血吸虫Sj14FABP和Sj26GST双价膜锚定表达DNA疫苗pIRES-Sj26-Sj14的构建和表达
发布时间：2019-02-24 17:01
【摘要】： 目的:构建日本血吸虫Sj14FABP和Sj26GST膜锚定共表达质粒pIRES-Sj26-Sj14,并检测其在体外的表达。 方法:利用RT-PCR法,以日本血吸虫成虫总RNA为模板,扩增获得Sj14FABP与Sj26GST的全长基因。并先将其克隆到真核表达质粒pVAC中,分别获得重组质粒pVAC-Sj14、pVAC-Sj26。然后分别以pVAC-Sj14、pVAC-Sj26质粒为模板,采用PCR技术扩增出含人白介素2(IL-2 )23个氨基酸的信号肽与人胎盘碱性磷酸酶(PLAP)COOH-末端32个氨基酸的膜锚定序列在内的Sj14、Sj26修饰基因。并将该两个修饰基因共同构建到真核表达载体pIRES上获得共表达质粒pIRES-Sj26-Sj14,将重组质粒转染Hela细胞,通过RT-PCR的方法及间接免疫荧光技术检测Sj26, Sj14基因的膜锚定表达。 结果:经过酶切鉴定、PCR、测序证实所克隆的Sj14FABP、Sj26GST基因与报道结果完全一致,重组真核表达质粒pVAC-Sj14、pVAC-Sj26、pIRES-Sj26-Sj14构建成功。并且pIRES-Sj26-Sj14质粒在体外转染Hela细胞后可表达膜锚定蛋白Sj14与Sj26。 结论:成功构建了日本血吸虫Sj14FABP和Sj26GST膜锚定双表达质粒,该质粒转染人子宫颈癌Hela细胞后可正常表达这两种蛋白,为下步对其免疫原性免疫反应性及免疫保护作用的进一步的研究奠定了基础,也给血吸虫疫苗的研究拓宽了思路。
[Abstract]:Aim: to construct the co-expression plasmid pIRES-Sj26-Sj14, of Schistosoma japonicum Sj14FABP and Sj26GST and to detect its expression in vitro. Methods: the full-length genes of Sj14FABP and Sj26GST were amplified by using total RNA of adult Schistosoma japonicum as template by RT-PCR method. The recombinant plasmid pVAC-Sj14,pVAC-Sj26. was obtained by cloning it into eukaryotic expression plasmid pVAC. Then the pVAC-Sj14,pVAC-Sj26 plasmids were used as templates, The Sj14,Sj26 modified genes including the signal peptide containing 23 amino acids of human interleukin 2 (IL-2) and the membrane anchoring sequence of 32 amino acids of (PLAP) COOH- terminal of human placental alkaline phosphatase (PLAP) COOH-) were amplified by PCR. The two modified genes were co-constructed into eukaryotic expression vector pIRES to obtain the co-expression plasmid pIRES-Sj26-Sj14,. The recombinant plasmid was transfected into Hela cells. The membrane anchoring expression of Sj26, Sj14 gene was detected by RT-PCR and indirect immunofluorescence technique. Results: PCR, sequencing confirmed that the cloned Sj14FABP,Sj26GST gene was in good agreement with the reported results. The recombinant eukaryotic expression plasmid pVAC-Sj14,pVAC-Sj26,pIRES-Sj26-Sj14 was successfully constructed. Moreover, pIRES-Sj26-Sj14 plasmid can express membrane anchoring protein Sj14 and Sj26. after transfection of Hela cells in vitro. Conclusion: the double expression plasmids of Schistosoma japonicum Sj14FABP and Sj26GST membrane anchoring have been constructed successfully. These two proteins can be expressed normally after transfected into human cervical cancer Hela cells, which is the next step to identify the immunogenicity of Schistosoma japonicum and Schistosoma japonicum. The further study of immunoreactivity and immune protection has laid a foundation for the research of Schistosoma japonicum vaccine.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
[Abstract]:Aim: to construct the co-expression plasmid pIRES-Sj26-Sj14, of Schistosoma japonicum Sj14FABP and Sj26GST and to detect its expression in vitro. Methods: the full-length genes of Sj14FABP and Sj26GST were amplified by using total RNA of adult Schistosoma japonicum as template by RT-PCR method. The recombinant plasmid pVAC-Sj14,pVAC-Sj26. was obtained by cloning it into eukaryotic expression plasmid pVAC. Then the pVAC-Sj14,pVAC-Sj26 plasmids were used as templates, The Sj14,Sj26 modified genes including the signal peptide containing 23 amino acids of human interleukin 2 (IL-2) and the membrane anchoring sequence of 32 amino acids of (PLAP) COOH- terminal of human placental alkaline phosphatase (PLAP) COOH-) were amplified by PCR. The two modified genes were co-constructed into eukaryotic expression vector pIRES to obtain the co-expression plasmid pIRES-Sj26-Sj14,. The recombinant plasmid was transfected into Hela cells. The membrane anchoring expression of Sj26, Sj14 gene was detected by RT-PCR and indirect immunofluorescence technique. Results: PCR, sequencing confirmed that the cloned Sj14FABP,Sj26GST gene was in good agreement with the reported results. The recombinant eukaryotic expression plasmid pVAC-Sj14,pVAC-Sj26,pIRES-Sj26-Sj14 was successfully constructed. Moreover, pIRES-Sj26-Sj14 plasmid can express membrane anchoring protein Sj14 and Sj26. after transfection of Hela cells in vitro. Conclusion: the double expression plasmids of Schistosoma japonicum Sj14FABP and Sj26GST membrane anchoring have been constructed successfully. These two proteins can be expressed normally after transfected into human cervical cancer Hela cells, which is the next step to identify the immunogenicity of Schistosoma japonicum and Schistosoma japonicum. The further study of immunoreactivity and immune protection has laid a foundation for the research of Schistosoma japonicum vaccine.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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