Cloning And Expression Of Capsid Protein P27 Gene Of Avian Leucosis Rous Sarcoma Virus And Development Of Immunology Diagnostic Methods

Avian Leukosis is a malignant neoplasitc disease caused by avian leucosis/sarcoma virus. Infection with ALV is widespread in poultry flocks in the world with high morbidity. Although sporadic cases of neoplasitc disease occur in most flocks and the incidence of death is usually low, heavy losses from AL are uncommon. Based on Epidemiological data, AL is highly prevalent and occurs ubiquitously in China. Coupled with the fact that vaccines or medicines against avian leucosis virus have not been successful up to now, AL was controlled primarily by the successful application of ALV eradication program to chickens. The eradication of exogenous avian leukosis virus (ALV) from infected lines of chickens depends on the exclusion of congenitally infected chicks from groups of hatched chicks and the prevention of reinfection from extraneous sources. Therefore obviously desirable, the development of a rapid, and exact diagnostic technique for detecting exogenous virus has been necessary precondition to control ALV.At present, serologic diagnosis of ALV infection has been developed by variety methods. These test, however, have some disadvantages in practical application. The capsid protein p27, primary group specific antigen of ALV, is main component in virion. Amino acid sequence of p27 protein shares an about 90% identity between exogenous ALV subgroup A, B, C, D, J. Therefore, p27 is the ideal target protein for detecting ALV antigen. The purpose in this study was to clone and express ALV p27 gene in prokaryotic and eukaryotic cell, Further, to develop a rapid, exact, sensitive and specific diagnostic technique for detection of ALV antigen and antibody using recombinant p27 as antigen.RNA was extracted from CEF cell artificially infected with avian leucosis SR-RSV-E and the gag-p27 gene was amplified by RT-PCR with p27 specific primers. Then the gene was cloned into plasmid pMD18-T and sequenced. Nucleotide sequence analysis indicated that the identity between cloned p27 gene and that published in GenBank (J02342) was over 97.3%. The whole coding region of p27 gene was subcloned into prokaryotic expression vector pGEX-6p-l. Then the recombinant plasmid carrying p27 gene was transformed into BL-21 E.coli. Through induced with IPTG, a soluble recombinant protein with molecular weight of 56kD expressed in E.coli was identified by SDS-PAGE. The content of recombinant p27 protein was 23% of the total cellular proteins at optimal conditions. The recombinant p27 was purified with Glutathion Spharose 4B affinity chromatography. Western-blot verified the antigen reactivity of recombinant p27 protein and an indirect Enzyme-Linked Immunosorbent Assay (E1ISA) will be developed using recombinant p27 as antigen for the detection of ALV infection in place of the whole ALV Compared with the former method for purification of p27, that is through extraction, disruption and Polyacrylamide gel electrophoresis (PAGE) of whole ALV, Antiserum was produced from rabbit immunized with purified recombinant p27 protein, then, An agar gel immune precipitation test (AG1P) using p27 antiserum as antibodies was developed to detect ALV antigen in the feather pulp of infected chickens, which was a simple and specific method suited for practical application.Further, in this study, p27 gene was expressed in Sf9 insect cells by BAC-TO-BAC?expression system. And the antigenic reactivity of recombinant p27 protein was identified by Western blot with antiserum against whole ALV. The recombinant p27 protein obtained from insect cells was more similar to nature p27 protein in structure and function than that from prokaryotic cells. With purified recombinant p27 protein as antigen, monoclonal antibodies against p27 will be produced afterward. Based on the monoclonal antibodies against p27 and available polyclonal antibodies against p27 above-mentioned, a DAS-EL1SA will be developed to detect group specific antigen p27 from chicken infected with ALV. This DAS-ELISA would be more specific, sensitive and applicable for the detection for ALV antigens in a large...