Cloning, Sequencing And Contructing Plasmid Of Yeast Expression Of Nucleoprotein And Glycoprotein Genes Of Rabies Virus From Yunnan

The direct immunofluorescent assay (DFA) was developed by importing 3 fluorescent labelled monoclonal antibodies raised against nucleoprotein of rabies virus from British. 4 pairs of detect primer and 2 clone primer were designed and synthesized based on Nucleoprotein (N or NP)gene and Glycoprotein gene (G) of known rabies viruses, the reverse transcriptase - polymerase chain reaction (RT-PCR) assay and the nested polymerase chain reaction (nested PCR) assay were developed. 56 dog brains of Yunnan province was detected by the diagnostic method, 12 was positive samples, positive rate is 21.4%. The results of the RT-PCR and nested PCR is same as the DFA. Meanwhile, the N and G genes of rabies strains from Qujing (YNQJ07), Zhaotong (YNZT07), Chuxiong (YNMD06) and Baoshan (YNTC06) of Yunnan province during 2006 to 2007, were amplified by reverse transcription– polymerase chain reaction (RT-PCR) and cloned into pMD18-T vectors for sequencing respectively. The sequence data were done alignment and phylogenetic analysis with known representative strains. The results showed Yunnan strains of rabies virus all belonged to genotypeⅠ. The nucleotide homologies of N and G genes between YNQJ07, YNZT07, YNMD06 and recent strains isolated from Guangxi, Zhejiang, Jiangsu, Hubei, Anhui, and Jiangxi provinces etc. were 97.0～99.3% and 97.4～9.3% respectively. The homologies between YNQJ07, YNZT07, YNMD06 and other genotypeⅠs trains were 86.3～90.2%,82.9～93.0% respectively. The homologies among YNTC06, Thailand and some Guangxi strains were 95.0～95.8% and the homologies between YNTC06 and other genotypeⅠstrains were 82.9～92.9%. This result of the nucleotide sequence analysis indicated that YNQJ07, YNZT07, YNMD06 belonged to subgroupⅡstrains and YNTC06 belonged to subgroupⅢstrain. There were some specific mutations of amino acid on nucleoprotein and glycoprotein of Yunnan strains.The purpose gene was enzyme digestion by the EcoRⅠa nd XbaⅠ(N gene)or KpnⅠ(G gene),after purification and recovery oriented cloned into pPICZαA vectors, successful constructed the donator plasmids of Yeast Expression pPICZαAN and pPICZαAG.