Cloning And Expression Of Porcine Cytomegalovirus GB And Preparation Of Polyelonal Antibodies

Porcine cytomegalovirus disease (PCMVI) is an opportunistic infection caused by porcine cytomegalovirus (PCMV). PCMV is a betaherpesvirus which causes generalized infection in newborn piglets. It has a worldwide distribution and is present in90%of herds in the United Kingdom, many of which are from well-managed, high-healthstatus farms. The disease was first discovered in UK in1955. Presently PCMV is found throughout the world and infection is ubiquitous with herd prevalence greater than90%in Europe, North America, and Japan. Within infected herds, more than98%of pigs are seropositive.In this study, samples from Jiangsu provinces were collected, including nasal swab and lung from clinically affected birds. A pair of primers was designed according to the gB gene of PCMV from GenBank to detect PCMV, and then PCR and RT-PCR methods were also used to detect PCV-2, PRRSV, PRV and CSFV. The result showed that PCMV infection rate of nasal swabs from Dongtai is9%(9/100), as the rate of Baoying is31%(31/100). There were14of26clinical samples PCMV positive with an infection rate of53.8%. Analysis also showed that the infection rate of lung was15.4%while nasal swab was38.5%. Infection rate of PCMV and PRRSV was46.2%as coinfection rate was23.1%of PCMV and PCV-2, coinfection rate was4%of PCMV and CSFV. Multi-infection rate of PCMV, PRRSV and PCV-2was15.4%; Multi-infection rate of PCMV, PRRSV and CSFV was4%.Partical gB gene of PCMV was amplified by PCR method, and then it was been cloned and sequenced. Sequence result showed that the nucleotide homology was98.0%-100%compared with the PCMV gB from GenBank. The evolutionary tree of PCMVgB gene was analyzed by MEGA5.05. Immunity reactivity of PCMV gB protein was also predicted in this study. The partical gB gene was recombinated into prokaryotic expression vector pGEX-6p-1. The recombinant plasmids were transformed into E.coli BL21(DE3) and then induced by IPTG. The result of SDS-PAGE analysis showed that gB protein was successfully expressed by E.coli BL21(DE3) as molecular weights was48Ku (named GST-gB). The recombination protein GST-gB was expressed in form of inclusion body. The immunity reactivity of the recombinant proteins was confirmed by Western-blot method. New Zealand rabbits were immunized with purified protein for producing polyclonal antibody. The available dilution potency was1:25600. The high-titer antibody was the first precondition of authenticating gene expression and researching gene function. The assay facilitates us to study the immunologic functions of the protein and it provided a useful tool for epidemiological investigation of PCMV in swine.