Recombinant Goose IL-17Enhancing Mucosal Immune Response Against Goose Parvovirus VP3

The mucosal surfaces of the gastrointestinal and respiratory tracts represent the principalportals of entry for most pathogens. Therefore the animal mucosal immune system plays a veryimportant role in the resistance to the invasion of pathogenic microorganisms. Effectivestimulation of the mucosal immune system needs to two basic conditions. First, the antigen in thevaccine can be effectively delivered to the mucosal lymphoid tissue; the second is a safe andeffective mucosal adjuvant to enhance the immune response. Immune adjuvant does not have theantigenicity, but it can promote vaccine antigen-specific immune response when incorporated intovaccine formulations. Until recently, IL-17-producing effector T helper cells, called Th17cells,have been discovered and characterized. IL17is an important and unique cytokine resistance tospecific pathogens. Because IL-17induces neutrophil trafficking to the site of inflammation, aswell as the promotion of the T cell response.Relative to the study of mammalian mucosal immune, poultry mucosal immunity is relativelyslow, and most studies have focused on the study of the chicken. In this study, IL-17, CD4andCD8α gene of goose were cloned from thymocytes. The recombinant GoIL-17（rGoIL-17）expressed in baculovirus expression system was used as a mucosal immune adjuvant combinedwith goose parvovirus （GPV） VP3virus-like particles, or goose IL-17and VP3fusion expressionto immunize geese. The results showed that the goose IL-17as mucosal immune adjuvants caneffectively induce the body’s mucosal immune and system immune. It has a very positive sense ofgosling plague prevention and the development of effective mucosal vaccine.1cloning and sequence analysis of goose IL-17, CD4and CD8α genecDNA was synthesized with total RNA from goose thymus by using SMART （SwitchingMechanism at5’ End of RNA Transcript） Reverse Transcriptase. IL-17, CD4and CD8α specificprimers were designed according to the sequence of chicken IL-17（Genbank ID: NM₂04460.1）,duck CD4（Genbank ID: AF378701）, and duck CD8α （Genbank ID: AF378373）, which were usedto amplify the whole real sequence of goose IL-17with touchdown PCR. Amplified fragmentswere inserted into the pEASY-Blunt vector, and sent to the Genomics Institute for sequencing.Sequences were initially analyzed using Blast search to confirm the correct gene was cloned.The sequences were submitted to NCBI and get a registration number, respectively. The CLUSTALW program was used to align the sequences and a phylogenetic tree was also created using the Neighbour-Joining method within the software MEGA5. The online software SignalP4.1Serverand TMHMM Server v.2.0were used to analyze signal peptide, transmembrane region predictionof IL-17, CD4and CD8α protein, in order to express mature extracellular region of goose IL-17（mGoIL-17） and geese CD4, CD8α.2Prokaryotic expression of mGoIL-17and goose CD4, CD8α extracellular domain andpreparation of polyclonal antibodyAccording to signal peptide analysis of goose IL-17, and extracellular area analysis of gooseCD4and CD8α, the expression primers were designed. The PCR fragment was digested andligated into the pET32a expression vector. The vector was transformed into an E. coli Rosetta（DE3） pLysS strain, and the expression of recombinant protein was induced by IPTG. Therecombinant protein was purified with Ni-NTA His column. After purification, the recombinantprotein was immunized into rabbits to generate antiserum. The immunoreactivity of antiserum wasdetermined by Western blot. The antiserum was the foundation of the follow-up test.3Eukaryotic expression and activity determination of the mGoIL-17and mGoIL-17fusionGPV-VP3proteinAfter digestion of pEASY-Blunt-mGoIL-17, the mGoIL-17fragment was ligated into thepFastBac HTB donor vector of the baculovirus expression system. The fusion fragment ofGPV-VP3, mGoIL-17, and the insertion of a flexible Linker （G4S）3was obtained by fusion PCRmethod. The fusion gene was sub-cloned into pFastBac HTB donor vector. The recombinantvectors were then transformed into DH10BAC bacterial cells. The final recombinant bacmidDNAs were obtained by blue and white, and three antibiotics （kanamycin, tetracycline, gentamicin）screening. Positive recombinant bacmid were used to transfect Sf9insect cells of logarithmicphase. All procedures were performed according to the manufacturer’s manual. The transfectedcells were continued to train until significant cytopathic. The culture supernatants were collected,namely, the primary generation virus （P1）. P1virus was inoculated monolayer Sf9cells with1:10dilution, and cultured at27°C until90%CPE of the cells. The culture supernatants were collectedand the recombinant baculovirus DNA were extracted for identification by PCR. Recombinantbaculovirus underwent four rounds of amplification （72h each） by infecting Sf9monolayers togenerate of high titer recombinant virus. Proteins expression was analyzed on SDS-PAGE andwestern blot. The recombinant protein was purified with Ni-NTA His column.The goose embryo fibroblasts were prepared from10-day-old goose embryo. Cells （1×107）were stimulated with1μg,5μg,10μg,20μg and50μg of rGoIL-17for12h in a DMEM medium.The PBS was used as control. The biological activities of rGoIL-17were assessed by RT-PCR. Theresults showed that, refold mGoIL-17/mGoIL17-VP3induced synthesis of IL-6and IL-8mRNArather than the control, and the expression of geese IL-6and IL-8was positively correlated withmGoIL-17/mGoIL17-VP3dose.4Assessment of the immunity effect of mGoIL-17mucosal adjuvant 1401-month-old Northeast White Geese were purchased from a goose farm in pingshan ofHeilongjiang Province. The geese were divided into seven groups, n=20, respectively. The geesewere immunized with VP3, IFN-α+VP3, LTB+VP3, mGoIL17+VP3, fusion mGoIL17-VP3andPBS through eye and intranasal immunization, and immunized with the commercialization vaccinethrough intramuscular immunization as a positive control. Each set of blood （10parts） werecollected weekly to detect the titer changes of serum immunoglobulin （Ig） and neutralizingantibody. Two geese each group were killed every other week, and the respiratory andgastrointestinal lavage fluid were collected for detection of lavage Ig, and peripheral bloodlymphocytes were separated to detect the change of CD4and CD8cells. The results showed that,（1）, mGoIL-17+VP3group mGoIL17-VP3group and the vaccine group had the similar antibodydynamic, and the overall level of the antibodies was vaccine group> mGoIL17-VP3group>mGoIL-17group> other groups;（2）, The trend of neutralizing antibody titer of each group wassubstantially the same, as the highest antibody titer was in the7th week. mGoIL17-VP3andmGoIL-17+VP3group were much the same, and both higher than the vaccine group;（3）, Total Igantibodies titer of respiratory lavage detected with indirect ELISA had significant differences withother groups, indicating that mGoIL-17+VP3and mGoIL17-VP3could obviously irritate theairways to produce antibodies;（4）, Total Ig antibodies titer of gastrointestinal lavage detected withindirect ELISA had significant differences with other groups, indicating that mGoIL-17+VP3andmGoIL17-VP3could also play a role of stimuli of specific antibodies in immune distal digestivetract;（5）, The differences of CD4+cell content among each group were tested by cell ELISA fromisolated peripheral blood lymphocyte cell. Compared with control group, IFN-α, mGoIL-17+VP3,mGoIL17-VP3, and the vaccine group could cause the increase of the content of peripheral bloodlymphocytes, but LTB group could not;（6）, The differences of CD8+cell content among eachgroup were tested by cell ELISA from isolated peripheral blood lymphocyte cell. Compared withcontrol group, only mGoIL-17+VP3, mGoIL17-VP3, and the vaccine group could cause theincrease of the content of peripheral blood lymphocytes, but IFN-α and LTB group could not.