Construction And Identification Of Recombinant Infectious Bovine Rhinotracheitis Virus With Bovine Parainfluenza Virus Type 3 HN Gene

Infectious bovine rhinotracheitis virus(IBRV)and bovine parainfluenza virus type 3(BPIV3)are the important pathogens of bovine respiratory disease complex(BRDC).BRDC seriously hinders the healthy development of the world's cattle industry,which has the characteristics of high incidence and high mortality.IBRV has a double-stranded linear DNA genome of about 130 kb.Foreign genes can be stably inserted into the genome of IBRV,which is a candidate virus vector of recombinant live vector vaccine.Several recombinants expressing immunogenic foreign proteins have been constructed as vaccines for other infectous disease.Bovine parainfluenza virus type 3,is one of the main pathogens causing pneumonia calves.The virus causes infection worldwide.HN gene of BPIV3 is protective antigen of virus,that can induce neutralization antibody and widely used in vaccine reseach,such as recombinant adenovirus vector vaccines,subunit vaccines,DNA vaccines and nanoparticle vaccines.Therefore,construction of recombinant IBRV live vector vaccine with BPIV3 HN gene can prevent and control the mixed infection of IBRV and BPIV3 in BRDC.The study found that infectious bovine rhinotracheitis could be spread through the air.Because of the characteristic of fast spread,long distance and high efficiency,it was difficult to defend IBR.With the development of intensive,large-scale livestock production,IBR aerosols play an important role in the spread of epidemics.The IBR aerosol fluorescence quantitative PCR method was established in this study for rapid diagnosis of disease in cowshed environmental aerosol.TK/g E gene deletion of IBRV live vector was constructed.On the basis of this,the recombinant bovine infectious rhinotracheitis virus with BPIV3 HN gene was constructed,which established the foundation for the prevention and control of IBRV and BPIV3.Main work includes:Objective: infectious bovine rhinotracheitis can be spread through the air.This disease has the characteristics of high speed of propagation speed,far distance and high efficiency.So it is difficult to defend.With the development of intensive,large-scale livestock production,IBR aerosols play an important role in the spread of epidemics.(1)to establish infectious bovine rhinotracheitis population monitoring techniques,that canprovide technical support for forecasting and early warning infectious disease risk assessment;(2)to construct live infectious bovine rhinotracheitis virus vector;(3)to prepare bovine parainfluenza virus type 3 HN protein polyclonal antibody.This research will provide material support for the recombinant virus;(4)to construct a recombinant infectious bovine rhinotracheitis virus expressing bovine parainfluenza virus type 3 HN gene,providing a platform to develop live vector-viral vaccine expressing exogenous gene and lay the foundation for IBRV BPIV3 prevention and control.Method:(1)An assay was carried out using two specific primers designed to amplify a highly conserved sequence of IBRV g D gene.A recombinant plasmid containg the target gene g D was constructed as a standard control.Various conditions in the amplification process,including the concentrations of fluorescent dyes and primer concentration,were optimized to establish a airborne infectious bovine rhinotracheitis pathogen detection method which can be used for continuous monitoring of farm environment and large-scale dairy farm aerosol samples.(2)p EGFP-N1 and IBRV DNA were used as template to obtain GFP expression cassette and TK(g E)gene fragment.After splicing,targeting gene deletion recombinant transfer plasmids were construced with GFP expression cassette.The recombinant transfer plasmid and genomic DNA IBRV were co-transfected MDBK cells.By homologous recombination,IBRV live vector was obtained.(3)Using BPIV3 genome as a template,HN gene was obtained by RT-PCR method.HN gene prokaryotic expression vector was constructed.Fusion protein was obtained by induction and purification.New Zealand rabbits were immunized with purified recombinant protein to prepare polyclonal antibody,and to evaluate its ability to induce neutralizing antibody.(4)Using BPIV3 DNA and IBRV DNA as template,the g E deletion transfer vector containing HN expression cassette was constructed.The transferred plasmid and IBRV g E-/EGFP+ live vector genomic DNA co-transfected cells.By homologous recombination,the recombinant virus IBRV with HN gene was obtained.Results:(1)Through the optimization of SYBR Green I real-time quantitative PCR conditions,the optimal amplification reaction system was 20 ?L: 2 × SYBR Premix Ex Taq II 10 ?L,template 2 ?L,the upstream primer and downstream primer(0.25 ?mol/L)for each 0.5?L,RNase Free dd H2 O 7?L.Optimum reaction conditions was: the initial denaturation 95? 30 s,the denaturation 95? 5s,63? annealing 30 s,40 cycles of elongation,dissolution curve 95? 5s,65? 60 s,95? Continuous,50? 30 s and finally the reaction was completed.Temperature conversion rate of 20?/s.Fluorescence signal is detected at the end of each cycle extension.Using SYBR Green I fluorescence quantitative PCR method established to test BVDV,BPIV-3,BCo V and BRV,nucleic acid detection of these pathogens were negative.Low copy number of template nucleic acid can be detected in the detection of 101copies/?L,and the lowest positive plasmid copy number nucleic acid that can be detected in conventional PCR were 102 copies/?L.Continued monitoring of aerosol samples of 247 samples of the seven large-scale dairy farm,there are two ranch repeatedly to monitor the nucleic acid positive of infectious bovine rhinotracheitis virus.(2)Using the DNA of p EGFP-N1 and IBRV as template,GFP expression cassette and fragments of TK gene and g E gene were obtained by PCR.A variety of technical means,including overlapping PCR,enzyme digestion,recovery of the target fragment,connection and conversion were used.Finally,the recombinant transfer plasmid pc DNA3.1-TKup-GFP-TKdown and pc DNA3.1-g Eup-GFP-g Edown containing GFP expression cassette were obtained.The recombinant transfer plasmid and IBRV genomic DNA were co-transfected into MDBK cells,using green fluorescent protein and virus plaque clone screening.The IBRV live vector were obtained.PCR test results showed that the GFP gene has been inserted into the recombinant virus IBRV genome.(3)Using BPIV3 genome as a template to amplify HN,the prokaryotic expression vector were constructed and transformed into E.coli BL21(DE3).The optimal concentration of IPTG were 1mmol/L.The optimal induction temperature was 37?.The optimal induction time was 6h.After induction and purification,the protein concentration was measured by Pierce BCA Protein Assay Kit for 412?g / ml.After emulsified with an equal volume of freund adjuvant or incomplete freund adjuvant,the purified BPIV3 HN protein and p ET32a(+)protein were used to immunize New Zealand rabbits to obtain the antiserum of high titer and specificity.(4)Using IBRV DNA as template,g E gene fragments were obtained.g Eup-HN-g Edown was obtained by overlapping PCR,and then was inserted into pc DNA3.1(+)to construct the transfer vector pc DNA3.1-g Eup-HN-g Edown containing HN expression box.The transfer plasmid DNA and IBRV g E-/EGFP+ mutant genomic DNA co-transfected MDBK cells.The mutant strain IBRV g E-/HN+ was obtained by reverse virus plaque screening method.PCR identification results showed that the HN gene was stably existed in the recombinant virus.The results of westernblotting with the pretared polyclonal antibodies showed that HN gene has expressed.The results of virus titer showed that the proliferation ablility of the recombinant virus was similar to that of the parental virus.Conclusion:(1)The group monitoring technology of dairy farm environment aerosol has been established and optimized to provide technical support for the infectious disease risk assessment and early warning.Infected cattle herds and latent infection of cattle could be found,through continuous monitoring of aerosol samples of large-scale dairy farms.It provided key technologies for early warning and group monitoring of infectious bovine rhinotracheitis.(2)The IBRV live vectors carrying green fluorescent protein(GFP)was constructed,which provided material support for IBRV live vector vaccine.(3)The prokaryotic expression vector of BPIV3 HN gene was constructed,and the purified recombinant protein was obtained using p ET32 a prokaryotic expression system.The specific antibody serum was induced after immunization of New Zealand rabbits.(4)IBRV recombinant virus carrying the HN gene was successfully constructed,and Western blotting confirmed that the HN gene was expressed in recombinant virus-infected cells.The results of TCID50 assay indicated that the proliferation ability of the recombinant virus and the parent virus was similar.In conclusion,this study established IBR aerosol fluorescence quantitative PCR method with good sensitivity and specificity,providing technical support to assess the existence of IBR persistent infection or latent virus in the farms,and also providing forecasting and early warning information for cattle.The IBRV recombinant virus carrying BPIV3 HN gene was constructed,which provides support for the prevention and control of bovine respiratory disease syndrome.