Transformation And Expression Of P12A-3C Gene Of FMDV In Birdsfoot Trefoil And Its Immunogenic Activity In Guinea Pigs

Foot and mouth disease is a highly contagious disease to many kinds of animal, leading to great economic loss. Vaccination is the main methods to prevent and control FMD, conventional vaccines make great effective, but its disadvantages such as thermal instability, short-term nature protection, extra cost, particularly some outbreaks in the history were obvious. Thus, the new vaccines which are effective, low cost, easy storage and safe were urgently needed to develop. Transgenic plant vaccine becomes highlight since its so many advantages. Up to now, many scientists are engaging in the work and make great achievements.Our reports focus on the transgenic birdsfoot trefoil containing the gene P12A-3C of FMDV. A serials of trials were done including constructing the expression vector, re-resist screening for plants and its immune response to the guinea pigs as an experimental immunogen. Details on the experiments were as follows:1. Objective genes obtaining: Asial/YNBS/58 of sulking mouse virus passaged two generations, and then total RNA of virus was extracted. Gene P12A and 3C were amplified from viral RNA by RT-PCR using the specific primers and cloned into pMD18-T vector. Sequencing results showed that the gene P12A and 3C were successful obtained and sequencing demonstrated that the genes span fragment of 2247nt encoding 749 amino acids and 639nt encoding 213 amino acids respectively.2. Constructing the binary expression vector: The complete P12A-3C fragment was firstly sub-cloned into pcDNA3.1 (+), constructed the pcDNA3.1 (+) -P12A-3C plasmid, and then the P12A-3C DNA fragment was removed from pcDNA3.1 (+) -P12A-3C by digesting with the BamH I /Xba I and cloned into the pBin438 plasmid containing the D35S promoter obtaining the recombinant of pBin438P12A-3C. The recombinant plasmid was introduced in A.tumefaciens GV3101 by triparental mating, which was used in the plants transformation protocols.3. Gene transferring and protein analysis in plants: in this section we focus on transferring the gene into the plants and the transgenes analysis. Plants transformation with pBin438-P12A-3C was mediated by Agrobacterium tumefaciens GV3101, which was described by PanLi and others researchers. The presence of the gene P12A3C was detected in the total genome of the samples extracted from about 50 mg leave and stem tissues by PCR. This products was absent in nontransformaed plants. And the transcription of the transgenes was performed by RT-PCR. Plants transformed with pBin438-P12A-3C presented the active transcriptional activity however, the nontransformed plants showed nonactive. The expression productions were tested by sandwich-ELISA using the anti-FMDV serum;results showed that the genes were integrated with the genome of plants and antigen to specific FMDV antibody showed by ELISA.4. Immune response in guinea pigs by inoculating transgenic bidsfoot trefoil: this part we report the immunogenicity of the expression products was tested in a guinea pigs model. Five groups were divided, namely, A, B, C, D and E. A: transgenic plants extracts;B: non-transgenic plants extracts;divided, namely, A, B, C, D and E. A: transgenic plants extracts;B: non-transgenic plants extracts;C, D and E inoculating the pBin438, traditional vaccine and incomeplete fruend adjuvant, respectively. ELISA showed that the specific antibodies were elicited, Sera titers reached highest dilution to 1 '. 64.