幽门螺杆菌ureI核酸疫苗的构建及其免疫活性初步研究

发布时间：2018-04-01 10:21

本文选题：幽门螺杆菌　切入点：ureI　出处：《南华大学》2009年硕士论文

【摘要】：目的:构建幽门螺杆菌尿素通道蛋白UreI的真核表达载体pcDNA3.1(+)/ureI,并在HeLa细胞中进行表达。通过肌肉注射免疫C57BL/6小鼠,观察其所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。方法:用PRIMER5.0软件设计引物, PCR扩增ureI基因,将该基因酶切插入pcDNA3.1(+)真核细胞表达载体构建pcDNA3.1(+)/ureI重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后,将核酸疫苗pcDNA3.1(+)/ureI、对照空质粒pcDNA3.1(+)及PBS分组通过肌肉注射免疫6w龄C57BL/6小鼠,隔周免疫一次,共免疫四次。ELISA间接法测定小鼠血清中特异性IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL-4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测ureI在小鼠肌肉组织中的表达情况,通过PCR法检测小鼠肌细胞中ureI基因的存在。结果: (1)成功构建了pcDNA3.1(+)/ureI真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白,且免疫荧光组化法检测ureI在小鼠肌肉组织中能够有效表达。 (2)小鼠接种pcDNA3.1(+)/ureI核酸疫苗后能产生特异性IgG抗体,10w后ELISA测定血清抗体A450值为1.249,效价为1:2048。 (3)核酸疫苗pcDNA3.1(+)/ureI免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL-4含量明显升高[分别为(275.20±43.21)pg/mL,(436.5±68.97) pg/mL)],与空质粒组[分别为(18.30±5.32 )pg/mL,(40.10±18.54)pg/mL]之间差异具有显著性(P0.01)。 (4)脾淋巴细胞增殖反应测定:pcDNA3.1(+)/ureI核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数(1.76±0.16)明显高于空质粒组(1.20±0.14)和PBS组(1.14±0.12)(P0.01)。 (5)PCR检测ureI基因可在小鼠肌细胞中存在。结论: (1)成功构建了pcDNA3.1(+)/ureI真核表达载体且其能在真核细胞中表达。 (2) pcDNA3.1(+)/ureI核酸疫苗能刺激机体产生较强细胞免疫应答和体液免疫应答。 (3) pcDNA3.1(+)/ureI核酸疫苗接种C57BL/6小鼠后能在肌细胞中存在。
[Abstract]:Objective: to construct the eukaryotic expression vector pcDNA3.1 (urei) of urea channel protein UreI of Helicobacter pylori and express it in HeLa cells. The humoral and cellular immune responses of C57BL/6 mice were observed by intramuscular injection. New nucleic acid vaccine of Helicobacter pylori provides experimental basis. Methods: the ureI gene was amplified by PRIMER5.0 software and amplified by PCR. The gene was digested into pcDNA3.1 () eukaryotic cell expression vector to construct pcDNA3.1 (rrureI) recombinant vector. The recombinant vector was transfected into HeLa cells. The expression of ureI gene in eukaryotic cells was identified by Western-blot observation. PcDNA3.1 (control plasmid pcDNA3.1 () and PBS) were injected intramuscularly into 6w C57BL/6 mice and immunized once every other week. The level of specific IgG antibody in serum of mice was determined by four times. Elisa double antibody sandwich method was used to detect the level of IFN- 纬 -IL-4 in the culture supernatant of splenic lymphocytes and the proliferation of splenic lymphocytes was detected by MTT colorimetry. The expression of ureI in mouse muscle was detected by immunohistochemical method, and the presence of ureI gene in mouse muscle cells was detected by PCR method. Results:. The eukaryotic expression vector pcDNA3.1was successfully constructed, and the recombinant plasmid could effectively express the target protein in HeLa cells. The expression of ureI in mouse muscle tissue was detected by immunofluorescence histochemistry. Mice were inoculated with pcDNA3.1The specific IgG antibody was produced 10 weeks after inoculation. The A450 value and titer of serum antibody A450 were 1.249 and 1: 2048, respectively. Nucleic acid vaccine pcDNA3.1 (after stimulated by specific antigen in spleen lymphocytes of mice immunized with rureI), the level of IL-4 in the supernatant was significantly increased (275.20 卤43.21 卤43.21 pg / mL) pg-1 / mLrespectively), which was significantly different from that in the blank plasmid group [18.30 卤5.32 渭 g 路mL-1] (40.10 卤18.54)pg/mL), and the difference was significant between the control group and the blank plasmid group (P < 0.01), and the expression of IL-4 in the culture supernatant was significantly higher than that in the control group (P < 0.05. 05 卤43. 05 卤68. 97), which was significantly different from that in the blank plasmid group (P = 18. 30 卤5. 32). (4) the proliferative index of splenic lymphocytes in the mice treated with specific antigen was significantly higher than that in the empty plasmid group (1.20 卤0.14) and PBS group (1.14 卤0.12) and 1.14 卤0.12 (P 0.01), respectively. The proliferation of splenic lymphocytes in the mice treated with pcDNA3.1 was significantly higher than that in the control group (1.76 卤0.16) and in the PBS group (1.14 卤0.12, P 0.01). UreI gene can be detected in mouse muscle cells. Conclusion:. The eukaryotic expression vector pcDNA3.1 was successfully constructed and expressed in eukaryotic cells. PcDNA3.1 can stimulate strong cellular and humoral immune responses. PcDNA3.1 (P. R. UreI nucleic acid vaccine) was found in the muscle cells of C57BL/6 mice after inoculation.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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