The Construction Of Plant Expression Vector Of The Vp7 Gene Of Porcine Group A Rotavirus And The Study On Transgenic Plant

Rotaviruses, which form one genus of the family Reoviridae, are now recognized as one of the most important causes of severe viral gastroenteritis in young humans and animals. RV infect 90% children under 5 years of age worldwide, in industrialized and developing countries alike. Now there is no effective medicine to cure RV, thus rotavirus vaccines are considered the most promising means for disease prevention. Previous studies in naturally infected have shown that antibodies are important in protection from rotavirus reinfection. With the development of molecular biology and culturing of cells, the use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as an alternative methodology for the production of experimental vaccines.Now we have known the gene structure of rotavirus. The particle has three capsids, and Vp7 glycoprotein, which was an important composition of the outer capsid protein, was capable of stimulating protective immunity and plays a key role in protection. The research has done :(1) the construction of pBI121-Vp7 expression vector: we design a primer with the sequence of cloned Vp7 gene under the ground of pBI121. After PCR and the digest of BamHI 和 SmaI, we ligase Vp7 and pBI121 to construct pBI121-Vp7. We obtain the positive clones after selected by kanamycin and then transfer them in the Agrobacterium tumefacien-LBA4404 after sequencing to transform plants.(2) the study on transgenic tobacco: leaves of the Nicotiana benthamiana (NC) were transformed by Agrobacterium tumefacien-mediated method with the expression plasmid. After selected by kanamycin, fifty resistant lines were obtained. Forty lines were positive through PCR with total DNA and twenty-five lines were positive through RT-PCR with total RNA from fresh leaves tissue. We extract the total protein of the positive lines for SDS-PAGE and then Western blot, the resultshows that the combined protein has the immunity.(3) The genetic transformation of alfalfa: using cotyledon and hypocotyl as explants to induce and differentiate callus, and a lots of resistant callus with green prominence were created, the completely differentiated transgenic alfalfa was not yet obtained.