Construction And Immune Evaluation Of Recombinant Adenovirus With H Gene Replication Defect Of Canine Distemper Virus From Giant Panda

Canine distemper(CD)is an acute,high fever and highly infectious infectious disease caused by canine distemper virus(CDV),and the mortality rate can exceed 80%.In recent years,the range of CDV infection of hosts has been expanding.In addition to common mammals such as dogs and cats,wild boars,seals and anteaters have also been reported.From the end of 2014to 2015,the outbreak of canine distemper in captive giant pandas occurred in Shaanxi Wild Animal Breeding and Protection Center,resulting in the death of 5 giant pandas,indicating that we should always be vigilant against the risk of canine distemper in giant pandas.At present,the inoculation against canine distemper for captive wild animals in China mostly relies on imported vaccines,and there is no stable and effective canine distemper vaccine for giant pandas.In order to solve this problem,it is urgent to develop a novel,safe and effective canine distemper vaccine for giant panda.CDV is a member of paramyxoviridae and measles virus genus.The capsule membrane of CDV is composed of a matrix membrane protein M and two surface glycoproteins H(hemagglutinin)and F(fusion)proteins.H protein is a type II glycoprotein encoded by 1824nucleotides and composed of 607 amino acids,with a molecular weight of 76-85k D,located on the surface of the virions and distributed with many epitopes of antigens.It is the main structural protein that induces the body to produce neutralizing antibodies and plays the role of the main inducer of humoral immune response.In addition,H protein also contains cytotoxic T lymphocyte(CTL)epitopes,which can produce specific CTL activity,so it is often used as an important candidate gene for the research of genetic engineering vaccine for canine distemper.Adenovirus vector is the most commonly used viral vector for vaccine development and gene therapy.This viral vector has good safety,wide host range,high titer of recombinant virus,high expression of foreign protein,and high immunogenicity.In this study,a replication-deficient human adenovirus type 5,which was unable to encode early adenovirus proteins due to the absence of regions E1 and E3,was selected as the presentation and expression system for the H protein of giant panda canine distemper virus.Combined with Gateway technology,a recombinant adenovirus capable of expressing the H protein of giant panda/SX/2014 canine distemper virus was constructed.Reverse transcription polymerase chain reaction(RT-PCR)was used to amplify the H gene of giant panda/SX/2014,which was cloned into the entry vector p ENTR/D-TOPO by TOPO linkage.Then,the vector p ENTR/D-TOPO-CDV-H was recombined with the adenovirus skeleton vector p Ad/CMV/V5-DEST by LR to obtain the recombinant plasmid p Ad-CDV-H.The recombinant plasmid was linearly digested by Pac I enzyme and transfected into 293A cells to obtain the recombinant adenovirus rAd-CDV-H.After successive passage,the obtained recombinant adenovirus was identified by RT-PCR,viral titration,immunofluorescence and Western blot,respectively.The results of RT-PCR showed that the target bands of the first,fifth,seventh and eleventh generation of recombinant adenovirus could be amplified,indicating that the recombinant adenovirus could be stable transcription.The results of immunofluorescence and Western blot analysis showed that the recombinant adenovirus rAd-CDV-H could successfully express H protein after infecting 293A cells and Vero cells,and the titer of the obtained recombinant adenovirus was as high as10¹⁰IFU/m L.In order to evaluate efficacy and safety of recombinant adenovirus,the recombinant adenovirus rAd-CDV-H was intramuscular inoculated into eight-week-old BALB/C female mice at a high dose of 10⁹IFU and a low dose of 10⁸IFU,respectively.DMEM control group was also set up.Sera were collected for neutralizing antibody and ELISA antibody levels,as well as for antibody subtype detection.The fur color and body weight of mice were observed for 15 consecutive days.The results of antibody detection showed that rAd-CDV-H10⁹IFU and rAd-CDV-H 10⁸IFU could induce neutralizing antibodies in the body,and the former induced higher neutralizing antibodies.ELISA antibody level and subtype detection results showed that the specific antibody levels of rAd-CDV-H 10⁹IFU and rAd-CDV-H 10⁸IFU in the high-dose and low-dose groups were 2.63-fold and 2.17-fold higher than those in the control group,respectively,with statistically significant differences(P<0.0001).It can be obtained from the ratio of Ig G1/Ig G2a,the specific Ig G2a antibody response induced by rAd-CDV-H tends to be Th1 type immune response.The body weight of mice inoculated with the recombinant adenovirus was basically the same as that of the control group,their hair was smooth,and the status was not abnormal,indicating that the recombinant virus was safe.In summary,the recombinant adenovirus rAd-CDV-H carrying the H gene of canine distemper virus from giant panda was successfully constructed using Gateway technology in this study.The recombinant adenovirus rAd-CDV-H could not only express CDV H protein effectively in vitro,but also in non-replicating cell lines(Vero cells).The results of animal experiments showed that the recombinant virus rAd-CDV-H could produce specific humoral immunity in one single immunization.It has good safety,and has the potential to be a candidate CDV vaccine strain for giant panda.