| aSubgroup J avian leukosis is a kind of myeloid leukosis disease in meat-type chickens, caused by a retrovirus named avian leukosis virus subgroup J (ALV-J). ALV-J was first recognized in the late 1980s and subsequently developed a disease concern for the meat-type poultry industry worldwide by horizontal and vertical transmission. The serious economic loss caused ALV-J is often due to mortality in meat-type breeder chickens, body weight suppression and immunosuppression. Eradication program has been enacted in some countries in recent years to control ALV-J by removing infected breeding stock from flocks, thereby reducing virus spread by vertical transmission. Therefore, isolation and genetic analysis of viruses, development of diagnositics for detecting antibodies are important for ALV-J control and eradication in China.In the study, two ALV-J strains designated as ALV-J JL-2 and Hrb-1 were isolated and identified from two flocks of commercial broilers in Jin Lin province and Harbin, respectively, by RT-PCR for detecting virus RNA, PCR for detecting provirus from ALV-J infected CEF cells.JL-2 and Hrb-1 env gene which including gp85, gp37 and E-element were cloned and amplified by PCR from SPF CEF cells 7 days postinfection with ALV-J by a pair of primers designed according to the sequence of prototype ALV-J virus HPRS-103 cDNA gene, The pMD18-T-JL2/env and pMD18-T-Hrb-l/env were obtained by inserting the env genes of two strains into pMD18-T vector. Genetic and antigenic analysis of gp85 and gp37 of Hrb-1 and JL-2 strains were performed on level of the amino acid and nucleic acid sequence, the result showed that the two viruses were related to ALV-J ADOL-HC-1 and UD3 strains, respectively.The JL-2 provirus gp85 gene was amplified and cloned into vector pGEX-6P-l, and the recombinant plasmid was obtained. The recombinant plasmid was transformed into E.coli BL21 (DE.O and expression was induced with IPTG The result of SDS-PAGE analysis showed that the JL-2 gp85 gene was efficiently expressed, the target protein (57KD) is 31.8% of the total bacterial protein. The immune reactivity of the expressed gp85 was also determined by western-blot, the result showed that the expressed product have a positive reaction with anti-JL-2 serum.An ELISA method was developed based on the recombinant gp85 to detect sera of ALV-J infected chickens. The P/N value of ELISA was determined by detecting 51 SPF chicken sera and the procedure of ELISA test was established after a series of tests on reaction conditions, specificity, repetition and sensitivity. The result of research on specificity showed that there was no cross reactivity to 10 kinds ofpositive sera against other avian viruses. The ami ALV-J antibody could be detected from 10 to 85 days after chickens inoculated. The total of 480 serum samples collected from 3 field flocks were comparatively tested with the gp85 ELISA and a commercial kit (Avian Leukosis Virus Antibody Test Kit-Subgroup J, IDEXX), the results indicated that the coincidental rate between the two tests is 89%^ 95.5%, 86.4%, respectively.In conclusion, two ALV-J strains, JL-2 and Hrb-1 from northeastern part of China were isolated and identified, related molecular epidemiologic data were obtained by the genetic analysis and the gp85 ELISA was established with the expressed gp85 as antigen. We believe that all above progresses in this research will provide effective technical support for the eradication of ALV-J in China in near future.
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