Generation And Identification Of Monoclonal Antibodies Against Transmissible Gastroenteritis Virus Of Swine

Transmissible Gastroenieritis (TGE) of swine is a kind of contagious disease of pigs, causing watery diarrhea, vomiting, weight loss, anorexia, which pathogen is swine Transmissible Gastroenteritis Virus (TGEV).In clinical diagnosis,it is difficult to distinguish TGEV with other diarrhea virus, so it has brought great difficulties in the diagnosis and prevention. Because of specificity for antibody-antigen identification, it is advantageous for using4E2to diagnosis this pathogen, and localizing the epitope, which can help to reveal the stricture and function of TGEV.In this study, we used differential centrifugation method to purified TGEV,then used purified virus immunized BABL/c mice in multi-point injection for three times.After that,we performed cell fusion between SP20cell and splenetic cells by using PEG.We used PK-15cells infected with TGEV to detect antibody by performed indirect immunofluorescence, screened positive hybridoma cell lines,and using positive cell lines were cloned by limiting dilution,after three rounds of screening positive,we expand in Cell culture and cryopreservation,then BABL/c mouse produces ascites.Finally,have got an TGEV antibody secreting hybridoma cell line,named4E2.Western blot analysis showed monoclonal antibody4E2could specifically recognize of approximately47kD N protein.To further determine the4E2monoclonal antibody identified viral proteins, we cloned the whole N gene of TGEV,and split N gene into two fragments named N1and N2,cloned into the pGEX6p-1vector, and pET-30a vector, then expressed vector pGEX6p-N and pET-30a-N1in E. Coli.We used TGEV positive mouse serum expression to performed Western blot analysis of the product, confirmed the expression of the two fusion proteins with well reactivity. Furthermore ELISA and Western blot analysis showed that MAbT-4E2can specifically recognize N protein and N1protein, we have obtained4E2monoclonal antibody against TGEV N protein exactly, they recognize epitopes located in the N protein N-terminal192amino acids.The results provided an effective method for the future clinical diagnosis for TGEV in the future. To clarify the reason of diarrhea outbreaks in piglets in Shanghai early2013,9stool samples were randomly collected from the diarrhea-strucken pig farms in this study.By using RT-PCR,4of9samples were PoRV positive (44.4%),while TGEV or PEDV were negative.Based on sequences of VP6and VP7of reference strain in GenBank.two pairs of primers were designed to amplify the target genes.The full-length of VP6gene and VP7gene were1300bp and1100bp.These results showed a reliable basis for the prevention and control to the piglet diarrhea epidemic.