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

【摘要】：目的:在大腸桿菌中表達(dá)1型單純皰疹病毒(HSV-1)囊膜糖蛋白gD,純化重組蛋白并對(duì)其免疫活性進(jìn)行鑒定。方法:將HSV-1 gD基因克隆入原核表達(dá)載體p ET-28b,利用異丙基-B-D-硫代吡喃半乳糖苷(IPTG)誘導(dǎo)重組質(zhì)粒轉(zhuǎn)化的大腸桿菌,探討IPTG濃度、誘導(dǎo)時(shí)間、誘導(dǎo)溫度對(duì)重組蛋白表達(dá)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(duì)純化后的蛋白進(jìn)行透析復(fù)性;Western blot和ELISA檢測(cè)gD蛋白的免疫活性。結(jié)果:酶切和測(cè)序結(jié)果表明gD基因克隆入p ET-28b載體。該重組質(zhì)粒轉(zhuǎn)化的大腸桿菌經(jīng)IPTG誘導(dǎo)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(dǎo)表達(dá)的最佳條件為0.5mmol/L IPTG于37℃誘導(dǎo)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(fù)性后獲得的gD蛋白總量為1.3mg/L,復(fù)性率為41.37%。Western blot及ELISA檢測(cè)表明表達(dá)的gD蛋白具有免疫活性。結(jié)論:在大腸桿菌中表達(dá)并純化獲得具有免疫活性的HSV-1 gD蛋白,為進(jìn)一步制備HSV-1診斷試劑和預(yù)防疫苗奠定了基礎(chǔ)。
[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(hào)：2005216