Genotyping And Expression Of Capsid Protein Of Bovine Papillomavirus In Northern Xinjiang

Bovine papillomatosis (BP) is a kind of infectious cancer disease caused by the infection ofbovine papillomavirus (BPV), which is characteristized by chronic proliferative tumor in themucosa and surface. BP is widely prevalent in many cattle-farming areas in the world, which hascaused great economic losses in cattle industry for decreasing leather quality and damaging animalhealth. So far,13BPVs and21putative new BPVs have been reported, and there are also manyunclassified assumed genotypes. So far, there is no effective vaccine to prevent bovinepapillomavirus. Xinjiang is one of five major pastoral areas in China, where dairy ARE about2.8million. The BPV-like infections have frequently occurred in dairy cattle. To date, however,surveys of the infection and the genotyping of BPVs in cattle in the farming areas in Xinjianghave not been conducted. It is very important to carry out genotyping of BPV and developeffective vaccine for the prevention and control of BP.The aim of this study is to survey the genotypes of BPVs in several cattle-farming areas innorthern Xinjiang. In this study, histopathological examination and detection by polymerase chainreaction (PCR) were conducted. The genotyping of BPV strains in Xinjiang was then carried out.The whole genome of BPV2was cloned and BPV L1gene was then subcloned into pGEX4T-1vector for expression. And BPV L1gene was subcloned into pcDNA3.1(+) vector for expressionin BHK-21cells. The expression of L1gene was confirmed by indirect immunofluorescence assay,which laid a foundation for the development diagnostic reagents and vaccine of bovinepapillomatosis. The research methods and results are as follows:1. Detection and genotyping of bovine papillomavirus in northern XinjiangA total of56cases of cattle with BP-like disease occurring in10dairy cattle farms located inseveral cattle-farming areas in northern Xinjiang (including Changji, Shihezhi, Tacheng and Yili)were detected by polymerase chain reaction with degenerate primers targeting the conservedregion of L1gene. Histopathological examination was then conducted. Genotyping on theprevalent types of BPVs in Xinjiang was conducted through comparing their DNA sequences. Theresults showed that all56cases of suspected BPV infection were all positive for PCR, amongwhich, BPV1(66.07%), BPV2(94.64%), BPV3(14.29%), BPV7(8.93%), BPV8(41.07%) and apresumed new type of BPV(named BPV/CHI-SW1)(1.79%) were detected. BPV2was majorgenotype in northern Xinjiang.2. Cloning and sequencing of the genome of bovine papillomavirus type2Xinjiang epidemicstrainAccording to genotyping results,6pairs of specific primers of BPV were designed and usedto amplify the genome of BPV epidemic strains (BPV2-SW01). The genome of BPV2-SW01lengths7944bp, including10open reading frame (E6、E7、E1、E8、E2、E4、E3、E5、 L2、L1). Phylogenetic analyses suggested that BPV2-SW01belongs to the genus Deltapapillomavirus, which is most closely related to BPV-1and BPV-13.3. Expression of L1gene of BPV2Xinjiang epidemic strain in prokaryotic cellsBPV2L1gene of Shawan was amplified from sample of skin les ions by PCR and sequencedafter cloned into pMD18-T (simple) vector. BPV2L1gene was then subcloned into pGEX4T-1ofprokaryotic expression vector. The expressed L1protein was analyzed by SDS-PAGE and westernblot after induced by IPTG. SDS-PAGE revealed that recombinant protein with a molecular weightof80KD was successfully expressed after induction. Western blot demonstrated that the proteingenerated an immune response with anti BPV2of polyclonal antibody, which confirmed thatrecombinant L1protein has good reactogenicity.4. Expression of L1gene of BPV2Xinjiang epidemic strains in eukaryotic cellsBPV2L1gene was cloned into pMD18-T vector, sequenced and then subcloned intoeukaryotic expressing vector pcDNA3.1(+). The recombined vector was transfected into BHK-21cells with lipofectin and the expression of L1gene was detected by indirect immunofluorescenceassay. The results showed that eukaryotic expressing vector encoding BPV2L1was successfullyconstructed, and L1protein correctly was expressed in BHK-21cells, which laid a foundation forthe development of DNA vaccine against BPV2infection.