Cloning And Expression Of The GB Gene Of Porcine Cytomegalovirus Of SiChuan Strain And The Analysis Of Its Antigenicity

Porcine cytomegalic inclusion disease caused by porcine cytomegalovirus.The virus was first diseovered in UK in 1955,but now the disease is found throughout the world,and the infection is ubiquitous with herd prevalence greater than 90% in Japan,North America and Europe. The gB protein is an envelope algcoprotein and it's a necessary factor in the viral adsorption,penetration,Cell-fusion and Cell-invading.In addition the GlycoproteinB has good immunogenicity and Protective Immunity.Following jobs have been done in this study to gB gene of PCMV:1. Cloning gB gene of PCMV and constructing pET30-gB expression plasmid(1) Design primers for PCR to amplify the all gB gene of PCMV and the targert gene fragment to expression.(2) Bioinformation analyses were used to analyses gB gene and its protein The results showed that:The PCMV gB gene was composed of 2580 nucleotides encoding for a polypeptide of 860 amino acidresidues.Phylogenetic tree analysis of gB gene showed that there is the closest phylogenetic relationship between HHV-6 and HHV-7.Including the signal peptides of 20 amino acids at N end. In the729aa to 751aa have transmembrane domains.The whole protein have high Hydrophilic Property. Antigenic sites well distributed on the gB protein.(3) Using EcoRⅠand SalⅠdouble digest target fragment and pET30a(+). Connecting gB target fragment with pET30a(+) and transfer recombinant plasmid pET30-gB into E.coli Rossta(DE3) using the method of calcium chloride.2. inducing expression and protein purification of prokaryotic expression vector of pET30a(+) of PCMV gB geneThe polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 80KD) were highly expressed with 0.6 mM IPTG at 37℃,the expressed fusion protein was purified by one-step histidine affinity resin.3. The antigenicity analysis of the expressed proteinsPurified protein was used to immunize rabbit for the gB anti-serum preparation. Agar diffusion reaction showed that the antibody titer was up to 1:16,and reveal the protein has nature immunity. The protein purified had nicer reaction activity by analysis of Western blot.In this study,The author has clonined the whole gene of PCMV gB and has analysed the gB gene and its protein by bioinformation software,and has realized the recombinant gB expressed in E.coli may useful for development of more sensitity and more specificity detection of PCMV infection, and t established the foundation of the research in subunit vaccine field of PCMV.