The Epidemiological Investigation Of ALV Infection In Jiangsu And Anhui Provinces And Expression Of Env Gene Of Subgroup A Of Avian Leukosis Virus

Avian Leukosis is one of the important epidemic diseases of poultry, which is caused by Avian Leukosis Virus and Avain Sarcoma virus. According to the difference of the host range and cross neutralization, ALV is divided into ten subgroups, A to J. Now subgroup J is the most popular, it has already spread from meat type chickens to layers, yellow broilers and local chickens. ALV has resulted in huge losses to the poultry industry. During past years, we found that the positive rate of some flocks up to68.4%. Some flocks showed positive to p27antigen, while other flocks showed antibody positive to ALV. Some flocks showed both antigen and antibody positive to ALV. In this study, we investigated the epidemiology of ALV in Jiangsu and Anhui provinces. An ALV-A strain was identified by PCR and sequence, which envelope gene was expressed in prokaryotic and eukaryotic expression system.1. The epidemiological investigation of ALV infection in Jiangsu and Anhui provincesIn order to know the epidemic of ALV in Jiangsu and Anhui provinces, we investigated5lines of more than50000chickens with p27antibody and p27antigen detection kit. The results showed that5lines of chickens showed both antibody and antigen positive, and the individual positive rate of antibody and antigen was25.44%and24.93%respectively. We found that only a few chickens showed antigen and antibody positive at the same time. The positive rate in Jiangsu provinces was49.77%, and only13.06%in Anhui. It was interesting that rooster positive rate was much higher than hen’s positive..2. The ampilication of PCR and analysis of subgroup A of avain leukosis virusOn the basis of the autopsy tissue, we detect ALV by PCR. A subgroup A of ALV was identified, which was named ALV-A JSYC130902. The of env gene was amplified with specific primers and sequenced. The results showed that the env gp85contained1020nucleotides. The genetic evolution analysis showed86.1-95.6%amino acid homology to other7viruses ALV-A published in GeneBank and87.1-96.6%for the nucleotide homology. The highest homology was to that of SDAU09C3. Compared with RAV-1, JSYC130902showed5substitution mutation (they were at147、157、217、325、328).3. Cloning and expression of gene env of ALV-AThe env gene of ALV-A was amplified by PCR and RT-PCR. Then it was cloned into pFastBacl to construct the plasmid pFastBacl-env. pFastBacl-env was transformed into the competent cells DH10Bac to construct the recombinant shuttle plasmid that was transfected to sf9cells to get the recombinant baculovirus. Immunofluorescence assay showed that the protein could be recognized by antiserum. At the same time, we also cloned the env gene of ALV-A into the prokaryotic expression vector pET-32a and successfully constructed recombinant plasmid pET-32a-env which was transducted into E.coli cells [BL21(DE3)] and expressed under the induction of IPTG SDS-PAGE and western blot results showed that the fusion protein band was about73KD of molecular weight.