Expression Of Small Regions Of Infectious Bovine Rhinotracheitis Virus Glycoprotein B In E.coli, Development And Application Of An Indirect Enzyme-Linked Immunosorbent Assay For Detection Of Anti-IBR Antibodies

Infectious bovine rhinotracheitis(IBR) is an acute, feverish and contagious infectious disease of cattle, caused by infectious bovine rhinotracheitis virus (IBRV). Virus genome encodes eleven glycoproteins. Among them glycoprotein B is one of major proteins presented on the envelope of virus. It is the major immunity protein as well. In this study, Plasmid T-gB was constructed by PCR amplification of 1776-(named gBI) and 1551-(named gBII) bp fragments from IBRV Bartha Nu/67 strain, using two pairs of specific primers designed according to IBRV DNA sequence in Genbank(sequence access number: NC₀01847). The fragments amplified were cloned into pGEM-T vector(Promega) to obtain plasmid T-gBI and T-gBII. Fragment gBII was released with XhoI&NsiI site from T-gBII and inserted into the XhoI&NsiI sites presented in T-gBI. Plasmid T-gB was obtained.Based on hydrophilicity, antigenity and surface probality analysis of the amino acids of gB using biosoftware DNAstar, the three outstanding small regions (named gBB, gBC and gBD, corresponding to Ala₁₉₂-Leu₂₆₈ Met₃₀₈-Leu₄₀₂ and Ala₄₉₄-Leu₅₉₈ were selected for expression in E.coli. The gBB, gBC and gBD gene fragaments obtained by PCR amplification from template T-gB with 3 pairs of specific primers, containing BamHI site in up ones and HindIII in lower ones, were inserted into pGEM-T vector. The plasmids of T-gBB, T-gBC, T-gBD were constructed. Confirmed by digestion of restriction endonuclease enzymes, BamHl&Hindlll and sequencing of the inserted fragments, the cohesive-ended fragments with BamHl&Hindlll sites cut from the three plamids were directionally subcloned into the expression vector pET32a(Novagen). The positive ones including gBB, gBC and gBD were named rpET32a-gBB, rpET32a-gBC and rpET32a-gBD, respectively. They were transformed into component bacteria BL₂₁(DE3). The positive bacteria colony at mid-log growth was induced by 0.6 mmol/L IPTG in LB medium. All the three proteins successfully expressed with 6-His tag fusion protein at N-terminal end were partially in soluble.formation, molecular weights are 29.2 KD, 31.2 KDand 32.4 KD as expected. They were named as r32a-gBB, r32a-gBC and r32a-gBD, respectively and expression efficiency are 40.9%, 41.2% and 61.4% respectively. The soluble proteins were purified using affinity chromatograpy under nature condition from crude preparations. The concentricity of purified proteins is 0.200 mg/mL, 0.240 mg/mL and 0.200 mg/mL respectively, and the purities is 79.2%, 88.2% and 76.4% respectively. The recombinant proteins were checked by indirect ELISA. They react to positive serum of IBR, do not to negative serum, and do not to positive serum of other six kinds of diseases, including BEF(Bovine ephemeral fever), CBPP(Contagious bovine pleuropneumonia), BVD-MD(Bovine viral diarrhea-mucosal disease), BTB(Bovine tuberculosis), BPB(Bovine paratuberculosis) and BAD(Bovine Akabane disese), meanwhile the fusion protein expressed by pET32a plasmid itself does not react to the positive and negative serum of IBR, showing that the recombinant proteins retain antigenicity of the nature proteins and possess the specificity of reaction.The Indirect ELISA for detection of antibodies of IBRV was established using r32a-gBB as coated antigen with the optimal working parameters, including 5 μg/mL of the r32a-gBB protein antigen for coating, testing sera dilution at 1:40, second antibody(Sigma) dilution at 1:5000, the standard of determining as positive sample S/P≥0.395 and others. The reaction between the antigen and the positiveserum can be blocked by IBRV and there is no any cross reaction to the positive sera of the other diseases above stated, demonstrating specificity of the diagnosis method is excellent. The variation coefficient of inter- and inner- batch antigen production testing quality control serum which can guarantee the quality of antigen and the accuracy of testing results is below 10 percents, demonstrating that repeatability of the antigen is good. Compared with the virus neutralization testing, the speciality of the test is 83.3%;the sensitivity, 90.9%;the agreement, 88.9%. Compared with the IBR ELISA kit from France, the three indexes are 92%, 92.7% and 92.5%, respectively. 158 of 220 serum samples collected from some area and provinces of China are positive tested by this diagnosis method and the positive rate is 71.81%. The assay was confirmed to be specific and sensitive by the results above, suggesting an application prospection.In conclusion, IBRV gB gene was successfully cloned and three small regions were expressed in soluble forms in E.coli and purified. They lay the foundation for further studies of the protein structure and function of glycoprotein B. The simple and fast diagnosis method using r32a-gBB expressed as diagnosis antigen can be a technique support for further developing the commercial kit of diagnosis, prevalence survey and control & eradication program of IBR in China.