| Jaagsiekte sheep retrovirus (JSRV), a member of betaretroviruses genus, is the etiological agent of ovine pulmonary adenomatosis(OPA).OPA is a chronic, progressives, contagious neoplasm of the sheep lungs. The disease extensively distributed in many of the sheep-rearing countries and areas except Australia, New Zealand and Iceland and seriously affected the development of the sheep-rearing. OPA strongly resembles human bronchiolo-alveolar carcinoma(BAC) in the clinical, macroscopic, histopathologic and ultrastructural features. The common characteristics between BAC and OPA suggest that OPA could be a unique experimental model for BAC. Now the research about OPA becomes a hot point, but progress in etiological agent research has been hampered because of the lack of an in vitro culture system. The source of virus was therefore limited to experimentally or naturally infected sheep lung washes and difficult to isolate the virus using the common method. Interestingly, no detectable circulating antibodies against JSRV antigens have been detected in affected animals, a very unusual finding in retroviral infection. So it was diffcult to diagnose the disease using the common immune method. Therefore, it is very important for constructing an in vitro culture system and establishing a particular diagnostic method during the lifetime. However the results of studying OPA has been rarely reported in our country . The primary lung cells of 3-mouth sheep embryoes were cultured and infected by JSRV from 4 natural sheep pulmonary adenomatosis cases , and the JSRV were detected by electron-microscope observation and PCR. The results showed that there were some scattered virions in the 4 natural OPA lungs, but none in the cultured virus fluid . The diameter of virion was 100-125 nm. The lesions of lung cells of sheep embryo were observed from passage 2 to passage 9 postinfection and the virion-like particles were found. However , the results of PCR were negative. It was the first reseach about JSRV culture system in vitro in china. In order to amplify the complete genomies of Jaagsiekte sheep retrovirus Inner Mongolia Strain, eight pairs of primers were designed according to the published genebank sequence . The eight fragments were obtained by polymerase chain reaction(PCR) and each fragment was cloned into pMD-18 T vectors respectively and identified by PstI, EcoRI, SalI , EcoRV digestion and PCR. The recombinant plasmids were obtained and the sequences were analyed by DNAstar. The complete genome of JSRV-NM strain was determined. The results showed that the complete genome is 7430bp in length and contain four open reading frames representing gag, pro, pol and env genes. The nucleotide and amino acid sequences of NM strain were compared with the counterpart sequences of South Africa strain (Accession No. NC-001494) and USA strain (Accession No. AF105220). The nucleotide and amino acid homology were 90.4%,90% and 89.1%,91% ,respectively. The NM strain is related to the South Africa strain and USA strain distantly. This is the first nucleotide sequence of exogenous JSRV reported in China. The exJSRV-2 specific fragment DNA probe was labeled with digoxigenin (Dig), Jaagsiekte retrovirus NM strain RNA and proviral DNA in the lung tissue of OPA were detected by in situ hybridization (ISH). The results showed JSRV-NM mRNA and provirus DNA in sheep lung tumor cells had very strong hybridization signals and the control group had no postive signals. A pair of primer including env gene was designed according to the JSRV-NM genome sequence, and particularly designed Hindâ…¢/ BamHâ… sites. env gene was amplified by PCR and cloned in the vector PET-30b. The expression recombiant plasmid was constructed in vitro by inserting env gene into vector PET-30b, and then transformed into E.coli BL21.The bacteria was induced with IPTG. SDS-PAGE analysis showed that the env fusion protein was expressed. The molecule is 69kD.This successful study will provide an important foundation in molecular biology for further study on the oncogen... |
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