Genotyping Of Bovine Papillomavirus Epidemic Strain And Expression Of L1 Gene In Guizhou Province

Bovine Papillomatosis(BP)is a disease characterized by bovine chronic proliferative neoplasm or part of the mucous membrane,the cause of the disease is bovine papillomavirus(BPV).BPV can be divided into 14 genotypes,and there are still considerable assortment of putative genotypes.Different genotypes of BPV can cause pathological changes in different parts of the body,and BPV are highly species-specific,thus it is difficult to infect other animals besides the host.In recent years,Guizhou Province has been vigorously developing the cattle breeding industry,and has continuously introduced fine breed cattle,and the scale of cattle breeding continues to expand.In 2015,there was a suspected case of bovine papilloma in a cattle farm in Zhenning County.In order to understand the epidemic situation of bovine papillomavirus in Guizhou Province,this experiment carried out a survey of pathogen infection of bovine papillomavirus,genotyping of BPV Guizhou epidemic strain and whole genome sequencing of BPV1 Guizhou strain,and carried out bioinformatics analysis and studies on prokaryotic and eukaryotic expression of the L1 gene of BPV.The objectives was to lay a foundation for the development of diagnostic reagents and vaccines for bovine papilloma disease.1.Survey of Papillomavirus Infection and Genotyping in Guizhou ProvinceIn order to clarify the BPV infection status and virus genotypes of cattle farms in Guizhou,this study was investigated the suspected cases of bovine papilloma infections and genotyping covered 32 cattle farms in Guizhou Province with several methods,including incidence surveys,clinical symptom observations,pathological section observations,PCR tests,and molecular typing methods The results showed that the majority of sick cattles had grayish white nodular or cauliflower-like body tumors.Histopathological observations revealed that the tumors had hyperkeratosisand cell vacuolation.The positive rate for BPV infection in all sick cattles was 100%(32/32)andall of them were used for BPV genotyping,in which 2 were BPV1(6.25%),28 were BPV2(87.50%),and 2 were BPV13.(6.25%).It can be seen that the cattles in Guizhou Province had a high risk of BPV infection,of which BPV2 is the major genotype of cattle infection in Guizhou.2.Cloning and related sequence analysis of full-base group of bovine papillomavirus type 1 Guizhou strainTo understand the whole genome structure and genetic variation of BPV1 Guizhou strain,7 pairs of specific primers was designed based on the BPV1 reference strain in Gen Bank and used for PCR assay and cloned the DNA extracts of six diseased cow skin tumors in Guizhou as a template.The result showed that 7 amplification products were consistent with the theoretical values(1248 bp,1461 bp,846 bp,817 bp,1475 bp,1488 bp,and 985 bp,respectively).The BPV1 Guizhou strain genome is 7946 bp long after sequence assembly,(A+T content is 54.73%,G+C content is 45.27%),containing 8 ORFs(E6,E7,E1,E2,E4,E5,L2,L1),consistent with the typical gene characteristics of the BPV1 genome The results of phylogenetic analysis showed that it is much closer to the BPV2,BPV13,and BPV14.3.Bioinformatics analysis of L1 gene of bovine papillomavirus type 1 Guizhou strainTo understand the molecular characteristics of L1 gene of bovine papillomavirus type1 Guizhou strain and to predict the biological function of the encoded protein,the L1 gene sequence of BPV1 Guizhou strain was selected.Bioinformatics software and methods were used for analysis.The results showed that: The homology of nucleotide and amino acid in the BPV1 Guizou strain and the BPV1 reference strain was the highest,which was99.8% and 100%,respectively;and the nucleotide and amino acid sequences of the reference strains of BPV2,BPV13 and BPV14 were highly homologous,being 84.3%,85.5%,73.4% and 92.1%,92.7%,80.6respectively,belong to the genus Delta with BPV2,BPV13 and BPV14.It is predicted that the secondary structure of protein L1 encoded by the BPV1 Guizhou strain mainly composed of irregular coiling,?-sheet and ?-helix There are multiple epitopes,no transmembrane and signal peptide regions,and it is anon-secretory protein.These findings provide a theoretical basis for the study of BPV1 L1 gene expression.4.Expression of L1 capsid protein in bovine papillomavirus type 1 Guizhou strainTo understand the expression of BPV1 Guizhou strain L1 gene in prokaryotic cells and eukaryotic cells,the target gene was first inserted into p Cold I vector.Then positive recombinant vector was transformed into BL21 competent cells,and the L1 protein was expressed efficiently by IPTG.Expressed products were comfirmed by SDS-PAGE and Western Blotting.L1 gene was also cloned into p EGFP-C1 and transfected into MDBK cells.The expressed products were identified by fluorescence microscopy and Western Blotting.The prokaryotic expression products were purified by His-tag nickel affinity chromatography and immunized rabbits to prepare polyclonal antibodies.The results showed that the Li gene was well expressed on prokaryotic cells,and the molecular weight of the expressed product was approximately 55.6 k D,and expressed in the inclusion body.The expressed protein reacted specifically with the His-tag antibody.After p EGFP-BPV1-L1 was transfected into MDBK cells,green fluorescence was observed under the fluorescence microscope.Theexpressed product could react specifically with the GFP-tag antibody and recognize by polyclonal antibodies prepared in rabbits...These results lay a solid foundation for the exploration of BP gene immunization targeted with the BPV1 L1 gene.In summary,this study confirmed for the first time that there was BPV infection in cattle in Guizhou and confirmed their genotypes,obtained the complete genome sequence of BPV1 Guizhou strain,and successfully constructed the prokaryotic expression plasmid p Cold I-BPV1-L1 and eukaryotic expression of BPV1 L1 gene.Plasmid p EGFP-BPV1-L1 was successfully expressed and rabbit anti-BPV1 L1 polyclonal antibody was obtained.