Molecular Diagnosis And Immune Studies Of Transmissible Gastroenteritis Virus Of Pigs

Transmissible gastroenteritis of pigs (TGE) , characterized by vomit and severe diarrhea, is a contagious disease induced by transmissible gastroenteritis virus of pigs (TGEV). It was a main task to study the molecular diagnostic method and immune activity and prepare the safety, utility and well immunogenically gene engineering vaccine. In the study, first, a TGEV strain named TS was isolated and identified by serology, electron microscope and reverse transcriptase polymerase chain reaction (RT-PCR), then, a double RT-PCR differential diagnosis method distinguishing TGEV from PEDV (porcine epidemic diarrhea virus) was established by amplifying the genes coding for the TGEV nucleocapsid protein and the PEDV membrane protein from feces. Furthermore, the gene coding for spike protein of TGEV TS strain was cloned and subcloned into shuttle vector pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/S, Linearized pAdTrack-CMV/S was electrotransformed into E. coli strain BJ5183 which is RecA~+ and perform homologous recombination with supercoiled adenoviral vector pAdEasy-1 which contained in BJ5183 cells, the resultant recombinant which exposed inverted terminal repeat (ITR) sequence by restriction enzyme digestion was transfected into AD-293 with lipoplast to produce the recombinant adenovirus (rAd-S). RT-PCR was used to detect the transcription of TGEV spike protein, enhanced green fluorescence (eGFP) was used to test the expression of foreign protein, and serial passage and PCR were used to detect the stability and safety of rAd-S. For evaluate the immune activity, rAd-S was inoculated to 28-day-old piglets by oral and HouHai acupuncture modality (it is lied in the umbilication which is between anus and root of tail) respectively, blocking enzyme linked immunosorbent assay (ELISA) was used to detect the titre of TGEV specific antibody;60 days after administration,, each piglet was challenged with TGEV virulent virus GS to test its shielding ability. To test the neutralization activity, 3-day-old piglets were challenged with virus incubated in the presence of control serum or incubated with rAd-S immune serum. Meanwhile, pregnant sows antepartum at 35 days were immunized with rAd-S and the 3-day-old offsprings obtained its colostrum were challenged with TGEV virulent virus GS to evaluate the protective lactogenic immunity. The results showed as follows:1) A strain of TGEV named TS was isolated, and identified by serology, electron microscope and RT-PCR.2) Double RT-PCR differential diagnosis method distinguishing TGEV from PEDV was established by established by amplifying the genes coding for the TGEV TS strain nucleocapsid protein and the PEDV QH strain membrane protein, and can directly detect the genes coding for the TGEV TS strain nucleocapsid protein and the PEDV QH strain membrane protein from feces, it take a feasible method for the molecular differential diagnosis of TGEV and PEDV.3) Sequencing analysis indicated the coding region of spike protein gene of TGEV TS strain is 4350bp long and 6bp longer than that of Purdue strain, its corresponding predicted protein has 1449 amino acids, the percentage of identity at the nucleotide level of the spike protein gene between TS and Purdue strain was found to be 98.4%. The nucleocapsid protein gene of TGEV TS strain is 1149bp long,its corresponding predicted protein has 382 amino acids, the percentage of identity at the nucleotide level is 98.2% with Purdue strain. The membrane protein gene of PEDV QH strain is 681bp long, its corresponding predicted protein has 226 amino acids, the percentage of identity at the nucleotide level with CV777 strain is 98.5%.4) Transfer vector pAdTrack-CMV/S, recombinant adenovirus vector pAd/S, and rAd-S which was stabilization, safety, and replication defective but packaging permitted recombinant adenovirus containing whole coding region of TGEV spike protein gene were successfully constructed, the virus titre of rAd-S was up to 1 X109 TCID50 per 0.2mL, RT-PCR test indicated that the TGEV spike protein gene was transcripted, eGFP detection show the foreign genes were expressed, it made an experiment foundation for the study of TGEV gene engineering vaccine.5) Recombinant TGEV spike protein has good immunogenicity;rAd-S recombinant that can express TGEV spike protein induced specific immune responses which neutralized TGEV infectivity in swine. In addition, porcine serum from Ad-TGEV-immunized animals provided passive protection against TGEV challenge when mixed with fully virulent TGEV GS and orally administered to highly susceptible 3-day-old newborn piglets. Protective lactogenic immunity also provided some effective protection to highly susceptible 3-day-old newborn piglets that were sucked the colostrum from rAd-S immunized pregnant sows.