关键词: 犬瘟热病毒; H基因; 原核表达; Western blotting; 活性

Abstract: The assay was aimed to highly express the H gene of canine distemper virus in E.coli and analyze the immunogenicity of the products. The fragment with main epitope sequences, amplified from Ondetstepoort vaccine strain via reverse transcription-polymerase chain reaction, was cloned into vector pET28b and expressed through IPTG introduction after transformed into Rosetta^TM strain. The product, after purification, was identified by SDS-PAGE, western blotting and indirect enzyme linked immunosorbent assay. After that, the antibody titers of mice immunized with the purified H proteins were determined with neutralization test.The 1113 bp fragment of interest was specifically amplified by RT-PCR and expressed with the introduction of 1.0 mmol/L IPTG at 37 ℃. The protein expressed, identified by SDS-PAGE and Western blotting with a size of 42.38 ku, could be recognized by anti-CDV antibody without nonspecific reaction. In the animal experiment, the serum antibody titer was determined to be 2^-3.3 by neutralization test.The target protein was expressed accurately and specifically, showing a good immunogenicity and antigenicity according to the animal experiment, and therefore could be employed as an antigen candidate for the Real-time examination of animal immune condition, which laid the foundations for the development of new-type canine distemper vaccines and ELISA kit for detecting the anti-CDV antibody.

Key words: canine distemper virus; haemagglutinin gene; prokaryotic expression; Western blotting; activity