Study On Porcine A Group Rotavirus VP6 Gene Transgenic Plant

Rotaviruse is the major cause of severe acute gastroenteritis in young piglets worldwide. Theclinical symptoms were fever, vomiting, sicchasia and diarrhea; if severely, it can cause young livestockto dehydrate. But for adult pigs were silent infection. It can also infect human, bovine, goat and so on.The disease can make young livestock to fall ill in very short time, and have high mortality rate. Untilnow, there are no specific medicament in prevention of rotavirus disease, It is the most important toprevent its occuring.In our country, Porcine rotavirus disease is distributed throughout this country and causes severeconomic loss for the animal husbandry. So, it is an effective prevention and cure for reducing themorbility of the disease of young piglets and increasing the efficiency of pig husbandry to effectivelyprevent this disease. In 1992, Mason put forward "transgenic plant vaccine". It is a technique by usingmolecular biology technique to transform foreign gene into plant cells, and the foreign gene can beexpressed largely in the plant.VP6 is encoded by genome segment 6 and is the major structural protein in virus particles, itconstitutes approximately 50% of the virion by weight. VP6 is both highly immunogenic and antigenic,and it is the most frequently targeted protein in diagnostic assays to detect virus particles. Althoughanti-VP6 antibodies do not exhibit virus neutralizing activity, but Esquivel FR etal reported VP6 primesfor an enhanced neutralizing antibody response. Subsequently Feng N etal thought the levels of theneutralizing antibody response do not always correlate with protection. Non-neutralizing IgAmonoclonal antibodies directed against the intermediate capsid protein vp6 can mediate immunityagainst rotavirus in vivo. The induction of efficient protection against rotavirus correlated with the levelof intestinal IgA but not with the level of rotavirus-specific serum IgG.According to the above content, Based on the nucleotide sequence of its encoded gene VP6 andprokaryotic expression vector pET5a, a pair of specific primers RO1/RO2 containing two restrictionsites of enzymes BamHI and NdeI. were synthesized. Using RO1/RO2 as primers and plasmidpGEM-T-Easy-VP6 as template, 1.194kb VP6 gene fragment was obtained by PCR amplification. Afterdigested by enzymes BamHI and NdeI, the VP6 gene fragment was ligased into a linear plasmid whichwas digested by the same restriction enzymes, then transformed into E.Coli BL21(DE₃)Plyss.When thecells were induced by the IPTG, the production of the gene was expressed and 44KD protein bandgenerated in SDS-PAGE.Based on the nucleotide sequence of its encoded gene VP6 and plant expression vector pBI121, apair of specific primers RO3/RO4 containing two restriction sites of enzymes BamHI and SacI. weresynthesized. Using RO3/RO4 as primers and plasmid pGEM-T-Easy-VP6 as template, 1.194kb VP6 genefragment was obtained by PCR amplification. After digested by enzymes BamHI and SacI, the VP6 genefragment was ligased into a linear plasmid which was digested by the same restriction enzymes, thentransformed into E. Coli DH5a and selected by kanamycin(kan). The positive clones weresequenced, theresult showed that a plant expression vector pBI121-VP6 was obtained.The recombinant plasmid pBI121-VP6 was transformed into Agrobacterium tumefaciens strainLBA4404 by freeze-thawing method and the introduced into the sterile tube culture shoot of tobacco NC89 and baoding alfalfa by leaf disc infection method. Via callus-induction, differentiation cultivationand kanamycin selection, 80 kanamycin resistance transgenic regenerated plants of tobacco NC89were obtained, but transgenic alfalfa plants were not obtained.Plant total DNA and RNA was extracted from kanamycin resistance transgenic regeneratedplants of tobacco NC89, and then analysed by PCR, RT-PCR, PCR-Southern and Dot blotting, theresults showed that VP6 gene was successfully integrated into the genomes of tobacco plants NC89and transcribed correctly.Plant total protein was extracted from the positive transgenic regenerated plants of tobacco NC89digested by above detection, and then detected by western-blotting, the results indicated that VP6protein was successfully expressed and the protein expressed had reaction activity.