Cloning And Expression Of Foot And Mouth Disease Virus(FMDV) VP1 Gene And Cytokine Measurement

This study is to express FMDV-VP1 protein using the expression system and analysize the protein structure .In this study Bioinformatic method is also used to research the mutation and epidemiology principles of the FMDV.First, a pair of PCR primers was designed to isolate FMDV-VP1 gene according to the published FMDV-VP1 sequence. After PCR of DNA isolated from the tissues , the FMDV-VP1 gene was cloned into cloning vector . Positive clones were analysed with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the FMDV-VP1 has mutated compared with the published sequences. And at the same time the 3 dimension protein structure was built for research .Second,a prokaryotic expression construct, obtained from Invitrogen transformed into prokaryotic and induced to express VP1 protein. The expressed VP1 fusion protein was purified by affinity chromatography using glutathione-agarose resin and used in ELISA and WESTERN BOLT analysis as the antigen.The ELISA and WESTERN BLOT results showed that the anti-FMDV antibody was elicited specifically against VP1 antigen. Third, the cloned FMDV-VP1 gene was subcloned into an eukaryotic expression vector, pSuperY-VP1. To evaluate the immune responses of this construct, animals sera were used to analyzed their specific antibodies against VP1 using WESTERN BLOT analysis.Cytokine productions play a central role in modulation of immune responses upon infection or immunization. To determine cytokine profile would be the critical to understand host immune system. To quantitatively measure the each cytokine levels from the spleens obtained before or after immunized mice, the total RNA were extracted and generated into cDNA by RT reaction. The native cDNA was subjected into a competitive PCR reaction with a competitor that is a slightly larger in size. The level of native cDNA of cytokine is determined by comparison with the known amount of competitor in agarose electrophoresis. The results from the expressions of cytokine from normal and immunized mice may be used to analyze the immune response profile or guide to design a better vaccine.