Prokaryotic Expression Of Infectious Bovine Rhinotracheitis Virus SH7 Isolate Glycoprotein B And Glycoprotein D And Establishment Of ELISA Methods

IBR(Infectious Bovine Rhinotracheitis)is an contagious disease of cow caused by IBRV(Infectious Bovine Rhinotracheitis Virus),which belongs to Alphaherpesvirinae,Herpesviridae.It is list as B class disease by OIE and the key quarantine disease for animal import in our country and international trade.Currently,gB and gD are the preferred protein of IBRV testing.The research starts on the gene of gB and gD deriving from IBRV SH7 in the previous stage,aiming to compare with the classic IBRV isolates found at home and abroad,meanwhile confirm the homoeologous relationship between IBRV SH7 and the above mentioned classic IBRV isolates.It also amplifies gene sequences of gB and gD with the size of 2802 bp and 1254 bp.In the homoeologous comparison,the gene sequences of gB and gD of IBRV SH7 share 100% homeology with that of Swiss BHV Cooper isolate(GenBank:Z78205.1).Through the analysis on the gene sequences of gB and gD,we have amplified the target gene with the size of 1461 bp and 1005 bp.Then we have cloned the new gB and gD gene on the prokaryotic expression vector pCold Ⅱ and pET-28 a,successfully constructed prokaryotic expression recombinant plasmid pCold Ⅱ-gB and pET-28a-gD.Next,we put pColdⅡ-gB and pET-28a-gD into BL21(DE3)and induce its expression with IPTG as well as purify the protein through Ni affinity chromatography.The result of SDS-PAGE shows that the recombinant pColdⅡ-gB expressed in inclusion body with relative molecular mass of 55.7kDa while the recombinant pET-28a-gD expressed in soluble form with relative molecular mass of40.6 kDa.The protein concentration of purified pColdⅡ-gB reaches 0.701mg/mL and protein concentration of purified pET-28a-gD reaches 0.518mg/mL.Using the purified recombinant pColdⅡ-gB and pET-28a-gD as coating antigen,we established three ELISA methods to test the antigen of IBRV,namely gB-ELISA,gD-ELISA and gB+gD-ELISA.Through the optimization experiment on various aspects of ELISA,we confirm the best coating concentration of the antigen of gB-ELISA and gD-ELISA are 2.804μg/m L and 4.144μg/mL.The serum dilution of gB-ELISA,gD-ELISA and gB+gD-ELISA are 1:80、1:40 and 1:80,respectively;the best serum response time is 1h,37℃;the best blocking solution are 1% BSA;the best HRP-conjugated antigen concentration are 1:12000,1:15000 and 1:12000;the best reactive time for HRP-conjugated antigen concentration is 1h 37℃;the best reacitvetime of substrate is 10 min and the positive and negative critical value are 0.311,0.346 and 0.292.The experiments show that specificity and repeatability in the three ELISA methods prove to be good for all.The utilization of these three methods on341 clinical samples testing,comparing with the SVANOVA IBR-Ab ELISA testing kits,show that the coincidence rates are 93.427%,92.965% and 95.673%respectively.The study analyze the homology of gB and gD gene in IBRV SH7 and set three indirect ELISA testing methods for IBRV antigen,which enjoys high coincidence rate comparing with foreign testing kits.Among them,the gB+gD-ELISA has the highest coincidence rate,providing new methods and technical support for further research and development on testing kits.