Functional Study On Glycoprotein GG Of Bovine Herpesvius Type 5

BHV-1, BHV-5 belong to herpesvirus family, a subfamily of herpes virus, varicella-zoster virus genus. BHV-1 is currently popular worldwide, and BHV-1 have been isolated all over the world except for some South America countries. This virus could cause not only the nose bronchitis, high fever, abortion, conjunctivitis, contagious respiratory disease characterized by pustules, but also Outside vaginitis, the general systemic infection. Consequent annual losses to the cattle industry are over one billion U.S. dollars. BHV-5 is a fatal cattle meningitis pathogens. BHV-5 could enter the central nervous system (CNS), causing severe fatal calf meningoencephalitis and sub-clinical symptoms of adult cattle, hence causes great losses of cattle industry.BHV gG is one main glycoprotein and relatively conservative in a herpesvirus. gG is able to affect BHV proliferation in host cells, cell proliferation and in vitro virus infection on cells effectively, and block cell Apoptosis. But chemotaxis of the BHV-1gG and BHV-5 gG is not clear. Whether chemotaxis of BHV-1gG and BHV-5 gG differ is unknown. The extracellular gG of BHV-5 could bind to chemokines, but the specific functional domain is unclear.In this study, several mutants were constructed, study gG to the pathogenicity of BHV-5 by means of animal experiments, sub-fragments of BHV-5gG are expressed by baculovirus expression system, and chemotaxis experiments are done further, to explore whether chemotaxis of BHV-1 gG and BHV-5 gG differ as well as whether BHV-5 gG has the region of trend capabilities. The main contents are: 1. MutantIn this experiment, we use bovine herpesvirus type 5 genome (gene accession number is AY261359) as template and design primers accordingly, PCR amplified upstream and downstream of gG gene, approximately 1.0 kb and the 1.1kb fragments are cloned into PMD18-T vector as a homologous recombination arm. Extracted downstream homologous arm cutted by responsible restriction enzymes is cloned into the cloning vector containing the upstream, and the intermediate transfer vector was constructed, named as pZF09-13. Then EGFP reporter gene of a complete promoter was inserted in the upstream and downstream, hence the recombinant transfer plasmid was got and named pZF09-14. After pZF09-14 was linearized, the sequence of upstream and downstream homologous arms was recyclied. The recyclied product, the plasmid pBICPO and BHV-5 genomic DNA cotransfect bovine kidney (MDBK) cells using calcium phosphate transfection method, and then homologous recombination was successfully completed after 4 rounds of selection. At last we obtained purified BHV-5△gG/EGFP mutant strains. we successfully obtained BHV-1 AgG/EGFP revertant strains and BHV-5 AgG/EGFP revertant strains of the same cotransfection way. 2. BHV-5 gG expression of baculovirus expression systemThe BHV-5 gG protein sequence was analysed by software, divided into three sections, primers were designed. The fragments amplified using pZF09-40 as template, were cloned into pMD18-T vector. After identified by sequencing, the fragments were cloned into pFastBacTM1 vector, and extracted recombination bacmid transfects sf-9 cells. The second generation of recombinant baculovirus were verified by PCR, and then infected High-five cell. gG fragments were expressed, verified by western. The result was 27KD for gGl, and 55 KD for gG3, the gG2 wasn't expressed.3. Chemotaxis assaysMDBK cells was inoculated with BHV-5 wt,BHV-5 AgG/EGFP,BHV-5△gG/EGFP rev,BHV-1 wt,BHV-1 AgG/EGFP and BHV-1 AgG/EGFP rev by 1MOI, and after 24h, the supernatant samples was harvested as a chemotactic specimen. The chemotactic specimen and Chemokines were added into the chemotaxis chamber, incubatied for 30min, and neutrophils was added into above room.45min later, count the number of the cells in the below room. Due to some uncertain factors, BHV-1 and BHV-5 three virus strains in the group have no significant difference in chemotaxis. The ability of chemokine binding for different segments of BHV-5 gG protein were detected, the results show that the gG2 binding ability is strong.4. Animal experimentWe use New Zealand rabbits as experimental animal models, and the animals were divided into two groups. One group was infected by BHV-5 wt, another group was infected by BHV-5△gG/EGFP.Everyday,we took detailed notes of every rabbit.7dpi, the two groups appeared the same neurosis. It suggested that gG had an effect on virulent of BHV-5 in acute infection period.