Expression Of Hepatitis B Surface Antigen In Nicotiana Benthamiana Using Potato Virus X Vector

In addition to great advantage in studing plants and plant pathogeny molarcular biology, and interaction between plant and virus, plant-virus-based vectors can be used as an alternative to stable genetic transformation for the introduction and expression of foreign genes in plants. The potato virus X(PVX)vector, containing a CaMV35S promoter, a dupicated coat protein promoter, and Nos terminator, was used instead of the traditional method of transcription in vitro, as the latter was time-consuming, and difficult to construct. It would be a potential and prospective way to produce plant vaccines.Hepatitis B virus is one of the alarming diseases, which is commonly cause of persistent viraemia in humans. More than 2 billion people are infected with hepatitis B virus (HBV) in the world, and 15–17% of them become chronic carriers. The expense of the available recombinant vaccine makes it difficult to be immunized in a mass in developing countries. The expression of vaccine in plants has been increasingly used as an alternative to the classical methodologies.Basing on Agrobacterium tumefaciens transformation and syringe inoculation, technological platform of virus-mediating foreign gene transformation was established. During the system,β-glucuronidase (GUS) was used as a report gene. The characterised results were that the color of inoculated leaves tissue was blue and the color of inoculated leaves tissue was none.It was reported that the expression of Hepatitis B virus surface antigen (HBsAg) in Nicotiana benthamiana by using PVX in the thesis. Primes were designed according to Genbank sequnce AF223961 of HBsAg(s), so 681bp DNA fragment was obtained by PCR method. The HBsAg gene was inserted into PVX expression vector, and transformed into Agrobacterium tumefaciens GV3101+PLICSa (helper plasmid).And gene transfer was accomplished by inoculating leaves, stems and roots of Nicotiana benthamiana with the plasmid DNA of PVX vector containing HbsAg gene by syringe. The expression level of the recombinant HBsAg protein was determined by ELISA at 11th,21 th, and 45th , its molecular weight was confirmed by SDS-PAGE and the immunity was assayed by Western blot analysis.The results showed that :(1) The age of leaves significantly affects the expression of HBsAg protein, as that the younger leaves accumulating 267.81ng/ml which is 2-60 times the concentration in older leaves;(2) The accumulation of recombinant protein slightly changed in a given leaf as over time the days post-inoculation (dpi), but was not significantly different between11 and 21 days;While the concentration of HbsAg had declined to a level below the limits of detection of the ELISA assay at 45th ,which shows the PVX-based vector is a transient system;(3) It was observed that the highest expression level was 796.81 ng/mg of total soluble protein(tsp);(4) SDS-PAGE analysis showed the recombinant protein with an apparent molecular mass 25 kDa, corresponding to the predicted size of the HBsAg;(5) Western blot analysis indicated that the recombinant HBsAg protein had the antigenicity to mouse anti- HBsAg monoclonal antibody.Using Potato virus X vector, technical system of expressing foreign proteins had been established, which lays a basis for further research on transiently expressing foreign genes and rapidly producing new pharmaceutical proteins of high expression levels, lower costs and greater flexibility.