The Clone And Sequence Analysis Of Porcine Transmissible Gastroenteritis Virus M Gene

Porcine transmissible gastroenteritis virus (TGEV) belongs to the family coronaviridae.The virus causes transmissible gastroenteritis (TGE) which does very much harm to the pigfarms. The normal medicines such as antibiotic and antitoxins have no effect to the disease.Vaccination is the effective means for the prevention of TGE.The study isolated RNA fromenteritis tissue. Two pairs of primes were designed to amplify cDNA sequence of M gene by aTranscription-nested Polymerase chain Reaction (RT-PCR)and a Reverse Transcription-nestedPolymerase chain Reaction (RT-nested PCR) . The products of PCR were sequenced andhomology compared .Then they were analysed deeply . The following was the mainexperiments and results: 1. The study isolated RNA from enteritis tissue by Trizol method. After formaldehydegel denaturation electrophoresis, we can see clearly two light bands(28s and 18srRNA) and avague band(5srRNA).The result indicated that the integrality of the isolation RNA was verygood and hardly was degradated.It may be used in RT-PCR. 2. According to the published cDNA sequence of TGEV M gene, with Prime5.0 twopairs of primes were designed to amplify the products which were 1037 bp and 1328 bprespectively by RT-nest PCR based on common RT-PCR. The result demonstrated that themethod has higher specialist and repeation. It can detect correctly virus and avoid falsepositivism. Compared with tranditional methods, such as electron microscopy andimmunization fluorescence technology and ELISE, it is more convenience andcredibility .The results showed that RT-nest PCR may be one method which is neoteric andreliable to detect the RNA virus especially. 3. The assay amplified the full seqence of M gene which is 1 328 bp nucleotide acids byRT-PCR . The amplified product was extracted , transformed and cloned into pGEM-TEasy vector. The recombinant plasmid was tested by restricted digestion of EcoRâ… and thesequencing. The sequence analysis indicated that the sequence homology of nucleotide acidswas 97.2%, 97.3% and the sequence homology of amino acid was 97.3%,98.0% which wereanalyzed by CLUSTUL(1.82) when it was compared with corresponding sequencing of thedifferent strains in GeneBank. There were twenty-four nucleotide acids mutation and sevenamino acids mutation. And the further analysis indicated that these mutations of nucleotideacids were no meaning and did not affect the fuction of M protein. The study provided not only the important basis for further research of TGEV molecularproperties and the preparation of vaccine, but also an useful viral strain discrimination method.In brief, it provided vital practical value for molecular study of TGEV , development ofvaccine and prevention of TGE as well.