Construction Of The High Efficient Maize Expression Vector With Codons Optimized VPI Gene Of The Foot-and-Mouth Disease Virus Type O

Foot-and-mouth disease virus (FMDV) causes a highly contagious infectious disease foot-and-mouth disease (FMD) in cloven-hoofed animals. This disease is widely epidemic all over the world and often results in considerable economic losses and dangers in the health of people. There are seven serotypes diversification and high potential for variation of the virus, so the protection against FMD with the inactivated vaccine is limited. The inactivated vaccines are unsafe due to nonstandard operation, and cause some serious risk. It is necessary to develop a safe and effective transgenic plant vaccine against FMD. Up to now, many scientists are engaging in the work which becomes highlight to prevent and control FMD.In this study, we modified the codons optimized VP1 genes of FMDV stereotype O according to the codons of the maize with the bias and synthesized the optimized genes. Then constructed the plant expression vector containing the codons optimized VP1 genes of FMDV stereotype O .Details on the experiments were as follow:1. Obtain the objective genes containing the codons optimized VP1 genes: based on the published sequence of the VP1 genes of FMDV stereotype O/China/99 in the Gene bank and the regulation of the codons usage of the maize with the bias, we optimized the codons of VP1 genes and synthesized the optimized genes: the sequences of Kozak,Linker and CTB genes was cloned into the up end of the coding genes of VP1 ; and added the endocytoplasmic reticulum leader peptide gene to the 3'end of the modified sequence; finally , inserted the restriction enzyme digestion sites X maI and ClaI to the 5'and 3'ends of sequence respectively. After the whole modified sequence was synthesized by the Shanghai Sangon Biological Engineering Technology and Service Co, Ltd. And the sequence was cloned into the puc plasmid. We sent the recombinant plasmid puc-HW055 to be sequenced by TaKaRa Biotechnology (Dalian) Co, Ltd. the sequencing result showed that the HW055(CTB-linker-VP1/O)sequence was obtained successfully.2. Construct the binary expression vector: the recombinant plasmid puc-HW055 and the expression vector pC1300SOD3.1 were digested by the same restriction enzymes of XmaI and ClaI respectively. Then the purified and collected the HW055 fragment was cloned into the digested pC1300SOD3.1 plasmid. We obtained the recombinant plasmid of pC1300-HW055; selected the positive recombinant plasmid by Kan liquid culture; checked it by PCR of the genes containing the genes VP1 and demonstrated by digestion analysis of XmaI and ClaI restriction enzymes. The recombinant plasmid Mini-expression vector pC1300-HW055 was introduced in Agro bacterium tumefactions GV3101 by triparnetal mating with coli.DH5a containing the helper plasmid T i. The construction of the binary expression vector was correct, which was checked by PCR of the HW055 gene and the triparnetal mating Agro bacterium tumefactions was selected by culture medium containing many antibiotics.