杆菌经IPTG诱导后重组蛋白主要以包涵体形式存在,大小约40kDa。gD蛋白诱导表达的最佳条件为0.5mmol/L IPTG于37℃诱导8h。镍柱亲和层析法纯化获得的gD蛋白总量为3.1mg/L,透析复性后获得的gD蛋白总量为1.3mg/L,复性率为41.37%。Western blot及ELISA检测表明表达的gD蛋白具有免疫活性。结论:在大肠杆菌中表达并纯化获得具有免疫活性的HSV-1 gD蛋白,为进一步制备HSV-1诊断试剂和预防疫苗奠定了基础。

Expression,Purification of Recombinant Herpes Simplex Virus 1 Glycoprotein D in E.coli and Identification of Its Immune Activity

Objective: To express and purify herpes simplex virus 1 (HSV-1) glycoprotein D (gD) in Escherichia coli, and analyze its immune activity. Methods: The gD gene was amplified by PCR from HSV-1 genome and then cloned into the prokaryotic expression vector pET-28b. Then the recombinant vector pET28b-gD was transformed into E. coli cells, and the recombinant gD protein was induced by Isopropyl-β-D-1-Thiogalactopyrano-side (IPTG). The influences of induction conditions including IPTG concentration, period and temperature on the gD expression were analyzed. The inclusion body was lysed by the Guanidine HCl, and purified by Ni-affinity chromatography,and refolded by dialysis. And then the immune activity of gD was detected by Western blotting and ELISA. Result: Restriction endonuclease analysis and DNA sequencing confirmed that HSV-1 gD gene had been cloned into pET-28b. After analysis by SDS-PAGE, the gD protein was expressed in inclusion bodies form and the size of gD protein was 40 kDa. The best expression condition for gD protein involved induction with 0.5mM IPTG for 8 hours at 37℃. The total gD protein purified with Ni-affinity chromatography was 3.1 mg/L, and the total gD protein was approximately 1.3mg/l after refolding, so the refolding yield of the gD protein was 41.37%. Western blot and ELISA analyses showed that the gD possessed immune activity. Conclusions: HSV-1 gD protein with immune activity was expressed and purified in E.coli,which lays a foundation for the preparation of HSV diagnosis reagent and prophylactic vaccine.