幽门螺杆菌Lpp20核酸疫苗的构建及其免疫活性的初步研究

发布时间：2018-07-07 22:54

本文选题：幽门螺杆菌 + Lpp20　；参考：《南华大学》2007年硕士论文

【摘要】： 目的:构建幽门螺杆菌脂蛋白Lpp20基因的真核表达载体pcDNA3.1(+)/Lpp20,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Lpp20全基因;将PCR产物纯化后插入pGEX-6P-2载体,将重组质粒pGEX-6P-2/Lpp20转化入表达宿主菌BL21(DE3)进行诱导表达,纯化重组蛋白并分析其免疫反应性。再将Lpp20基因克隆至pcDNA3.1(+)真核细胞表达载体构建pcDNA3.1(+)/Lpp20重组体,并转染HeLa细胞,用免疫细胞化学及Western-blot观察鉴定其在真核细胞得到表达后,将核酸疫苗pcDNA3.1(+)/Lpp20、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,隔两周免疫一次,共免疫四次。ELISA间接法测定小鼠血清中抗Lpp20 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水平,MTT比色法检测脾淋巴细胞增殖反应。通过PCR法检测小鼠肌细胞中Lpp20基因的存在。结果:成功克隆了Lpp20基因,并将其成功构建到了pGEX-6P-2原核表达载体上,SDS-PAGE分析显示,在IPTG诱导下,重组工程菌表达了一相对分子量(Mr)约为44 KDa的目的蛋白条带, Western-blot检测显示重组蛋白有良好的免疫反应性。成功构建了pcDNA3.1(+)/Lpp20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。小鼠接种pcDNA3.1(+)/Lpp20核酸疫苗后能产生特异性IgG抗体,6w后ELISA测定血清抗体A450值为0.74,效价为1:1024。核酸疫苗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含量明显升高(410.36±56.23pg/mL),与空质粒组(25.26±10.85pg/mL)之间有显著性差异(P0.01)。脾淋巴细胞增殖反应测定,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(2.37±0.22)明显高于空质粒组(1.53±0.47)和PBS组(1.20±0.13)(P0.01)。PCR检测Lpp20基因可在小鼠肌细胞中存在。结论: (1)成功克隆了幽门螺杆菌Lpp20基因,并构建了pGEX-6P-2/Lpp20原核表达载体,且其可在原核细胞内表达具有良好免疫反应性的重组蛋白。 (2)成功构建了pcDNA3.1(+)/Lpp20真核表达载体且其能在真核细胞中表达。 (3) pcDNA3.1(+)/Lpp20核酸疫苗在小鼠体内可诱导较强的特异性体液免疫和细胞免疫应答。 (4) pcDNA3.1(+)/Lpp20核酸疫苗接种C57BL/6小鼠后能在肌细胞中存在。
[Abstract]:Aim: to construct eukaryotic expression vector pcDNA3.1 () / Lpp20 of Helicobacter pylori lipoprotein Lpp20 gene and express it in HeLa cells. C57BL / 6 mice were immunized intramuscularly to observe their humoral and cellular immune responses, which provided experimental basis for the development of a new type of nucleic acid vaccine for Helicobacter pylori. Methods: the whole Lpp20 gene was amplified by primer 5.0 primer, then purified and inserted into pGEX-6P-2 vector. The recombinant plasmid pGEX-6P-2 / Lpp20 was transformed into BL21 (DE3), and the recombinant protein was purified and its immunoreactivity was analyzed. Then Lpp20 gene was cloned into pcDNA3.1 () eukaryotic expression vector to construct pcDNA3.1 () -Lpp20 recombinant, and transfected into HeLa cells. The expression of Lpp20 gene in eukaryotic cells was identified by immunocytochemistry and Western-blot. The nucleic acid vaccine pcDNA3.1 () / Lpp20, the control plasmid pcDNA3.1 () and PBS were injected intramuscularly to C57BL / 6 mice at the age of 6 weeks. The level of anti-Lpp20 IgG antibody in serum of mice was determined by indirect immunosorbent assay. Elisa double antibody sandwich method was used to detect IFN- 纬 level in the culture supernatant of splenic lymphocytes. MTT colorimetric assay was used to detect the proliferation of splenic lymphocytes. The presence of Lpp20 gene in mouse muscle cells was detected by PCR. Results: Lpp20 gene was successfully cloned and constructed into pGEX-6P-2 prokaryotic expression vector. SDS-PAGE analysis showed that Lpp20 gene was induced by IPTG. A target protein band with a relative molecular weight (Mr) of about 44 KDa was expressed in the recombinant engineering strain. Western-blot analysis showed that the recombinant protein had a good immunoreactivity. The eukaryotic expression vector pcDNA3.1 () / Lpp20 was successfully constructed, and the recombinant plasmid could effectively express the target protein in HeLa cells. Mice inoculated with pcDNA3.1 () -Lpp20 nucleic acid vaccine could produce specific IgG antibody for 6 weeks. The serum antibody A450 value was 0.74 and the titer was 1: 102424 by Elisa after 6 weeks inoculation with pcDNA3.1 () -Lpp20 nucleic acid vaccine. The level of IFN- 纬 in the culture supernatant of mice immunized with nucleic acid vaccine was significantly increased (410.36 卤56.23 PG / mL), which was significantly higher than that in the blank plasmid group (25.26 卤10.85 PG / mL) (P0.01). The proliferation of spleen lymphocytes in the nucleic acid vaccine group (2.37 卤0.22) was significantly higher than that in the blank plasmid group (1.53 卤0.47) and PBS group (1.20 卤0.13) (P0.01). PCR assay showed that Lpp20 gene could be detected in mouse muscle cells. Conclusion: (1) Helicobacter pylori Lpp20 gene was cloned successfully and pGEX-6P-2 / Lpp20 prokaryotic expression vector was constructed. It can express recombinant protein with good immunoreactivity in prokaryotic cells. (2) pcDNA3.1 () -Lpp20 eukaryotic expression vector was successfully constructed and expressed in eukaryotic cells. (3) pcDNA3.1 () / Lpp20 nucleic acid vaccine could induce strong specific humoral and cellular immune responses in mice. (4) pcDNA3.1 () / Lpp20 nucleic acid vaccine was inoculated with C57BL / 6 mice. Can exist in muscle cells.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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