Clone, Expression And Identification Of Transmissible Gastroenteritis Virus Spike Protein Gene

Porcine transmissible gastroenteritis(TGE)is an acute, highly contact intestinal infection,caused by porcine transmissible gastroenteritis virus (TGEV). Main clinical features are vomiting, diarrhea and dehydration. Different ages and different varieties pigs are susceptible with highly infectious, especiallly less than two week old piglets, and its mortality rate could reach 100%. Currently the disease spread in each country which raise pigs. China, being as the biggest pig breeding country, constantly had reports about happening and epidemic of the disease. Owing to the mixed infection and bacteria secondary infection, it is usually hard to diagnose,and lack of the effective vaccine .It brings a seriously harmful effect to the swine industryThe study firstly analyzed a suspected case of porcine transmissible gastroenteritis in changchun suburb. A TGE virus strain (TGEV JL) was successfully isolated from mesenteric lymphnodes of died piglets with clinical diarrhea symptom from a pig farm by passaging in PK-15 cells. The virus was identified as TGEV based on morphology, PCR results and animal regression test. The isolation of TGEV JL strain provide basic conditions for gene cloning of contagion enterogastrtisetc virus in vitro and plants vaccine development.It's reported that,TGEV had four types of structural proteins: the Spike glycoprotein (S), the inner membrane protein (M), small membrane protein (sM) and nucleocapsid protein (N). The length of S gene, which encoded S protein, was about 4300bp. It decided the host's tropism, hemagglutination and pathogenicity, while it is the most important structure protein which induced the organism to produce the neutralizing antibody. On the basis of getting TGEV JL strain, the whole sequences of S gene (main immunogenicity gene) TGEV was obtained by RT-PCR amplification technology, which connected to the pMD18 - T vector.And sequenced and analyzed sequential in this research. Through compared the gene sequence and the amino acid sequence to seven different sources TGEV strains,and done some homology analysis, it showed that S gene of the isolate strain TGEV was highly conservative. It could be used as the purpose gene for the transfer of plant vaccines. Inserted this gene into plant expression vector pBI121, downstream of CaMV35S promoter , building high efficient plant expression vector pBI121-S.Corn is the main food for human beings and animals. Feed consumption is the most important channel of corn consumption, accounts for about 70 percentages. Corn as feed consumption has two kinds of cases in China . One as compound feed and the other one is use to feed consumption. This research is in basis of acquired pBI121– S of Agrobacterium tumefaciens Corn and use the embryogenic calli of "Dan 598" and "H99" to make the experiment for kanamycin sensitive test. This study makes sure that 40-56mg/L is the best selection pressure. The pBI121-S ,TGEV S gene's plant expression vector, was transferred into corn inbred line by using gene gun method. Conversion condition of the gene gun method had been studied. After being induced differentiation emergence, inversive callus was detected by PCR and RT-PCR.The result of PCR showed that exogenous gene had been transfered into the maize genome,while RT- PCR showed that the target gene could transcribe in the transgenic maize plants .At the same time, through the means of mediated transferred the TGEVS gene of Agrobacterium tumefaciens into the maize inbred line, and studied its mediated condition. The research showed that when the value of the EHA101 Agrobacterium bacteria at 600nm OD was from 0.5 to 0.6 and 20min infection was the optimum condition for Agrobacterium tumefaciens transformation. The inversive callus, induced differentiation emergence, detected by PCR amplification, Sorthern and Nouthern blot.It turned out that the target gene had already been diverted to the maize genome.The research results provides a basis for further studying, not only the transgenic plant vaccine of transmissible gastroenteritis virus of swine,but also using it in veterinary clinic.