Gene Mutation Detection Related To OPA And Sequence Analysis Of Goat’s And Sheep’s EnJSRV

BACKGROUND:Jaagsiekte sheep retrovirus (JSRV) is a type βretrovirus specifically associated with a contagious lung tumor of sheep, ovine pulmonary adenomatosis (OPA).Incubation period of OPA cause in nature is2～24months, incubation period of OPA cause by experiment is3～7months. OPA is the B-type infectious disease according to the disese classification from OIE.The JSRV envelope glycoprotein (Env) functions as a dominant oncoprotein in vitro and in vivo. Several reports suggested that the phosphatidylinositol3-kinase/AKT pathway and RAS-MEK-MAPK pathway are important for transformation of cells. Establish specific diagnosis method is the foundation to control and prevention the disease. mechanism of JSRV is key point to understand the disease.OBJECTIVE AND METHOD:Jaagsiekte sheep retrovirus should be detected by using specific PCR and invested the relationship between OPA and gene mutation. The specific primers were designed according to sequences of JSRV in GenBank. And the peptide sequence of env, YXXM was take into count in designing of primers. The spefic PCR method could used in detection of JSRV,and the clinical sambles were detection by the method. High mutation rate site in gene nras、kras、pik3ca、egfr、p53was confirmed by search SANGER data bank.Exon of gene nras、kras、pik3ca、egfr、p53was confirmed by search data bank of UCSC and GenBank. Gene mutation site was detection by using technique of PCR-SSCP and the samples were sequencing. Three pairs of primers were designed according to the sequence of strain enJSRV-20. Sequences of sheep’s and goat’s enJSRV were amplified by PCR technology and these PCR products were cloned into pMD18-T vector and then sequenced.RESULTS:(1)The method of spefic PCR showed high sensitivity, With high specificity and repeatability. The lowest level of10～20copies of the virus.DNA or cDNA can be detected by the PCR method.(2)10clinic samples were positive detected by the method.10positive samples and10negative samples were detection by the method of PCR-SSCP,The mutation was detection in the second exon of kras in2positive sample. The mutation site was in the12th genetic code, Which nucleotide G was mution to T,amino Glycine was mutivon to valine.The mutation of7th exon of gene p53was detectied in one sample. Which nucleotide T mution to C, amino was not mutivon.(3)The complete genome of goat’s and sheep’s enJSRV were obtained successfuly. Analysis results showed that the sequence of sheep’s enJSRV was99.4%similarity with enJSRV-20and90.9%similarity with strain JS7. Analysis results show that the sequences of goat’s enJSRV was94.2%similarity with enJSRV-5and88.1%similarity with strain JS7. The sequence of sheep’senJSRV was93.1%similarity with goat’s enJSRV. enJSRV promoter methylation analysis of sheep and goat tissue showed that:methylation and unmethylation enJSRV promoter was detection in all the organization DNA.(4)The gene pro、env were link to eukaryotic expression vector confirm by the enzyme cut reaction, Protein immune fluorescence and Western Blot test showed that constructuring plasmid EGFP-enpro%pcDNA3.1-enenv can the instantaneous expression in eukaryotic cells.CONCLUSIONS:(1)The specific RCR detection method of JSRV was established in this study, and it is important for studying OPA control measures.(2)Gene mutations of kras and p53was detection in JSRV infection tumor tissue will be useful in the molecular pathogenesis and further study of OPA.(3) The complete genome of goat’sand sheep’s enJSRV were amplication and sequence analysis, methylation and unmethylation enJSRV promoter were detection in all the organization DNA.(4) The construction of the enJSRV pro and env eukaryotic express plasmid will provides an important experimental platform to study of enJSRV function and inhibiting virus infection.