摘要
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。
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-der/ved hepatitis bivalent edible vaccine. Hepatitis B Virus S gene was connected 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 results showed that the fusion gene （BC） was successfully constructed. The fusion gene （BC） was cloned to the plant expression 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 fight signal. The results confirmed that the fusion gene had been integrated into genome of tomato.
出处
《长江蔬菜》
北大核心
2008年第07X期41-44,共4页
Journal of Changjiang Vegetables
基金
吉林省科技厅项目"乙肝丙肝转基因番茄植物双价口服疫苗的研究"(20050402-5)
关键词
乙肝病毒S基因
丙肝病毒核心蛋白
融合基因
农杆菌
转基因番茄
Hepatitis B Virus S gene
Hepatitis C Virus core protein
Fusion gene
Agrobactrium tumefaciens
Trans- genic tomato
