本文選題：幽門螺桿菌　切入點：ureI　出處：《南華大學(xué)》2009年碩士論文

【摘要】：目的:構(gòu)建幽門螺桿菌尿素通道蛋白UreI的真核表達載體pcDNA3.1(+)/ureI,并在HeLa細胞中進行表達。通過肌肉注射免疫C57BL/6小鼠,觀察其所產(chǎn)生的體液免疫和細胞免疫應(yīng)答水平,為研制高效、新型的幽門螺桿菌核酸疫苗提供實驗依據(jù)。 方法:用PRIMER5.0軟件設(shè)計引物, PCR擴增ureI基因,將該基因酶切插入pcDNA3.1(+)真核細胞表達載體構(gòu)建pcDNA3.1(+)/ureI重組載體,并轉(zhuǎn)染HeLa細胞,用Western-blot觀察鑒定其在真核細胞得到表達后,將核酸疫苗pcDNA3.1(+)/ureI、對照空質(zhì)粒pcDNA3.1(+)及PBS分組通過肌肉注射免疫6w齡C57BL/6小鼠,隔周免疫一次,共免疫四次。ELISA間接法測定小鼠血清中特異性IgG抗體水平,ELISA雙抗體夾心法檢測脾淋巴細胞培養(yǎng)上清中IFN-γ、IL-4水平,MTT比色法檢測脾淋巴細胞增殖反應(yīng),免疫熒光組化法檢測ureI在小鼠肌肉組織中的表達情況,通過PCR法檢測小鼠肌細胞中ureI基因的存在。 結(jié)果: (1)成功構(gòu)建了pcDNA3.1(+)/ureI真核表達載體,且重組質(zhì)粒能在HeLa細胞內(nèi)有效表達目的蛋白,且免疫熒光組化法檢測ureI在小鼠肌肉組織中能夠有效表達。 (2)小鼠接種pcDNA3.1(+)/ureI核酸疫苗后能產(chǎn)生特異性IgG抗體,10w后ELISA測定血清抗體A450值為1.249,效價為1:2048。 (3)核酸疫苗pcDNA3.1(+)/ureI免疫組小鼠脾淋巴細胞經(jīng)特異性抗原刺激后,培養(yǎng)上清中IFN-γ、IL-4含量明顯升高[分別為(275.20±43.21)pg/mL,(436.5±68.97) pg/mL)],與空質(zhì)粒組[分別為(18.30±5.32 )pg/mL,(40.10±18.54)pg/mL]之間差異具有顯著性(P0.01)。 (4)脾淋巴細胞增殖反應(yīng)測定:pcDNA3.1(+)/ureI核酸疫苗組小鼠脾淋巴細胞經(jīng)特異性抗原刺激后,刺激指數(shù)(1.76±0.16)明顯高于空質(zhì)粒組(1.20±0.14)和PBS組(1.14±0.12)(P0.01)。 (5)PCR檢測ureI基因可在小鼠肌細胞中存在。 結(jié)論: (1)成功構(gòu)建了pcDNA3.1(+)/ureI真核表達載體且其能在真核細胞中表達。 (2) pcDNA3.1(+)/ureI核酸疫苗能刺激機體產(chǎn)生較強細胞免疫應(yīng)答和體液免疫應(yīng)答。 (3) pcDNA3.1(+)/ureI核酸疫苗接種C57BL/6小鼠后能在肌細胞中存在。
[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- 緯 -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 鹵43.21 鹵43.21 pg / mL) pg-1 / mLrespectively), which was significantly different from that in the blank plasmid group [18.30 鹵5.32 渭 g 路mL-1] (40.10 鹵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 鹵43. 05 鹵68. 97), which was significantly different from that in the blank plasmid group (P = 18. 30 鹵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 鹵0.14) and PBS group (1.14 鹵0.12) and 1.14 鹵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 鹵0.16) and in the PBS group (1.14 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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