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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。
OBJECTIVE: To express HSV-1 glycoprotein gD in Escherichia coli and purify the recombinant protein and identify its immunocompetence. Methods: The HSV-1 gD gene was cloned into the prokaryotic expression vector p ET-28b. The recombinant plasmid was transformed into E.coli using isopropyl-BD-thiogalactopyranoside (IPTG) to investigate the effects of IPTG concentration, induction time, The effect of temperature on the expression of recombinant protein was studied. The inclusion body of guanidine hydrochloride and denatured by nickel column were used to purify gD protein. The purified protein was dialyzed and refolded. The immunogenicity of gD protein was detected by Western blot and ELISA. Results: The digestion and sequencing results indicated that the gD gene was cloned into p ET-28b vector. The recombinant plasmid transformed E. coli induced by IPTG recombinant protein mainly in the form of inclusion bodies, the size of about 40kDa. The optimal conditions for gD protein induction were 0.5mmol / L IPTG induced at 37 ℃ for 8h. The total amount of gD protein purified by nickel column affinity chromatography was 3.1 mg / L, the total amount of gD protein obtained after dialysis refolding was 1.3 mg / L, the refolding rate was 41.37%. Western blot and ELISA showed that the expressed gD protein was immunoreactive. Conclusion: The expression and purification of HSV-1 gD protein in Escherichia coli has provided the basis for further preparation of HSV-1 diagnostic reagent and vaccine.