| Infectious laryngotracheitis(ILT)is an acute respiratory disease of poultry caused by Infectious laryngotracheitis Virus(ILTV).The typical clinical symptoms are depression,dyspnea,eyelid swelling,Spit out bloody mucus,etc.At present,the main way to prevent and control the disease is vaccination.The commercial vaccines on the market are mainly attenuated vaccines,but they have the risk of returning to strong virulence,so the development of new ILTV vaccines is extremely important.gB protein is the main protective antigen of ILTV.In this study,the ILTV gB gene was inserted into the heat-resistant strain of Newcastle disease virus(TS09-C strain)by reverse genetic manipulation technology,and the recombinant Newcastle disease virus r TS-gB was obtained,and its biological characteristics and immune efficacy were determined;At the same time,an indirect ELISA detection method for ILTV antibody was established.The specific research contents and results are as follows:1.Construction of recombinant Newcastle disease virus expressing chicken infectious laryngotracheitis gB gene and its immune protection experimentThe gB gene of the ILTV WG strain was amplified by PCR and inserted between the P and M genes of the NDV TS09-C strain genome to rescue the recombinant virus r TS-gB.Indirect immunofluorescence and Western-Blot results showed that gB protein could be expressed in cells infected with recombinant virus.The mean lethal time(MDT)of the minimum lethal dose of the recombinant virus was greater than168 hours;the intracerebral pathogenic index(ICPI)of SPF chicks at 1 day old was 0;the thermal stability test at 56 ℃ showed that the r TS-gB and TS09-C strains had the same effect.The thermal stability was similar;it indicated that the recombinant virus r TS-gB maintained the biological characteristics of attenuated and heat-resistant of the parental TS09-C strain.In order to evaluate the immunization effect of the recombinant virus r TS-gB,the immunization experiment of 1-month-old SPF chickens was carried out.14 days after immunization,the hemagglutination inhibitory titer of NDV antibody in chickens immunized with r TS-gB was greater than 4 log2,and the positive rate of ILTV antibody detected by indirect ELISA method was 100%(consistent with the immunization effect of commercial vaccine).After 14 days of immunization,the ILTV WG virulent strain was used to carry out the challenge test,and the clinical symptoms were observed.The results showed that the protection rate of the r TS-gB test group reached 100% as the commercial attenuated vaccine control,and the unimmunized group was all morbid and had two Chickens die.The above results indicate that the recombinant Newcastle disease virus r TS-gB expressing the gB gene of chicken infectious laryngotracheitis can be used as a dual vaccine candidate for the prevention and control of ILT and ND.2.Establishment of an indirect ELISA method for the detection of ILTV antibodiesThe ILTV gB truncated fragment gB2(aa327-665)was expressed and purified using the E.coli expression system.Using purified ILTV gB2 protein as the coating antigen,the reaction conditions were optimized,and an indirect ELISA method for detecting ILTV antibody was established.The method has no cross-reaction to avian influenza virus,avian leukemia virus,chicken infectious bronchitis virus,avian Escherichia coli and Salmonella positive sera,indicating that the method has good specificity.Clinical samples were detected by this method and a commercial ILTV antibody ELISA detection kit,and the coincidence rate of the two methods was 94%.The above results indicated that the indirect ELISA method for ILTV antibody was successfully established,and it could be used as a method for detecting ILTV antibody in this study and clinically.In conclusion,this study constructed a recombinant Newcastle disease virus r TS-gB with gB gene of chicken infectious laryngotracheitis.The recombinant virus has similar characteristics to the parental strain and can induce chickens to produce higher levels of NDV and ILTV antibodies,ILTV.The challenge protection rate was100%,and the protective effect was similar to that of commercial vaccines.In addition,an indirect ELISA detection method for ILTV gB protein was established.to provide technical support for ILTV prevention and control. |
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