绵羊肺腺瘤病相关基因突变检测及绵羊、山羊内源性反转录病毒扩增-detection of gene mutations related to sheep pulmonary adenomatosis and amplification of endogenous retroviruses in sheep and goat.docx

Gene Mutation Detection Related to OPA and Sequence Analysis of Goat，s and Sheep，s enJSRV Abstract 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 is 2 ～ 24 months, incubation period of OPA cause by experiment is 3～7 months. 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 phosphatidylinositol 3-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、p53 was confirmed by search SANGER data bank.Exon of gene nras、kras、pik3ca、egfr、p53 was 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 of 10～20 copies of the virus .DNA or cDNA can be detec