Detection Of JSRV And Study On Tumorigenic Mechanisms Of JSRV LTR

Ovine pulmonary adenomatosis (OPA), a contagious tumour originating in the distal lung is caused by the infection of Jaagsiekte Sheep Retrovirus (JSRV), a betaretrovirus. And OPA is the B-type infectious disease according to the disese classification from OIE. At present, the detection and diagnosis of OPA mainly depend on classic pathology methods in China. Which hindered the rapid diagnosis of OPA and harmed its prevention and control. And there are lots of puzzles present in the pathogenesis of JSRV.Aim to investigate the pathogenesis of JSRV and diagnosis method of OPA, JSRV ca gene was divided into four overlapping fragments and expressed in E. coli BL21 respectively in this paper. Pepscan technology was employed to screen the antigen epitope through the expressed fusion proteins were detected respectively with three McAbs by western blot. Then three liner epitopes recognized by three McAbs were preliminary identified, and the functional affinities of anti-CA McAbs were assessed with non-competitive ELISA method. Since Jaagsiekte sheep retrovirus can proliferate in the lung of sheep or goat, high content of JSRV virion can be find in the serous rhinorrhea and lung tumor tissues from infected animals, this paper take positive and negative sample as research object to compare the discrepancy of blocking value between positive sample and negative sample. Then, the blocking-ELISA method for detecting JSRV was developed and being used to check the clinic tissue samples.Meanwhile, Ovine pulmonary adenomatosis is a naturally occurring contagious lung tumor of sheep which is caused by an exogenous retrovirus of sheep, jaagsiekte sheep retrovirus (JSRV). Although no specific circulating antibodies against the virus can be detected in infected sheep, exogenous JSRV proviral DNA sequences (exJSRV) and JSRV RNA transcripts can be detected in lung tumors, lymphoreticular system and peripheral blood mononuclear cells (PBMC) from sheep affected by JSRV. The sheep genome carries 15 to 20 copies of endogenous retrovirus loci (enJSRV) that were similar to JSRV in structural genes but divergent in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for use in the specific PCR and nested PCR (n-PCR). Sensitivity between specific PCR assay and n-PCR assay was compared by using serial dilutions of positive plasmid pJSRV LTR in a background of 700ng sheep genome DNA. Then, the PCR methods were developed and also being used to check the clinic tissue samples.As research objects, ATⅡcell and LTR sequences were necessary for investigating the pathogenesis of JSRV. It is described removing leukocyte to purify and culture ATⅡcell by IgG and magnetic resin in this paper,and then stained by alkaline phosphatase (AKP) and nuclear fast red (NFR) to identificate ATⅡcell, and the ratio of living cells was analyzed by trypan blue staining. Then, Cloning and sequencing exJSRV LTR from sheep, enJSRV LTR from sheep and goat respectively were done successfully.Homology and transcription factor binding sites were compared between exJSRV LTR and enJSRV LTR DNA by using Biology Software DNAstar and MatInspector, and phylogenetic tree analysis was obtained by using Biology Software Mega.Based on acquirement of ATⅡcell and LTR sequences, dual-luciferase gene reporter system was employed to compare the luciferase activity with a reporter plasmid driven by exJSRV LTR or enJSRV LTR sequences by transfecting 10 different cell lines; Then, through single element's deletion mutation analysis, this paper analysis diversification of promoter's activity in ATⅡcell when exJSRV LTR was deleted Gfi-Ⅰ, C/EBP or HNF-3βelement respectively.The result show that blocking value 37% is the demarcation point between positive sample and negative sample in the blocking ELISA assay; Sensitivity of n-PCR is tenfold to specific PCR, 1 to 10 copies exogenous JSRV sequences can be detected by PCR methods, the detection results of serological method or PCR methods were in full accord with histopathological diagnosis. Specially, the n-PCR is the only available method for detecting blood sample from JSRV infected alive sheep, and may be useful in epidemiological studies and in control programmes of OPA.The Proportion of ATⅡcell to cell mixture is more than 90 after staining by alkaline phosphatase (AKP) and nuclear fast red (NFR), and the ratio of living cells was analyzed to 95% by trypan blue staining. All this may be helpful in virus isolation and JSRV culture in vitro, which also lay a foundation on mechanism research of pulmonary fibrosis and pathogenesis of lung cancer. Phylogenetic tree analysis showed that exJSRV LTR and enJSRV LTR could be classified into two clusters. Meanwhile, exJSRV LTR can be divided into two types and enJSRV LTR also can be divided into two groups. And the Inner Mongolia JSRV strain has close phylogenetic relationship with the virus strains from Europe and America.The promoter activity of exJSRV LTR and enJSRV LTR didn't show discrepancy in 9 cell lines except for ATⅡcell; Inspiring, the exJSRV LTR's relative luciferase activity transfecting ATⅡcell was 24-230 times to others, which unveil the truth exJSRV LTR has cell tropism for ATⅡcell. By single element's deletion mutation analysis, this study established that the promoter activity of exJSRV LTR deleted C/EBP or HNF-3βelement respectively appear to decrease approximately 60-70% in ATⅡcell. This date showed C/EBP and HNF-3βare two important transcription factor binding sites which are responsible for cell tropism.The results reported above laid a certain foundation on the epidemiological studies of OPA and the tumorigenesis induced by JSRV, it will be useful or helpful for taking effective measures to control the disease.