Study On Expression Of The Carboxy (C)-terminal VP1 Gene Of FMDV And Porcine Interferon-alpha/beta Gene In Escherichia Coli

Foot-and-mouth disease virus (FMDV) is the causative agent of highly infectious and the economically most important animal viral disease in the world. The key of control and prevetion FMD is to develop sensitive diagnostic tests and effective vaccine. Now there are several diagosis methods, and ELISA is one of the most economical and effective methods. But conventional ELISA was based on inactive virus, and it had the risk of disseminating virus into the surroundings. An alternative to this is to identify type specific gene sequences, express them in safe heterologous systems and use them as diagnostic antigens. Moreover, researchs have indicated that that FMDV is highly sensitive to IFN-a/p. The stuy has been done as following two parts: 1. Expression of the 2VP1 Gene of FMDV and Development of ELISA Based on ItThe C-terminal half of VPI gene of FMDV was amplified by PCR, including B cell epitope and T cell epitope. The PCR products were cloned into pMD18-T and sequenced. The sequence analysis indicated that the C-terminal half of VPI gene was about 285 bp and encoded 75 amino acids. The homology analysis of the gene with O/HKN/12/91 and O/PEN/TAW/99 in GenBank indicated that it was relatively conservative. Then the PCR products were inserted into PGEX-KG and transformed BL21 (DE3) bacteria. The expression of GST-2VP1 and GST-VPI was induced with IPTG and confirmed by SDS-PAGE, the recombinant protein had a molecular weight of 45 kDa and 37kDa respectively. Their activity was confirmed by Western blotting. Based on expressed GST-2VP1 protein as antigen the ELISA to detect antibody against VPI was developed and was primarily used to detect serum samples. This reseach will be very useful in the development of digonosis tests and vaccinate.2. Study on Clone, Seqence Analysis, Directed-site Mutation and Expression of Meishan Porcine Interferon-alpha/betaThe completed interferon alpha/beta gene (MS-IFNa/p) was amplied from leucocyte genome DNA of Meishan porcine by PCR.The PCR products were cloned into pMD18-T and sequenced.The squence result indicated that the MS-IFNp gene was about 561 bp and encoded 186 amino acids.The homology analysis of the gene with S41178 in GenBank showed that it was highly conservative, only 128 and 177 sites nucleotide different; MS-IFNa gene was about 570 bp and encoded 189 amino acids, the homology analysis of the gene with M28623 in GenBank showed that it was highly conservative. Then IFNa/p gene was treated via site-directed mutation which lied in 326 sites and 113 sites respectively. IFNa/p gene encoding mature protein was subcloned into pGEX-KG and transformed BL21 (DE3) bacteria. The expression of IFNp was induced with IPTG andconfirmed by SDS-PAGE, the recombinant protein had a molecular weight of 47KDa and could restrain FMDV replication .It is very useful for further research of interferon.