Cloning And Expression Of Env And Capsid Protein Gene Of JSRV-NM Strain And Preparation Of The Monoclonal Antibody

Sheep pulmonary adenomatosis (SPA),a contagious lung neoplasia of sheep is caused by the Jaagsiekte Sheep Retrovirus(JSRV),a betaretrovirus. According to the OIE classification, SPA belongs to the B-type infectious disease.The aim of this study was to investigate the pathogenesis of JSRV and diagnosis method of SPA. The genomic DNA isolated from lung tumour tissues of sheep infected with JSRV-NM strain naturally was used for PCR amplification of surface protein(SU),transmembrane protein(TM) encoded by env gene and capsid protein(CA) encoded by gag gene. The PCR products of three independent PCRs were pooled and cloned into pGEM-T vector, and the recombinant plasmid were sequenced and compared with the JSRV-NM strain. The expression construction were engineered by inserting corresponding DNA into the expression vector and the recombinant plasmid were transformed into E.coli and eukaryotic cells, respectively. Then BALB/c mice were immunized with purified CA protein expressed in E.coli,and monoclonal antibody (McAb) against CA were obtained. Simultaneously,JSRV proviral DNA copies in peripheral blood leukocytes,lungs,mediastinal lymph nodes and nostrils fluid of SPA cases were detected by the established method,real-time quantitative PCR. The results showed that: (1) The amino acid sequences of the CA,SU and TM were 99.45%,97.36% and 80.2% identical to JSRV-NM strain, respectively. There were many mutations and stop codons in nucleotide sequences of the TM. The existence of incomplete proviral may be related to the protective immunity; There existed"YXXM"sequence specific for exJSRV in amino acid sequences comparison with enJSRV. (2) The recombinant plasmid pGEX-4T-1-CA,pGEX-4T-1-SU and pGEX-4T-1-TM were transformed into E.coli BL21 CodonPlus cells, and target protein was induced by IPTG. Results revealed that product yield approximately 37 Kda,68KDa and 46 KDa in size and fusion protein expressed accounted for 20%,25% and 30% of total bacterial protein, respectively. And the soluble protein and inclusion bodies were purified with glutathione-sepharose 4B and obtained high purity of fusion protein. (3) The CA gene was cloned into a plasmid pCDNA3.1(+), containing a cytomegalovirus promoter. When the recombinant plasmid pCDNA3.1(+)-CA-HA was transfected into 293 cells and Hela cells, indirect immunefluorescence and western blotting analysis with an anti-HA antibody resulted in detection of fluorescence and an expected protein. (4) Myeloma cells SP2/0 were fused with the splenocytes of the immunized mice, an indirect immunefluorescence with CA protein expressed in eukaryotic cells as antigen was used to detect antibody-producing hybridomas. And 3 hybridomas cells of them can product McAb against CA of JSRV-NM strain steadily. (5) The conservative sequence of env gene was amplified with specific primers and TaqMan probe designed as the JSRV-NM strain sequences by PCR from genomic DNA isolated from lung tumour tissues of the SPA natral case.The PCR product was cloned into pGEM-T and the recombinant plasmid was used as a standard quantitative template to develop real-time Q-PCR, including optimization of PCR system, and obtained the standard curve: Y=-3.308X+47.848,correlation coefficient: 0.991. The reproducibility and sensitivity of real-time Q-PCR were good,and 10-100 fold more sensitive than standard PCR. (6) Blood and tissues preparations from different sheep were tested for detection of JSRV proviral DNA by real-time Q-PCR. The results showed that the peripheral blood leukocytes,lungs and mediastinal lymph nodes and nostrils fluid of both B and C group were positive in the real-time Q-PCR,and have been found that proviral DNA more positive replicates in the lungs than those in peripheral blood leukocytes;In all sheep from group D, in which proviral DNA could be detected in the mediastinal lymph nodes but no clinical signs of SPA;One sheep from group E was positive in lung, and group A was negative.The results reported above laid certain foundation for explaining the pathogenesis of SPA, for establishing a sensitive diagnosis method of JSRV, and for taking effective measures to control the disease. It was the first time to study on the molecular cloning and expression of relative protein of JSRV and preparation of their monoclonal antibody, and on the real-time Q-PCR technique for the detection of JSRV in our country. Consequently, it has very important academic and practical significance for ensuring the security of stud sheep industry and the health development of sheep-rearing.