| Porcine transmissible gastroenteritis virus (TGEV) belongs to the family coronaviridae. The virus causes transmissible gastroenteritis (TGE) in all kinds and ages of pigs. Particularly, its mortality in pigs under 1 to 2 week old may reach 100%. TGE is a kind of acute, highly infectious disease. The clinical manifestations of typical TGE consist of omitting, serious diarrhea and loss of water. Vaccination is the effective means for the prevention of TGE.Since the discovery of TGE, many veterinary researchers devoted themselves to the preparation of vaccine against it. However, both attenuated and inactive vaccines exist inherit deficiencies. For example, attenuated vaccines could result in dissemination of virus, recovery of virulence and widespread of disease, even though its excellent vaccination effect; inactive vaccine is safer than the former, but it also exists unfavorable immunization effect, needing of many-times vaccination and adding of adjuvant etc. So the exploitation of novel vaccine, especially genetic engineering vaccine is the urgent task currently. With the development of immunology, virology and biology in molecular level, the task become more and more available, therefore, the following experiments are finished:Assay 1: Sa fragment encoding major antigenic sites of S gene was obtained from cloned recombinant plasmid pUC-S of TGEV Chinese isolate TH-98, and then was inserted into different prokaryotic expression vectors according to correct ORF (open reading frame). These recombinant vectors were identified and analyzed with RE (restriction endonuclease) and by nested PCR, which provided solid basis for further study.Assay 2: Complete S gene obtained with RE from pUC-S was subcloned into EcoRI and PstI sites of vector pFASTBACHTb in insect/baculovirus expression system in appropriate transcription sequence. The transposition of positive recombinant named BAC-S was achieved with competent cell DH10BAC. The recombinant named pBAC-S was verified with RE and by PCR with specific and universal primers according to DNASIS analysis results of S gene and transposition region characteristics of the vector. The result showed that theinfectious recombinant was successfully constructed, which could be expressed directly in insect cell.Assay 3: With Two pairs of primers PS1, PR1N and PS2N, PR3 (There are nucleotide changes led to a new Seal recognized site in sense primer PS2N and antisense primer PR1N. Besides, There are artificial nucleic acid modifications in PR1N, aiming at eliminating potential pre-termination codon of S gene. The deduced amino acid sequence is the same as original one due to utilizing nucleic acid degeneracy) and template PUC-S, two PCR products named SNa and SNb. of 1.3kb and 1.0kb were produced respectively. SNa was cloned into T vector and negative direction recombinant was partially digested with Seal and KpnI, and then about 3.9kb fragment named TSNa was obtained. SNb was inserted into corresponding sites of TSNa after digestion of the same RE and recombinant was named TSNab. The favorite fragment containing SNa and SNb was obtained with EcoRI and KpnI, and then subcloned into plasmid pUC-Sb derived from pUC-S. The recombinant named pUC-SN was verified by nest PCR and with RE and was . cloned into EcoRI and PstI sites of pCR3.1 cloning vector. Eventually, the recombinant SNPCR3.1 was digested with EcoRI and NotI and subcloned into the corresponding sites of Pichia yeast expression vector pPIC3.5 in correct ORF. which could be used to express in yeast cell.Assay 4: Through subclonal recombination technique, complete S gene and major antigenic site fragment of S gene named Sa were cloned into corresponding sites of eukaryotic expression vector PCI and pcDNA3 respectively. The nucleic acid vaccine SPCI and SaPCI as well as SPCDNA3 were constructed and animal experiments were finished.KunMing mice were divided into 3 groups randomly, group A, B, C were injected intramuscularly with SaPCI, SPCI and PCI control respectively for three times with 2 weeks interval.
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