Construction,Identification,Immunological Evaluation Of Recombinant Adenovirus Expressing E1 Of CHIKV And Its Residue Detection In Immunizedanimals

In 1952,Chikungunya Fever(CHIKF)was described in Africa for the first time.By 2017,CHIKF had been reported in Africa,Asia,Europe and America.CHIKFposes a serious threatto thepublic safety.There is no vaccine and effective therapeutic drug licensed both at home and abroad currently.Thus,the development of a safe and effective CHIKF vaccine needs to be further researched..In this study,thehuman adenovirusvector type 5 was chose as the vector and CHIKV E1 as the target gene toconstruct arecombinant adenovirus expressing CHIKV E1 glycoprotein.The recombinant adenovirus was used to evaluate the immunizationof miceand test the residue of the recombinant adenovirus.CHIKV E1 gene and EGFP gene were cloned to pacAd5 shuttle vector to construct recombinant shuttle plasmid Ad5-EGFP-CHIKV-E1.rAd5-EGFP-CHIKV-E1 recombinant adenovirus were packaged by cotransfection of HEK293 cell withthe linearized recombinant shuttle plasmid and pacAd5 plasmid.The CHIKV E1 gene was successfully inserted into the recombinant adenovirus via PCR.The results of Western blot and indirect immunofluorescence assay(IFA)showed that CHIKV E1 protein was successfully expressed in recombinant adenovirus.The Hexon structure of recombinant adenovirus was observed under the electron microscope.Extracted the gene of recombinant adenovirus at different generations and analyzed the recombinant adenovirus protein at different time to prove its stability of the recombinant adenovirus.Next,the mice were immunized with recombinant adenovirus of CHIKV E1 toevaluate the effectiveness of the recombinant adenovirus.Results showed that the levels of IL-4,IFN-? and TNF-? were significantly increased 35 days post immunization(dpi);spleen T lymphocyte proliferation and cell subsets in group CHIKV E1 recombinant adenovirus were higher than those in group PBS;the specific antibody level in the immune group reached the maximum value at 35 dpi,which was 2.67 timeshigher than the PBS groupsignificantly(P<0.05);the neutralizing antibody in the immune group was 3.66 times higher than that in PBS group(P<0.05).Above all,CHIKV E1 recombinant adenovirus could stimulate specific humoral and cellular immune response in mice.The recombinant adenovirus rAd5-EGFP-CHIKV-E1 was used to immunize mice,and PCR and Real-time Quantitative PCR were used to detect the residues of recombinant adenovirus.The PCR results showed that the recombinant adenovirus of CHIKV E1 was less than 14 d in mice;the results of real-time quantitative PCRanalysis showed that the copiesof rAd5-EGFP-CHIKV-E1 up to 109 in organs or tissues.Above all,recombinant adenovirus of CHIKV E1 rAd5-EGFP-CHIKV-E1 showed transient infection in mice.This experimentprovided the foundation for further study on CHIKV vaccine.