Studies On JSPV Envelope Protein And Its Subunits Induced Transformation Of NIH3T3Cells And Molecular Mechanisms

Background:Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a chronic infectious lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). JSRV, classified as a β retrovirus as its genomic organization contains only the essential genes characteristic of retroviruses:gag, pro, pol, and env. The env gene, an oncogene, encodes the surface (SU) and transmembrane (TM) domains of the envelope protein (Env), which could induce the transformation of NIH3T3cells. The receptor of JSRV was Hyaluronoglucosaminidase2(Hyal-2). NIH3T3, considered to be extremely sensitive to contact inhibition of growth, was an important cell line for studies in tumorigenic mechanism of JSRV because it was easy to recognize the transformed cell by losing contact inhibition. Subsequent experiments indicated that expression of the JSRV env gene alone was necessary and sufficient for inducing transformation of NIH3T3cells, while the mechanism of JSRV induced transformation of NIH3T3was still worth to being discussed.Objective:Studies on the molcear mechanisms of JSRV Env and its subunits in inducing transformation of NIH3T3cells were theoretical significance and potential applications for clarifying the pathogenesis and determining the prevention target of OPA.Methords:1. The Hyal-2coding sequence was amplified by RT-PCR from the rumen tissue of ovine, which was used to construct the eukaryotic expression plasmid contained ovine Hyal-2coding sequence. The eukaryotic expression plasmid was transfected NIN3T3cells to construct the cell line stable expressed ovine Hyal-2, which was screened and purified by G418.2. The eukaryotic expression plasmids contained JSRV Env and its subunits were used to transfect NIH3T3and the ovine Hyal-2stable expressed cell line. After transfection, the contact inhibition, the ability of anchorage-independent growth, proliferative activity and the protein content of phosphorylation of Akt were measure and compared with control group.3.kras, nras, egfr, p53and pik3ca tumor related genes mutations located in exon mutation detection primers were designed. PCR-SSCP and nucleic acid sequencing analysis were used to measure mutations in JSRV Env and its subunits induced transformation. 4. Total DNA from the lung tissue of a JSRV-infected sheep in Inner Mongolia was used to clone gag, pro and pol gene fragments and linked with LTR and env to obtain the recombinant plasmid including complete genomic sequence of the JSRV.Results:1. The eukaryotic expression plasmid pcDNA-Hyal2-HA, contained ovine Hyal-2, was constructed, and the NIH3T3-Hyal2-HA cell line which was stable expression of ovine Hyal-2, was constructed successfully.2. Env and TM could induce transformation and activate of Akt in NIH3T3cells, however, only TM had such ability in NIH3T3-Hyal2-HA cells.3. The mutations of tumor-related genes, kras, nras, egfr, p53and pik3ca, did not observed in the process of JSRV Env and its subunits induced transformation.4. The recombinant plasmid pMD-JSRV, contained full length genome of provirus of exogenous JSRV, was constructed, and homology analysis showed that the virus strain belonged to the JSRV type Ⅱ. pMD-JSRV and AF105220strain share a high nucleotide identification of95%.Conclusions:1. Cell line stable expressed ovine Hyal-2was constructed successfully.2. TM could induce transformation, and it played a major role in JSRV Env induced transformation and Akt activation in NIH3T3cells.3. The ability of JSRV Env in transformation was inhibited by ovine Hayl-2in NIH3T3cells.4. The mutations of tumor-related genes may not involved in JSRV Env and its subunit induced transformation.5. The recombinant plasmid contained full length genomic clone of JSRV will provide an experimental platform for studying the effects of structural proteins of JSRV in Env induced transformation.