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

doi:10.13523/j.cb.20141108

China Biotechnology

2014, Vol. 34

Issue (11): 54-59 DOI: 10.13523/j.cb.20141108

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

MAO Hong-yan, MA Zheng-hai

College of Life Science and Technology, Xinjiang University, Urumqi 830046, China

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Abstract

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.

Key words： Herpes simplex virus 1 Glycoprotein D Escherichia coli expression Immune activity

Received: 01 September 2014 Published: 25 November 2014

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MAO Hong-yan, MA Zheng-hai. Expression, Purification of Recombinant Herpes Simplex Virus 1 Glycoprotein D in E.coli and Identification of Its Immune Activity. China Biotechnology, 2014, 34(11): 54-59.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20141108 OR https://manu60.magtech.com.cn/biotech/Y2014/V34/I11/54

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