Prokaryotic Expression Of GB Gene Of Porcine Cytomegalovirus And Development Of Indirect ELISA Of Recombinant Protein

Porcine cytomegalovirus infection (PCMVI) is an opportunistic infection in easy infected herds, it can cause clinical symptom as fetus and young swine died, rhinitis, pneumonic fever and retarded growth and so on. Its infected animal limited in swine. PCMV was first discovered isolated by Done in UK in1955. The disease is epidemic in all over the world, the disease incidence is especially high in UK, America and Japan.In infected herds, more than90%of swines are seropositive. It indicated that the disease infection is ubiquitous with herds.According to the gB gene of PCMV from GenBank, two pairs of primers were designed by selecting conserve district in PCMVgB sequence, the one is detected primer, the other is total length of PCMVgB gene. Using detected primer to detect nasal swab specimens fromYangzhou, Huaian, Yancheng and Haian through PCR. The results showed that23of53clinical samples were positive with a positive rate of43.4%. Using total length primer amplified total sequence, its length was2627bp. The gene sequence was cloned into pUCm-T vector. The sequencing result showed that the nucleotide homology was97.8%-99.4%and amino acid homology was97.1%-99.3%compared with the PCMVgB from GenBank. The cladogram was analyzed by DNAStars, The result showed that the sample of this study was in the same cluster with UK strain, Germany strain and Hunan strain in our country. The biology characteristics and signal peptide of PCMVgB antigen protein were analyzed by bioinformatics software and DNAStars. The immunity reactivity of PCMVgB antigen protein was predicted by this study.According to the gB gene of PCMV from GenBank, two pairs of primers were designed by choosing immunodominant region of PCMVgB genes (named PCMVgB, and PCMVgB2). The gene sequence of gB1and gB2was amplified by PCR. Two gene sequences and expression vector pGEX-6p-1were digested by BamH I and sal I, And then they were recombinated into prokaryotic expression vector pGEX-6p-1(named pGEX-gB1and pGEX-gB2). The sequencing results showed that the nucleotide homologies were99.4%and100%compared with the PCMVgB from GenBank. The recombinant plasmids were transformed into E.coli BL21(DE3)and induced by IPTG. The result of SDS-PAGE analysis showed that gB antigen gene was successfully expressed in E.coli BL21(DE3). The molecular weights were39KDa and36KDa (named GST-gB1and GST-gB2). The recombination protein GST-gB1was expressed with the form of inclusion body, GST-gB2was expressed with the form of solubility. The immunity reactivity of the recombinant proteins was confirmed by Western-blot.The recombinant protein GST-gB1was purified by cutting gel, GST-gB2was purified by GSH-Sefinose, The recombinant protein GST-gB2was used as detected antigen to establish the indirect ELISA method. The coating concentration of recombinate protein and sera dilute strength of PCMV infected swine were2μg/mL and1:100, the positive critical value was0.224, the negative critical value was0.189, the sensitivity of detecting anti-PCMV sera was1:3200.