HBsAg/截短型HCV核心蛋白融合基因植物表达载体的构建及对番茄的遗传转化

探索乙肝病毒表面抗原/截短型HCV核心蛋白融合基因在植物中表达乙肝丙肝双价抗原的可能性.以期为进一步研制植物乙肝丙肝双价口服疫苗打下基础。利用重组PCR技术将乙肝病毒(HBV)S基因连接在丙肝病毒(HCV)截短型核心蛋白序列的5′端,二者通过柔性肽(Gly4Ser)2序列相连,构建成-融合基因BC,测序结果正确。将融合基因BC克降到植物表达载体pBin438上,获得pBin438BC,然后用冻融法将其转入根癌农杆菌EHA105中,采用叶盘法转化番茄。获得了15株抗卡那霉素的番茄植株。对抗性植株进行PCR及Southern检测,8株获得阳性信号,表明目的基因已整合到了番茄基因组中。提取阳性植株的总蛋白.经透析后.将适当浓度蛋白质点在PVDF膜上,用Dot-ELISA方法验证番茄中表达蛋白的活性。结果表明,转基因番茄中有重组蛋白的表达。

Construction of Plant Expression Vector with HbsAg/Truncated HCV Core Protein Gene and Transformation of Tomato

This research was to explore the feasibility of expressing HBsAg/HCV core protein fusion gene in plants for the purpose of developing a new type of plant-derived hepatitis bivalent edible vaccine.Hepatitis B Virus S gene was con- nected to the 5" end of Hepatitis C Virus core protein sequence with the technique of recombinant PCR,a designed DNA sequence which encoded a polypeptide linker (Gly4Ser)2 was used to splice the two fragments.The fusion gene (BC) was constructed.Tomato leaf discs were transformed via Agrobacterium tumefaciens mediated procedure.DNA sequencing re- suits showed that the fusion gene (BC) was successfully constructed.The fusion gene (BC) was cloned to the plant expres- sion vector pBin438.The recombinant plasmid pBin438BC was constructed and introduced into Agrobactrium tumefaciens EHA105 by freeze-thaw method.Transgenic tomato plantlets grew in induction medium and 9 plants resistant to kanamycin were obtained.The regenerated tomato plantlets were analyzed by PCR and PCR-Southern blot.In which 5 plants appear right signal.The results confirmed that the fusion gene had been integrated into genome of tomato.