Eucaryotic expression and bioactivity analysis of the recombinant HSV-gD1.
- Author:
Zhengmao WANG
1
;
Lin LI
;
Wenyan GUAN
;
Yuexi LI
Author Information
1. Department of Biochemistry & Molecular Biology, School of Preclinical Medicine, Nanjing Medical University, Nanjing 210029, China.
- Publication Type:Journal Article
- MeSH:
Animals;
HEK293 Cells;
Herpesvirus 1, Human;
immunology;
metabolism;
Humans;
Immunization;
Mice;
Recombinant Proteins;
biosynthesis;
genetics;
immunology;
Transfection;
Viral Envelope Proteins;
biosynthesis;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2010;26(5):657-663
- CountryChina
- Language:Chinese
-
Abstract:
Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.
