Antigenicity Analysis And Expression, Identification Of Extracellular Regions Of HSV1 Glycoprotein B, HSV1 Glycoprotein D And HSV2 Glycoprotein D

Herpes simplex virus,a common pathogen threatening human health with a high incidence rate,which can easily cause recurrent and latent infection,includes two subtypes:HSV1 and HSV2.HSV1 mainly results in oral surface and ocular infection as well as herpes encephalitis while HSV2 chiefly causes genital infection and is closely related with cervix cancer.In traditional drug treatment of HSV infection up to now, resistance frequently arises.Therefore,an effective and practical way to solve this problem is the development of vaccine,which can enable the organism to combine the functions of humoral immunity and cellular immunity in the fight against HSV infection.Envelope proteins of HSV play 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 proteins,glycoprotein B and D,with important epitopes,which can induce specific immune response,are the primary targets of humoral and cellular immunity of the host.Therefore, the genes of HSVgB and HSVgD are the ideal target genes in the development of HSV vaccine.In this research,epitopes of HSV1gD, HSV1gB and HSV2gD expressed through a genetic engineering method, are analysed and screened,which establishes a sound foundation for the development of HSV1 and HSV1 bivalence vaccine.In order to chemically synthesize the genes of HSV1gB,HSV1gD and HSV2gD with signal peptide sequence,extracellular region fragments of the three,glycoproteins with relatively abundant dominant antigenic determinants are screened based on the analysis of the epitopes,and meanwhile,codons preferred by both eukaryotic and prokaryotic cell are adopted.Extracellular region of the genes thus obtained serve as template for the synthesis of gene fragments of HSV1gB,HSV1gD and HSV2gD without signal peptide sequence through PCR,which are then inserted into plasmid vector pGEX-4T-2.Afterwards,the recombinant plasmid is transformed into E.coli TG1 and induced by IPTG for expression. SDS-PAGE analysis indicates that relative molecular weight of the expressed HSV1gB,HSV1gD,HSV2gD chimeric protein are about 96kD, 56kD and 56kD.Purified HSV1gB,HSV1gD,HSV2gD recombinant protein can be further obtained through affinity chromatography.To test antigenicity,the primarily purified chimeric protein HSV1gB/GST and negative control group are diluted with the same ratio with human-HSV1 ELISA Kit.At the same time,purified HSV1gD/GST and HSV2gD/GST are diluted with the ratio mentioned above to have their antigenicity tested via HSV1+HSV2 antibody marked by HRP. Results are that HSV1gB/GST,HSV1gD/GST and HSV2gD/GST all show good antigenicity and specificity.In the following experiment in vivo on Newzealand white rabbit, purified recombinant HSV1gD/GST is used as antigen to immune the rabbit after its GST tag have been cut by thrombin.Venous blood extracted form ear edge is tested with ELISA method,and the resulted valence of antibody(≥1:64 000)demonstrates good immunoreactivity.With the extracellular region of the chemically synthesized genes of HSV1gB,HSV1gD and HSV2gD containing signal peptide sequence served as template,gene fragments with signal peptide sequence 1～706aa,1～314aa and 1～314aa are proliferated through PCR method, which are inserted into eucaryotic expression vector pcDNA3.1(+)in to transfect HEK 293 cells after sequencing identification.48 hours after transfection,Western Blot method is used to test the expression of interest protein with His tag,and the expression of HSV1gD and HSV2gD is detected in the cell.