High Expression Of Hepatitis B Surface Antigen In Transgenic Ginseng Cell Line

Hepatitis B virus infection is one of the most common viral infections affecting humans, being particularly widespread in China. The intramuscular injection of CHO-derived HBsAg or yeast-derived HBsAg into healthy individuals generates effective immunization and protection from viral infection, however, there is still an urgent need for a more affordable and reliable vaccine. Over the past decade, the expression of subunit vaccine antigens in plants has emerged as a convenient, safe and potentially economical platform technology, with the potential to provide a novel biotechnological solution to vaccine production and delivery. As an alternative to administration of vaccines by needle and syringe, oral vaccines offer significant advantages: including simplicity of use, increase in compliance, enhanced immune responses at mucosal sites, and stimulation of humoral immunity. Hepatitis B surface antigen has been successfully expressed in plants and delivered orally in both animals and human.However, one of the major limitations of the expression of recombinant antigens in transgenic plants remains the achievement of antigen-yields high enough to confer total protection in humans. This has led us to develop a system for the high-level production of rHBsAg in ginseng cell cultures, which is vital for their use as potential vaccines.Panax ginseng C.A. Meyer, a perennial herb, commonly known as "ginseng"has been widely used as a tonic and herbal medicine since ancient times particularly in Korea and China. It also contains various saponins and sapogenins. It is effective against gastroenteric disorders, diabetes and weak circulation. It has been used as an adjuvant to prevent various forms of hepatitis. Due to the relatively long growing cycle of ginseng, four to seven years in the field, we have produced ginseng cells through plant tissue cell cultures.In this report, we describe a transgenic system based around the efficient omega leader element for the high-level production of HBsAg in ginseng callus (dicots). This represents an important technological development for the subsequent use of ginseng in vaccine delivery.In order to produce HBV oral vaccine, we chose the Panax ginseng C.A. Meyer cell line, which was cultured in 67-V medium without plant growth regulators, as a system to investigate the expression of recombinant HBsAg. It also served as a model system for the expression of foreign proteins with pharmaceutical value.The HBsAg fragment from CHO cell line which produced rHBsAg (adr subtype) for use in vaccines was subcloned into GUS-less pBI121. Synthesis of 5'-nontranslated leader, we changed CAU in nature nucleotides into CAA lest the open reading frame should exist in reverse nucleotide sequence. The CaMV 35S promoter linked to the forward and the reverse omega leader, respectively, acted as a translation enhancer.The recombinant binary vector prepared from an E. coli clone was transformed into Agrobacterium strain LBA4404 cells. The plasmids contained the left and right border regions, which denoted the limits of the DNA via Agrobacterium tumefaciens-mediated transformant, as well as the neomycin phosphotransferase gene, which allowed selection with kanamycin. The resulting plant cells infected with the transformed Agrobacterium were maintained on 67-V semi-solid medium composed of MS salt and G418. The cells were incubated at 23℃in the dark and subcultured every three weeks until the new cell aggregates grow.To verify that G418-resistant cell lines contained the target gene, genomic DNA extracted from callus cultures was subjected to PCR amplification and the products were electrophoresed through agarose. A diagnostic 678bp fragment amplified with the primers specific to HBsAg 's' gene was detected in pBI transformed cell lines. HBsAg gene linked with omega leader amplified with the primers specific to forward omega leader and HBsAg gene was identified by the presence of a product of 750bp, while it was absent in control. HBsAg gene linked with reverse omega leader was amplified with the primers specific to reverse omega leader and HBsAg gene was also identified, while they was absent in control. A 750bp fragment was consistent with the expected size, and owing to the 5' addition of the nontranslated leader sequence. Western blot analysis confirmed the presence of an HBsAg specific band of the expected 24kDa size in pBIo transformed cells.To assess the effects of the TMV 5'-nontranslated omega leader and its antisense RNA on translational efficiency, we measured expression levels in transformed cells by ELISA. Expression levels of 300ng HBsAg g-1 fresh weight of cells in pBIo transformed cell lines which carried the omega translational enhancer were observed.The 5'-nontranslated region (omega sequence) of TMV (strain U1) was used as a translational enhancer sequence. A detailed analysis of the leader sequence pointed to the importance of the primary structure by identifying two elements, three copies of an eight-base direct repeat and a (CAA)n region. Previous work had shown that leader length was one of the parameters known to influence translation efficiency in both animal and plant cells, increasing the length of the 5'-leader increases translational efficiency. The introduction of secondary structure into the 5'-nontranslated region reduced protein yield. Via the comparison, we therefore designed the reverse leader construct. Antisense RNA sequence of omega was used to generate a control construct, in which the leader varied in orientation but not in structure, since little secondary structure is predicted to exist within omega. Moreover the changed base from U (wild-type leader) to A (synthesized leader) was between the eight-base direct repeat and the (CAA)n region.The 5'-nontranslated leader of the TMV RNA contains a highly organized 68-base region which has been shown to act as an enhancer of both eukaryotic and prokaryotic translation in vivo and in vitro. This element, known as the omega leader, is thought to influence the effectiveness with which bound 40S ribosomal subunits migrate and recognize the translational start site. Moreover, the degree to which omega leader enhances translation is highly species-dependent. Enhancement is higher in dicotyledonous plant cells than in monotyledonous cells.Interestingly, the presence of the omega sequence in the reverse orientation in pBIro transformed cells also increased the efficiency of HBsAg expression. In the current study, the enhancing activity of forward omega was about three to four-fold higher in comparison with reverse omega leader. Translation enhancing activity associated with the antisense omega was about one to two-fold higher in comparison with the absent omega leader.As described above, the use of transgenic plant cells for the expression and delivery of recombinant HBsAg as edible vaccines has been an important recent development. The production of recombinant proteins in callus culture is an emerging technology. However, one of the main drawbacks of this method of vaccine delivery is the requirement for high-levels of consumption of the plant material in order to achieve effective immunization. Here we describe a transgenic system utilizing the TMV omega sequence as an enhancer capable of expressing high-levels of HBsAg. This should reduce the level of consummation required for effective immunization. The use of our expression system in combination with ginseng callus cultures offers an ideal system for the production of rHBsAg under certified good manufacturing practice.