Preparation Of Monoclonal Antibodies And Construction Of Infectious Clones Of Porcine Getah Virus

Getah virus(GETV)belongs to the Togaviridae family of Alphavirus.This insect-borne virus spreads among vertebrates through mosquitos,and can cause self-limiting diseases in certain animals.GETV mainly affects pigs and horses,resulting in reproduce,abortion and stillbirth of sows,causing fever,rash,swelling of hind legs and swollen lymph nodes in horses,etc.In recent years,strains have been isolated from mosquitoes and pigs in many regions of China,and the positive rate of GETV antibodies in pig farms has continued to increase in certain provinces.GETV has led to certain economic losses,however,few studies on GETV have been carried out in China,pathogenic mechanism of which remaining unclear.Therefore,this study conducted a preliminary study on the growth characteristics and protein function of the virus.? The growth characteristics of GETV on a variety of conventional passage cells were tested.? For requirement of identification of GETV and the study of E2 and 6K proteins,monoclonal antibodies were prepared,and the epitope of the E2 mAb was identified.?A full-length cDNA infectious clone of GETV HuNl strain was constructed using reverse genetics technology;The exogenous gene EGFP was further inserted in different sites of the viral genome to construct two recombinant reporter viruses,and the function of GETV vector was studied;At the same time,the 6K-gene-deleted strain was constructed,and the influence on the virus was explored.The work mentiond above provides an effective tool for the diagnosis and research of GETV.The exhaustive contents are described below:1 Identification of growth characteristics of GETV HuNl strainIn this study,GETV HuN1 strain was inoculated into BHK-21,Vero,ST,ST-R,PK15,LLC-PK1 and HEK293T cells for continuous passage,respectively.The F1 generation and the highest generation viruses on each cell line were collected,titer of which measured.The results showed that GETV can be passaged on BHK-21,Vero,ST and ST-R cells,with a titer of 107-108 TCID50/mL;However,it can be passaged no further after F4 on PK15 and LLC-PK1 cells,and no CPE appeared after.In addition,the virus was inoculated into ST cells and a growth curve was drafted.The results showed that the titer of GETV HuNl strain reached its peak at 24-36 hpi.,at a level of 2×108 TCID50/mL.2 Preparation of GETV E2 and 6K protein monoclonal antibodyTo prepare monoclonal antibodies against GETV E2 and 6K protein,the hydrophilic area of E2 gene and the full-length 6K gene were cloned into the prokaryotic expression vector pCold-TF for expression.The total protein in bacteria was collected for purification by Ni2+ affinity chromatography and gel-slicing before immunized to BALB/c mice.After 3 times of immunization,the spleen cells were collected and fused with SP2/0 myeloma cells.The positive hybridoma cells were screened by IFA method,and a strain of GETV E2 protein monoclonal antibody and a strain of 6K were obtained,named as 9E8(E2)and 25C5(6K)respectively,and the monoclonal antibody ascites were perpares.The results showed that both 9E8 and 25C5 can be applied in IFA and WB tests with fine immune responses,and 25C5 can also be used in IP tests.By WB analysis of truncated E2 protein for epitope identification,recognition epitope of 9E8 is found to be 66KIRYIAGHD74.Preparation and identification of monoclonal antibodies 9E8 and 25C5 lays the foundation for the isolation and identification,pathogenic mechanism and protein function of GETV.3 Construction of GETV infectious clones and exploration of 6K protein function(1)In this study,an infectious clone of GETV HuN1 strain was constructed using reverse genetics technology.A fragment of oligo DNA containing restriction sites was inserted into the pACYC-177 vector,then the CMV promoter and the viral genome fragments were inserted into the vector.The full-length cDNA infectious clone obtained was named as pAC-GETV,which was latterly transfected into BHK-21 cells.The supernatant was harvested and taken to inoculate ST cells for rescue of the virus.The result of IFA using E2 and 6K mAb showed that the virus was successfully rescued,which was named as rGETV-HuN1(also as rGETV).(2)On the basis of the full-length infectious clone pAC-GETV,a fragment of 26S-EGFP synthesized by Overlap PCR was inserted into either between the two ORFs of viral genome or betwteen the E1 gene and 3'UTR to construct two recombinant infectious clones containing EGFP(pAC-GETV-EGFP/C and pAC-GETV-EGFP/E1).The reporter viruses rGETV-EGFP/C and rGETV-EGFP/E1 were rescued.In cells infected of both viruses,EGFP expressed at high levels was observed.Results of RT-PCR showed that the exogenous gene has relatively better stability with the passage of the reporter virus,when inserted betwteen the El gene and 3'UTR.(3)A 6K-deleted strain of infectious clone(pAC-GETV-?6K)was constructed and the deletion strain rGETV-?6K was rescued.WB analyis of the viral structural protein showed that the deletion of the 6K protein did not affect the cleavage of GETV structural protein.However,results of titer measuring showed that deletion of 6K slowed down the replication of the virus.