Cloning And Expression Of Varicella Zoster Virus Glycoprotein E Extracellular Domain And It’s Application

Objective Varicella-zoster virus(VZV) glycoprotein E(g E) extracellular domain was cloned and expressed in E. Coli and COS-7 cells respectively, and the immunoreactivity of the recombinant proteins were studied by immunofluorescence and western blot. New Zealand rabbits were immunized with the recombinant proteins to produce anti-VZV g E polyclonal antibody. We used ELISA method to determine the antibody titers. Purified the extracellular domain of g E was used to develop a serological test for diagnosis of VZV infection as well as for the evaluation of VZV vaccination efficiency.Methods VZV clinical strains were isolated from skin lesions from patients with varicella or zoster in the Dermatology Clinic and the viral DNA was used the the template in PCR to obtain the DNA fragment coding for the extracellular domian of VZV g E. The DNA fragment was cloned to construct prokaryotic expression plasmid g E-p ET-32a(+) and eukaryotic expression plasmid g E-p CDNA3.1/myc-His(-). The authenticity of the insert was confirmed by DNA sequencing before it was transformated into E.coli BL21(DE3) or transfected into COS-7 cells. The fusion proteins were identificated by Western blot and purificated with Ni2+-NTA columns.New Zealand rabbits were immunized with the recombinant proteins. Antibody titers were determined by ELISA. Ni2+-NTA column purified g E was used to develop an ELISA assay to detect Ig G antibodies against VZV in 127 serum specimens from children between 0 and 10 years old of age.Results Using prokaryotic expression system, I was able to successfully induce the expresses the VZV g E fusion protein, the total g E protein was 3.01 mg/ml and the purity was nearly 90%. The titer of the rabbit anti-VZV g E polyclonal antibody was 1:6400~1:12800. Using eukaryotic expression, VZV g E extracellular domain protein was successfully expressed in COS-7cell line. It was demonstrated by Western blot that the recombinant g E fusion protein was expressed both in the cells and in culture supernatant of the COS-7 cells. Indirect immunofluorescence with commercial monoclonal g E antibodies showed g E was present in cell membrane and cytoplasm in the transfected COS-7 cells as it was in VZV infected cells from patients. The total g E protein was 0.632mg/ml, the purity was nearly 90%, and the rabbit anti VZV g E polyclonal antibody was 1:25600~1:51200. The serological test detected VZV-Ig G antibody in 81.89% of the 127 serum specimens from normal children. The specificity and the sensitivity of this ELISA test were 93.75% and 88.24%, respectively.Conclusion E. coli and COS-7 cell lines that efficiently express the extracellular domain of VZV dorminant protein g E were established. Purified recombinant protein was used to develop an ELISA assay for evaluation of VZV infection or immunity after VZV vaccination. Furthmore, the purified protein could be a canditate for VZV subunit vaccine. Highly specific and affinity of polyclonal anti VZV g E antibodies were obtained by immunization in New Zealen rabbits, which could be used to develop a method for VZV laboratory diagnosis.