High-efficient Expression Of Truncated Glycoprotein B Gene Of Infectious Laryngotracheitis Virus And The Development Of The TgB-ELISA

After analyzing glycoprotein B (gB) of infectious laryngotracheitis virus(ILTV) by DNASIS and GENE RUNNER, a truncated fragment of gB(403 to 686 aa), with better immunogenicity was chosen. The gene fragment of gB was amplified by PCR with two primers containing restricted enzyme sites EcoRI or SalI. The truncated gB was cloned into the expression vector pET30a(+) and generated recombinant plasmid pET-tgB. A 6xHis-tagged fusion protein (His-tgB) with an apparent molecular weight of about 37ku was expressed high-efficiently in E. coli. Western blotting analysis indicated that the expressed tgB is immunoreactive. The indirected ELISA for detection anti-ILTV antibody is established, employing the purified His-tgB. The optimal reaction condition is determined. The coated concentration of antigen is 0.5μg of protein one well, with 0.05M pH9.6 sodium bicarbonate buffer, the antigen-coated plates is incubated at 4℃ overnight. The dilution of serum samples is 1:100, with PBST, and incubated at 37℃ for 60 min. 90 sera from specific-pathogen-free(SPF) chicken are detected under optimal condition, and the optical density(OD) values are gathered. The results of statistics analysis showed that the average OD valued±3Sd is 0.154, therefore, the determined standard is obtained and OD≥0.154 is positive, OD≤0.154 is negative.The present study demonstrates that the indirected ELISA is sensitive and specific, showing virtually no cross reactivity to 11 kinds of positive sera from other diseases of chicken. The competitive-inhibition test show that this antigen is specific to the ILTV antibody.