本文選題：型單純皰疹病毒 + gD�。� 參考：《中國生物工程雜志》2014年11期

【摘要】：目的:在大腸桿菌中表達1型單純皰疹病毒(HSV-1)囊膜糖蛋白gD,純化重組蛋白并對其免疫活性進行鑒定。方法:將HSV-1 gD基因克隆入原核表達載體p ET-28b,利用異丙基-B-D-硫代吡喃半乳糖苷(IPTG)誘導重組質(zhì)粒轉化的大腸桿菌,探討IPTG濃度、誘導時間、誘導溫度對重組蛋白表達的影響;鹽酸胍裂解變性包涵體,鎳柱親和層析法純化gD蛋白,并對純化后的蛋白進行透析復性;Western blot和ELISA檢測gD蛋白的免疫活性。結果:酶切和測序結果表明gD基因克隆入p ET-28b載體。該重組質(zhì)粒轉化的大腸桿菌經(jīng)IPTG誘導后重組蛋白主要以包涵體形式存在,大小約40k Da。gD蛋白誘導表達的最佳條件為0.5mmol/L IPTG于37℃誘導8h。鎳柱親和層析法純化獲得的gD蛋白總量為3.1mg/L,透析復性后獲得的gD蛋白總量為1.3mg/L,復性率為41.37%。Western blot及ELISA檢測表明表達的gD蛋白具有免疫活性。結論:在大腸桿菌中表達并純化獲得具有免疫活性的HSV-1 gD蛋白,為進一步制備HSV-1診斷試劑和預防疫苗奠定了基礎。
[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，

本文編號：2005216