| Infectious bronchitis virus(IBV)is one of the important pathogens that seriously endanger poultry industry.IBV is a coronavirus prone to mutation during replication,resulting in many variants with different antigenicity and pathogenicity.Due to the large number of IBV serotypes,the serological diagnosis of IBV is particularly difficult.In this study,polyclonal antibodies and monoclonal antibodies against M protein were successfully prepared using the conserved region of IBV membrane(M)protein expressed in prokaryotic cells as immunogen.Finally,a double-antibody sandwich ELISA method for detecting IBV was established,which provided a simple and reliable choice for clinical diagnosis of IBV.1.Prokaryotic expression of IBV M proteinProtean software was used to analyze the amino acid sequence of M protein of IBV CK/CH/JS/0611 strain.The 158-223 amino acids with good antigenicity and relatively conservative among different serotypes were selected and the corresponding primers were designed.After extracting total RNA of IBV,RT-PCR was used to amplify the M gene.The M gene was cloned into pET-32a(+)with His tag and pGEX-6p-1 with GST tag respectively and constructed recombinant expression plasmids pET-32a-M and pGEX-6p-1-M.Two recombinant plasmids were transformed into BL21(DE3)and BL21 respectively,then induced by IPTG.SDS-PAGE showed that the size of the recombinant M-HIS and M-GST were 28 kD and 33 kD,respectively,which were consistent with the expectation.Western blot results showed that both recombinant M-HIS and M-GST could react specifically with IBV positive serum,indicating that both recombinant proteins had good immunogenicity.2.Preparation of polyclonal and monoclonal antibodies against IBV M proteinAdult rabbits were immunized by subcutaneous multipoint injection on the back with 200?g M-HIS recombinant protein per animal.After three immunizations,blood was collected from ear vein to prepare serum.The serum antibody titer of immunized rabbits reached 1:102400 detected by indirect ELISA.Using M-HIS and M-GST recombinant proteins as immunogen and detector respectively,5 hybridoma cell lines stably secreting monoclonal antibodies against M protein were obtained by hybridoma cell technology,named 1B2,1D4.3E5,10G3 and 10D7,respectively.Indirect ELISA showed that all 5 strains of hybridoma cells could secrete antibodies stably within 20 generations.The titer of ascites prepared by in v/vo-induced method reached 1:103.Western blot results showed that all 5 monoclonal antibodies could specifically bind to QX-type IBV M protein.Among them,1B2 had the broadest reaction spectrum.Besides QX type,it could also react with IBV strains of other four common serotypes,including TW-1,TC07-2,Mass and 793/B type.3.Establishment of double-antibody sandwich ELISA method for IBV detectionUsing polyclonal antibody and monoclonal antibody 1B2 as capture antibody and detection antibody respectively,and M-GST as antigen,a double-antibody sandwich ELISA method was established.The optimum conditions were as follows:the optimal coating concentration of capture antibody was 4 ?g/mL and overnight at 4?;the optimal working concentration of detection antibody was 2?g/mL;3%BSA solution blocked at 37? for 60 min;the antigen was incubated at 37? for 60 min;the working concentration of HRP labeled sheep anti-mouse IgG was 1:10000,incubated at 37? for 60 min;and the optimal substrate reaction time was 15 min.The linear correlation coefficient of the double-logarithmic regression fitting line established by this method was greater than 0.99,and the detection range of M protein for quantitative analysis was determined by regression equation from 50 ng/ml to 1.8 ?g/ml.The results of inter-batch and intra-batch repeated experiments showed that the coefficient of variation was less than 10%,indicating that the established method had good repeatability. |
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