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

发布时间：2018-06-11 12:17

  本文选题：型单纯疱疹病毒 + gD　； 参考：《中国生物工程杂志》2014年11期

【摘要】：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p ET-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p ET-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40k Da。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。
[Abstract]:Aim: to express the envelope glycoprotein gDof herpes simplex virus type 1 (HSV-1) in Escherichia coli, to purify the recombinant protein and to identify its immunological activity. Methods: HSV-1 GD gene was cloned into prokaryotic expression vector pET-28b. The recombinant plasmid was induced by isopropyl -B-Dthiopyranoside (IPTGG). The effects of IPTG concentration, induction time and induction temperature on the expression of recombinant protein were investigated. The GD protein was purified by nickel column affinity chromatography, and the immunological activity of GD protein was determined by dialysis renaturation Western blot and Elisa. Results: restriction endonuclease digestion and sequencing showed that GD gene was cloned into pET-28b vector. The recombinant protein of E. coli transformed by IPTG was mainly in the form of inclusion body. The optimal conditions for the expression of the recombinant protein was 0.5 mmol / L IPTG at 37 鈩，

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