Gao Yi, Ma Ying, Li Mei, Cheng Tong, Li Shao-Wei, Zhang Jun, Xia Ning-Shao
The Key Laboratory Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen University, , Fujian Province, China.
World J Gastroenterol. 2003 May;9(5):996-1002. doi: 10.3748/wjg.v9.i5.996.
To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests.
The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice's serum were examined by ELISA every week.
By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo, level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight. In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response.
The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.
研究重组乙肝表面抗原（rHBsAg）在转基因酸浆中的表达，通过动物免疫试验探索利用酸浆生产乙肝口服疫苗的可行性。
构建含乙肝表面抗原基因的重组植物表达载体。以根癌农杆菌介导，将乙肝表面抗原基因转入酸浆子叶。通过潮霉素延迟筛选获得转基因酸浆。通过潮霉素抗性筛选、Gus检测、聚合酶链反应（PCR）和斑点杂交分析确认转基因酸浆中的整合DNA。用酶联免疫吸附测定（ELISA）检测rHBsAg的抗原性，通过将转基因组织经口投喂给预先接种商用乙肝疫苗的BALB/c小鼠，确认转基因酸浆组织来源的rHBsAg的免疫原性。每周用ELISA检测小鼠血清中的特异性抗体滴度。
目前已获得10株含乙肝表面抗原基因的转基因酸浆阳性株系。在同一转基因酸浆的不同器官中，叶片中rHBsAg的表达水平最高，产量可达300ng/g鲜重。果实中rHBsAg的表达水平约为10ng/g鲜重。在动物免疫试验中，用转基因组织经口投喂预先接种商用疫苗的小鼠而非未免疫小鼠，引发了显著的免疫反应。
该动物免疫试验结果表明，转基因酸浆来源的rHBsAg具有正常的免疫原性。本研究证明了在转基因酸浆中产生口服免疫原性rHBsAg的可行性，并将为大规模利用转基因酸浆生产低成本口服疫苗提供一些实验方法。
