人结核杆菌Hsp65和Ag85B双价膜锚定表达DNA疫苗pIRES-MTHsp65-Ag85B的构建和日本血吸虫pIRE

发布时间：2018-03-18 19:58

  本文选题：结核杆菌　切入点：Hsp65　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:构建人结核杆菌Hsp65和Ag85B膜锚定共表达质粒pIRES-MTHsp65 -Ag85B,并检测其在体外的表达。 方法:利用PCR技术分别从质粒pBCG-SP-Hsp65和结核杆菌H37Rv的基因组中扩增出Hsp65和Ag85B基因。并将其克隆到真核表达质粒pVAC中,分别获得重组质粒pVAC-Hsp65、pVAC-Ag85B。然后分别以pVAC-Hsp65、pVAC-Ag85B质粒为模板,采用PCR技术扩增出5′端含编码人白介素2(IL-2)23个氨基酸的信号序列与3′端含编码人胎盘碱性磷酸酶(PLAP)COOH-末端32个氨基酸的膜锚定序列的Hsp65、Ag85B修饰基因。并将这两个修饰基因先后定向克隆到真核表达载体pIRES上获得共表达质粒pIRES-MTHsp65-Ag85B。将重组质粒转染Hela细胞,通过RT-PCR的方法检测转染细胞中Hsp65和Ag85B mRNA的表达。 结果:经过PCR、酶切鉴定和测序证实所克隆的Hsp65、Ag85B基因与报道结果完全一致,重组真核表达质粒pIRES-MTHsp65-Ag85B构建成功,并且转染Hela细胞总RNA中结核杆菌Hsp65和Ag85B mRNA为阳性。 结论:成功构建了人结核杆菌Hsp65和Ag85B膜锚定双表达质粒pIRES-MTHsp65-Ag85B,该质粒转染人子宫颈癌Hela细胞后获得表达,为进一步对其免疫原性和抗感染效应的研究奠定了基础。 目的构建含日本血吸虫相对分子质量为26 000的抗原(Sj26GST)基因的膜锚定表达DNA疫苗,观察其免疫BALB/c小鼠后的免疫应答效应。 方法用RT-PCR法,以血吸虫成虫RNA为模板,扩增获得Sj26的全长基因。利用重组PCR技术,在Sj26基因的5′端加上编码IL-2的信号肽核苷酸序列,3′端加上编码胎盘碱性磷酸酶的膜锚定序列,然后将其克隆入pIRES载体中,构建一个膜锚定型真核表达质粒pIRES-Sj26。将重组质粒转染HeLa细胞,通过RT-PCR及间接免疫荧光技术检测目的基因的表达。用构建的pIRES-Sj26疫苗肌肉注射免疫BALB/c小鼠后,用ELISA试剂盒检测小鼠血清中的总IgG抗体浓度和脾淋巴细胞培养上清的干扰素γ(INF-γ)含量,以淋巴细胞刺激指数(SI)反应淋巴细胞增殖能力,用流式细胞术检测脾细胞CD4/CD8亚群。 结果经过酶切鉴定、PCR及测序证实重组质粒pIRES-Sj26构建成功,经转染HeLa细胞及免疫荧光检测证明质粒pIRES-Sj26能在体外进行表达。免疫小鼠后检测结果表明pIRES-Sj26组的血清总IgG抗体浓度、INF-γ的含量明显高于对照组和空载体组(P0.01);其脾SI高于对照组和空载体组(P0.05);CD8+细胞百分比高于对照组和空载体组(P0.05), CD4+细胞百分比没有显著变化(P0.05)。 结论成功构建日本血吸虫膜锚定表达DNA疫苗pIRES-Sj26,表达的Sj26蛋白大部分锚定在细胞膜上。pIRES-Sj26疫苗能增强BALB/c小鼠的免疫应答反应。
[Abstract]:Aim: to construct the co-expression plasmid pIRES-MTHsp65-Ag85B of Hsp65 and Ag85B, and to detect its expression in vitro. Methods: the Hsp65 and Ag85B genes were amplified by PCR from the genome of plasmid pBCG-SP-Hsp65 and Mycobacterium tuberculosis H37Rv, and cloned into the eukaryotic expression plasmid pVAC. The recombinant plasmid pVAC-Hsp65 pVAC-Ag85B was obtained, and then the plasmid pVAC-Hsp65 pVAC-Ag85B was used as a template. PCR technique was used to amplify the signal sequence of 23 amino acids encoding human interleukin-2 (IL-2) and Hsp65Ag-85B modified gene with 3'terminal mooring sequence encoding 32 amino acids of human placental alkaline phosphatase (PLAPAPP) COOH- terminal. The co-expression plasmid pIRES-MTHsp65-Ag85Bwas cloned into eukaryotic expression vector pIRES. The recombinant plasmid was transfected into Hela cells. The expression of Hsp65 and Ag85B mRNA in transfected cells was detected by RT-PCR. Results: the cloned Hsp65 Ag85B gene was confirmed by PCR, restriction endonuclease digestion and sequencing. The recombinant eukaryotic expression plasmid pIRES-MTHsp65-Ag85B was successfully constructed, and Hsp65 and Ag85B mRNA were positive in the total RNA of Hela cells. Conclusion: the double expression plasmid pIRES-MTHsp65-Ag85B was successfully constructed by Hsp65 and Ag85B membrane anchoring. The plasmid was transfected into human cervical cancer Hela cells and was expressed successfully, which laid a foundation for the further study of its immunogenicity and anti-infection effect. Objective to construct the membrane anchored DNA vaccine containing Schistosoma japonicum antigen Sj26GST gene and observe the immune response of BALB/c mice immunized with Sj26GSTgene. Methods the full-length gene of Sj26 was amplified by RT-PCR and RNA of adult Schistosoma japonicum was used as template, and the recombinant PCR technique was used. The signal peptide nucleotide sequence encoding IL-2 was added to the 5 'terminal of Sj26 gene and the membrane anchoring sequence encoding placental alkaline phosphatase was added to the 3' terminal of IL-2 gene. Then it was cloned into pIRES vector. A membrane anchored eukaryotic expression plasmid pIRES-Sj26 was constructed. The recombinant plasmid was transfected into HeLa cells. The expression of the target gene was detected by RT-PCR and indirect immunofluorescence technique. The BALB/c mice were injected intramuscularly with the constructed pIRES-Sj26 vaccine. The concentration of total IgG antibody in serum and the content of interferon 纬 -INF- 纬 in the supernatant of spleen lymphocytes were detected by ELISA kit. Lymphocyte proliferation was measured by lymphocyte stimulating index (SI). The CD4/CD8 subsets of splenocytes were detected by flow cytometry. Results the recombinant plasmid pIRES-Sj26 was successfully constructed by PCR and sequencing. Transfection of HeLa cells and immunofluorescence assay proved that the plasmid pIRES-Sj26 could be expressed in vitro. The results of immunized mice showed that the serum total IgG antibody concentration of pIRES-Sj26 group was significantly higher than that of control group and empty vector group (P0.01), and the spleen SI of mice was higher than that of control group and empty vector group. The percentage of CD8 cells in control group and empty carrier group was higher than that in control group and empty carrier group, but the percentage of CD4 cells did not change significantly. Conclusion DNA vaccine pIRES-Sj26 was successfully constructed by membrane anchoring of Schistosoma japonicum. The majority of the expressed Sj26 protein was anchored on the membrane. PIRES-Sj26 vaccine could enhance the immune response of BALB/c mice.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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