Establishment Of Antibody Detection Method And Development Of Subunit Vaccine For QX-type Infectious Bronchitis Virus

Infectious bronchitis(IB)is an acute,highly contagious disease caused by infectious bronchitis virus(IBV).IBV is prone to mutation so that it has numerous serotypes,and cross-protection between different serotype strains is poor.It is important to develop a vaccine for the serotype of the epidemic strain and establish an immune evaluation method to match it.In recent years,QX-type IBV has been prevalent in Asia and Europe,accounting for more than 70%of the epidemic strains isolated and identified in China.So the purpose of this study was to develop a subunit vaccine against QX-type IBV and establish an indirect ELISA method for detecting the antibody level afte immunization,and to coordinate with the combined attenuated vaccine of Newcastle Disease and Infectious Bronchitis(La Sota strain+QXL87 strain)developed by our laboratory,which can provide a comprehensive scheme for the immune prevention and control of QX-type IBV.1 Establishment of an indirect ELISA method for antibody detection of infectious bronchitis virusThe S1 protein antigenicity of QX-type IBV CK/CH/JS/2010/12 strain was analyzed by DNAstar Protean software,and five sections with strong antigenicity were selected.The homologous gene fragments were named S1-A(61-525 bp).S1-B(454-933 bp),S1-C(835-1299 bp),S1-D(1453-1620 bp),S1-E(277-882 bp).The amino acid sequence of S1 protein is closely related to the serotype of IBV.Sl-A-S1-E 5 peptides are located in the hypervariable region of S1 protein,the amino acid sequence of those peptides were compared with that of S1 protein of six vaccine strains(H120,H52,MA5,M41,W93 and 4/91)respectively by DNAstar Megalign software.The comparison result showed that the mutation rate of Sl-E peptide was the highest,which mutation rate was 25%and 20.3%comparing to Mass vaccine strain and 4/91 vaccine strain respectively.It is speculated that S1-E peptide is the best coating antigen for establishing ELISA method.The five gene fragments were amplified by RT-PCR and cloned into pET-32a(+)vector for prokaryotic expression.The results of SDS-PAGE showed that the five peptides S1-A?S1-E were all successfully expressed,the molecular weight of which is 36.9kD,37.7kD,37.2kD,26.4kD,42.2kD respectively as expected.The five peptides purified by Ni-NTA affinity chromatography medium were used as the coating antigen to detect QX-type IBV positive serum and negative serum,through the value of P/N,it showed that the reaction between positive serum with S1-E peptide had the highest sensitivity.Therefore,S1-E peptide was decided to use as the ELISA coating antigen,and after a series of conditions optimization,the best working concentration was determined as following,the coated antigen was diluted at 1 ?g/mL,and the HRP-labeled rabbit anti-chicken antibody was diluted to 1:10000.And also the best working conditions were determined,the antigen was coated at 4? for 12h,excepting that,the remaining steps were all incubated at 37? for 1h.It was determined that the modified method did not cross-react with the positive serum of Newcastle disease virus,avian influenza virus and infectious bursal disease virus,and the variation coefficient of the intra-assay and inter-assay repeatability tests were less than 12%,indicating the good repeatability and specificity of this method,which can be further used for monitoring the antibody level of QX-type IBV vaccine.2 Development of S1 protein subunit vaccine for QX-Type infectious bronchitis virusThe S1 gene of QXL strain was amplified by PCR,and the signal peptide was truncated.The gene was cloned into pET-32a(+)vector for prokaryotic expression.The results of SDS-PAGE showed that the form of protein expression was mainly inclusion body,and the molecular weight of it is 70 kD as expected,which is named S1 protein.The S1 protein under denaturing conditions was purified by Ni-NTA affinity chromatography medium and renatured by protein dialysate.The purified renatured S1 protein was used as the aqueous phase of the vaccine,and the white oil was used as an adjuvant to prepare a water-in-oil dosage form subunit vaccine,containing 100 ?g/ml of vaccine.The three days old SPF chicks were randomly divided into four groups:S1 subunit vaccine group,QXL87 attenuated vaccine group,QXL87+S1 subunit vaccine group and non-immunization challenge control group,with 15 chicks in each group.At seven days of age,the QXL87 attenuated vaccine was used nasally and ocularly at the dose of 103 SEID50/0.1 mL,and the S1 subunit vaccine was used by chest muscle injection at 21 days of age with the dose of 0.5 mL.One week after subunit vaccine immunization,the antibody level of QXL87+S1 subunit vaccine group increased significantly and was higher than that of other groups.The antibody level in the subunit vaccination group increased obviously after 21 days of immunization.All chickens were challenged with QXL strain through the way of intranasal and intraocular dropping at the 49 days of age with the dose of 104 0 EID50/0.1 mL.After challenged,the incidence and mortality of each group were recorded daily and the results showed that the clinical protective efficacy of S1 subunit vaccine,QXL87 attenuated vaccine and QXL87+S1 subunit vaccine against QXL strain were 70%,90%and 90%respectively.The virus contend in cloaca swabs and throat swabs were deteced by using Real-time fluorescence quantitative PCR at 2,5,and 7 days after challenge.The amount of virus shedding of each immunization group began to decrease at the seven days after the challenge,and that of QXL87+S1 subunit vaccine immunization group was lowest.At 10 days after the challenge,all chickenss were sacrificed and necropsy,tracheal and renal lesions were observed and recorded.Lesions in the trachea and kidney of the chickens of each immunization group were not observed.And the cilia vatality of chickens trachea in each test group was observed and recorded.The results showed that the cilia activity of three immunization groups of the experimental group is from 67%to 100%.Comprehensive clinical protection efficacy,amount of virus shedding and cilia activity of tracheal,S1 subunit vaccine can provide certain protection against the challenge of QXL strain,and QXL87 live vaccine combined with S1 subunit vaccine can achieve adequate immune protection against QXLstrain.