Construction And Immunogenicity Of Recombinant Adenovirus Expressing 5' Terminal Coding Gene Of Spike Protein Of Transmissible Gastroenteritis Virus

Transmissible gastroenteritis virus (TGEV) is a coronavirus that infects the enteric and respiratory tissues of newborn piglets and can cause severe and often fatal diarrhoea in young pigs resulting in mortality of nearly 100%. The study of the induction of protective immunity to TGEV has focused on S protein because it is the major inducer of TGEV-neutralizing antibodies and it mediates binding of TGEV to its cellular receptor. The four major antigenic sites (A, B, C and D) of S protein have been described on the amino-terminal domain. And earlier studies indicated that the intact globular N-terminal half of the protein is sufficient to achieve a protective immune response equivalent to that induced by the full S protein.In this study, a recombinant replication-defective human adenovirus serotype 5(Ad-5) carrying a 1.0kb fragment (S1) containing the B and C antigenic sites of the transmissible gastroenteritis virus (TGEV) spike (S) gene was generated by homologous recombination in E. coli strain BJ5183 cells and subsequent transfection of HEK293-A cells. The DNA fragment was cloned by RT-PCR and then was inserted into the multiple cloning site of the Ad transfer vector pShuttle-CMV. The positive recombinant pShuttle-CMV vector was linearized by restriction enzyme Pmel. To obtain homologous recombination, the mixture of linearied pShuttle-CMV-S1 and adenovirus backbone vector pAdEasy-1 were co-transformed into E. coli strain BJ5183 cells by electroporation. The recombinant adenovirus pAd-Sl was confirmed by Pad digestion, and then transfected HEK293 cell with Transfast Transfection Reagent Kit. The cell CPE could be observed within one week after transfection. The expression of S of TGEV was identified by RT-PCR, indirect immunofluorescent assay (IFA) and the recombinant virus was analysised by Western blot analysis to decide the size of the recombinant protein. The virus titer was10^7.7 TCID₅₀/ml.