Construction Of Mammalian Gland Special Eukaryotic Expressing Vector For Swine's Transmissible Gastroenteritis Virus S Gene

Transmissible gastroenteritis (TGE) which is a kind of acute , highly infectious diseasewas caused by transmissible gastroenteritis virus of swine(TGEV)belongs to the familycoronaviridae, it's typically clinical manifestations consist of omitting, severe diarrhea andloss water. All kinds and ages of pigs can be infected. Particular, the mortality new bornpiglets may reach 100%. Currently, vaccination is the effective means for the prevention ofTGE. Traditional vaccine includes attenuated and inactive vaccine, however, they have manydeficiencies, such as recovery of virulence, dissemination of virulence, need for adjuvant, etc.Therefore, it is urgent for preventing fundamentally TGE to exploit a safe and effectivegenetic vaccine. In this study , taking the safe , effective and low cost prospect of mammarygland bioreactor producing medical protein as the starting point, the eukaryotic expressingplasmid or gene vaccine of TGEV was constructed by TGEV antigen spike protein gene andthe general gland expressing vector containing beta－casein protein promoter. 1. According to spike protein S gene sequence of transmissible gastroenteritis virus ofswine (TGEV) in Gene Bank, 5 primers were designed to amplify cDNA sequence of S geneby RT-PCR from enteritis tissue. The products of PCR were named 2 127 bp S1 and 2 450 bpS2, respectively. They were integrated into pMD18-T vector, recombinant plasmid pMD-S1and pMD-S2 were tested by restrict endonuclease (RE)and sequencing. The sequencingresult indicated that the sequence homology comparing with corresponding sequencing ofdifferent strains are 94﹪ or so. The sequencing of 4 496 bp S gene was amplified byrecombinant PCR, its template was the mixture of S1 and S2, its primers were 5′ flanking ofS2 fragment and 5′ flanking of S1 fragment. S gene was cloned into pGEM-T easy vectorand named pGEM-S. The result affirmed that recombinant plasmid containing S gene wasidentified by RE and PCR. 2. A pair of primer TS1′ and TA2′was designed according to multiple cloning sites ofⅲ AbstractpEGFP-C1 Vector and the restriction endonuclease site of the beta-casein promoter sequence,namely, primer 5′ flanking fragment of TS1 was added restriction endonuclease NheⅠsite, 5′flanking fragment of TA2 was added BamHⅠendonuclease site. S′ gene was amplified frompGEM-S plasmid and inserted into pGEM-T easy vector, The recombinant plasmid pGEM-S′was identified and analyzed with RE by restriction endonuclease and PCR. Complete S′ geneobtained with RE NheⅠand BamHⅠfrom pGEM-S′. S′ gene was subcloned into theplasmid pG－BBCP which contain beta-casein promoter sequence 5′ and 3′ flankingfragment.The recombinant plasmid was identified and analyzed with RE and PCR. BCP－S′ was endonucleased by SacⅠ/ MluⅠ, harvested and directed subcloned into pEGFP-C1vector. The recombinant expressing plasmid was identified and analyzed with RE and PCR,which shows that TGEV eukaryotic expressing plasmid constructed successfully, whichconsists of reporter gene (Enhancer Green Fluorescent Protein ), antibiotics gene (Neo gene),regulatory element (bovine beta-casein promoter 2.2 kb 5′ and 0.6 kb 3′ flanking fragment)and foreign target gene (TGEV S gene). The recombinant plasmid provided solid basis forfurther study.