Construction Of Efficiency Expression Vectors Including FMDV Or HIV Main Antigen Gene And Potato Transformation

On account of new plant-based vaccines production and application, we selected AIDS (Acquired immunodeficiency syndrome) and FMD (Foot and Mouth Disease) as object, which have significant impact on society and economy. The high efficiency plant expression vectors including main antigen sites of HIV-I and FMDV were constructed so as to investigate methods or models for producing new gene engineering subunit vaccines using transgenic plant as bioreactors.FMDV structure protein gene PI was cloned by RT-PCR in our laboratory on the basis of analyzing FMDV genome and antigen sites. PI gene closed to natural infection, which could induce more strong immunization response in comparison with VP1 fragment or peptide-based epitope reported in the past experiment. The PI gene was modificated through designing a series of middle vectors. Redundant and unnecessary enzyme sites were cut off. The sequences that could regulate or control the transcription or translation procedure were added. Afterwards the 3.2kb S-P1 expression cassette cloned. Then inserted this cassette into pBI131 comes into being recombinant expressing plasmidpB1131SPl.HIV-I (human immunodeficiency virus type I) is the main pathogen of AIDS. Its structure protein gene gag encodes core protein of virus, which could induce cellular immunity and humoral immunity, and develop ADCC(antibody-dependent Cell-mediated cytotoxicity) action or CTL (Cytotoxic T Lymphoid) response. Besides, extra-membrane protein gp120 has high affinity with CD4+, which play an important role in HIV cell pathological changes and kill cell procedure. According to correct ORF, gag and gp120 were fused by molecular biology techniques, and cloned these entire three genes into pBH21 to construct plant expression vectors pBI121gag, pBI121gpl20 and pBI121gg.Cytokine act as mediate and adjust immune and inflammation response. Interleukin18(IL-i8), a new discovered cytokine, has important effect on enhance NK (natural killer cell) and CTL activity. Plant expression vector pBI121IL18 was constructured by subcloning swine IL18 cloned by our laboratory into pBI121.After that we transfomed FMDV PI complete gene, HIV gag structure protein gene and HIV gag-gp!20 fusion gene into potato internodes or minituber via agrobacterium tumefaciens. 38 putative transgenic plantlets were obtained after resistant selection. The results showed that FMDV PI gene, HIV gag gene and HIV gag-gp120 fusion gene has been transformed into potato by polymerase chain reaction analysis, dot Blot and Southern Blot techniques of putative resistant plantlets. To our knowledge, this is the first report on engage in this study.Meanwhile, the research on potato transformation using gene-gun has been carried out. Potato internodes were bombarded with plasmid pBI131SPl. Six resistant putative plantlets were obtained after kanacyllin selection. And the positive result was obtained by PCR and PCR-Southern. This paper will provide a fast transformation technology to potato as well as expand potato heredity transformation methods.