Epitopes Identification Of Surface Protein Of JSRV And Detection Kit Of JSRV Preparation

BACKGROUND: The jaagsiekte retrovirus (JSRV) is the pathogen that causes ovine pulmonary adenocarcinoma, which is one of the major infectious viral diseases in sheep and is commonly used as a unique large-animal model for lung carcinogenesis. And OPA is the B-type infectious disease according to the disese classification from OIE. However, there is no immunological method for the detection of the JSRV infections in China due to the presence of endogenous JSRV. That hindered the rapid diagnosis of OPA and harmed its prevention and control.OBJECTIVE: To establish a rapid and specific laboratory diagnostic methods; screening the JSRV surface protein epitopes based on monoclonal antibodies.METHODS: Phage display and pepscan technologies were employed to screen the antigen epitope through the expressed fusion proteins were detected respectively with three McAbs. Then three liner epitopes recognized by three McAbs were preliminary identified, and the functional affinities of anti-surface protein (SU) McAbs were assessed with non-competitive ELISA method. Positive and negative samples were taken as research object to constructe the double antibodies sandwich ELISA method on the basic of anti-JSRV-SU McAb and multi-antibody. A method of loop-mediated isothermal amplification was developed and evaluated to detect JSRV infections. The genomic DNA isolated from lung tumour tissues of sheep infected with JSRV naturally was used for PCR amplification of ras gene and JSRV env gene. JSRV env was constructed using recombinant eukaryotic expression vector, and transfected by liposome establish stable cell lines.RESULTS:1,The affinity constants of three monoclonal antibodies 3B3, 3D6, 1D6 were 5.06×10⁹﹑ 5.82×10⁹﹑ 2.91×10⁹ L/mol; while their epitope were W¹⁵⁶APEGTPD¹⁶³,F⁷¹SYQSQHPHCI⁸¹,D⁹⁸EKTGKR¹⁰⁴;2,The sandwich ELISA method was established based on JSRV-SU monoclonal antibody,and the negative and positive criteria of ELISA was established (When the P / N≥2.21 , the result was thought as positive; when the P / N <1.85 as negative; when 2.21> P / N≥1.85 as suspicious);3,A LAMP assay specifically detected exogenous JSRV in the infected tissues of OPA sheep in reactions that were carried out for 60 minutes in a thermostatic water bath and quantified a minimum of 10 copies of the JSRV LTR/reaction in a testing plasmid. The sensitivity of this method is comparable to that of nest-PCR and is 10 times higher than that of standard PCR. Tissue samples from clinically diagnosed and experimentally infected OPA sheep and frozen stocked OPA organs were simultaneously examined by LAMP, nest-PCR and standard PCR. The coincidence of the results was 100% between LAMP and nest-PCR and 92% between LAMP and standard PCR;4,The amplified pMD-exJSRVenv1 gene was highly homologous with the U.S. strains JSRV21, which belongs to JSRV-II, while the amplified pMD-exJSRVenv2 gene was highly homologous with the South African strains, which belongs to JSRV-I. There was also a"YXXM"motif in the amino acid sequences , which was exogenous JSRV sequence unique compared with endogenous JSRV. Ras gene in sheep was relatively conserved compared with other species, especially with bovine;5,The recombinant pcDNA3.1-Env and pcDNA3.1-TM-HA were confirmed by sequencing and stablely expressed in HepG2 cells under G418. CONCLUSIONS: The specific detection of JSRV sandwich ELISA method and LAMP method was established in this study, and it is important for studying OPA control measures.The cloning and expression of JSRV env gene and the cloning of the ras gene of sheep will be meanful for riching the molecular pathogenesis of OPA and for further study of the pathogenesis of JSRV induce OPA provides an important experimental platform.