本文選題：幽門螺桿菌 + Lpp20��； 參考：《南華大學(xué)》2007年碩士論文

【摘要】： 目的:構(gòu)建幽門螺桿菌脂蛋白Lpp20基因的真核表達(dá)載體pcDNA3.1(+)/Lpp20,并在HeLa細(xì)胞中進(jìn)行表達(dá)。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(xì)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(kuò)增Lpp20全基因;將PCR產(chǎn)物純化后插入pGEX-6P-2載體,將重組質(zhì)粒pGEX-6P-2/Lpp20轉(zhuǎn)化入表達(dá)宿主菌BL21(DE3)進(jìn)行誘導(dǎo)表達(dá),純化重組蛋白并分析其免疫反應(yīng)性。再將Lpp20基因克隆至pcDNA3.1(+)真核細(xì)胞表達(dá)載體構(gòu)建pcDNA3.1(+)/Lpp20重組體,并轉(zhuǎn)染HeLa細(xì)胞,用免疫細(xì)胞化學(xué)及Western-blot觀察鑒定其在真核細(xì)胞得到表達(dá)后,將核酸疫苗pcDNA3.1(+)/Lpp20、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,隔兩周免疫一次,共免疫四次。ELISA間接法測定小鼠血清中抗Lpp20 IgG抗體水平,ELISA雙抗體夾心法檢測脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ水平,MTT比色法檢測脾淋巴細(xì)胞增殖反應(yīng)。通過PCR法檢測小鼠肌細(xì)胞中Lpp20基因的存在。 結(jié)果:成功克隆了Lpp20基因,并將其成功構(gòu)建到了pGEX-6P-2原核表達(dá)載體上,SDS-PAGE分析顯示,在IPTG誘導(dǎo)下,重組工程菌表達(dá)了一相對分子量(Mr)約為44 KDa的目的蛋白條帶, Western-blot檢測顯示重組蛋白有良好的免疫反應(yīng)性。成功構(gòu)建了pcDNA3.1(+)/Lpp20真核表達(dá)載體,且重組質(zhì)粒能在HeLa細(xì)胞內(nèi)有效表達(dá)目的蛋白。小鼠接種pcDNA3.1(+)/Lpp20核酸疫苗后能產(chǎn)生特異性IgG抗體,6w后ELISA測定血清抗體A450值為0.74,效價為1:1024。核酸疫苗免疫組小鼠脾淋巴細(xì)胞經(jīng)特異性抗原刺激后,培養(yǎng)上清中IFN-γ含量明顯升高(410.36±56.23pg/mL),與空質(zhì)粒組(25.26±10.85pg/mL)之間有顯著性差異(P0.01)。脾淋巴細(xì)胞增殖反應(yīng)測定,核酸疫苗組小鼠脾淋巴細(xì)胞經(jīng)特異性抗原刺激后,刺激指數(shù)(2.37±0.22)明顯高于空質(zhì)粒組(1.53±0.47)和PBS組(1.20±0.13)(P0.01)。PCR檢測Lpp20基因可在小鼠肌細(xì)胞中存在。 結(jié)論: (1)成功克隆了幽門螺桿菌Lpp20基因,并構(gòu)建了pGEX-6P-2/Lpp20原核表達(dá)載體,且其可在原核細(xì)胞內(nèi)表達(dá)具有良好免疫反應(yīng)性的重組蛋白。 (2)成功構(gòu)建了pcDNA3.1(+)/Lpp20真核表達(dá)載體且其能在真核細(xì)胞中表達(dá)。 (3) pcDNA3.1(+)/Lpp20核酸疫苗在小鼠體內(nèi)可誘導(dǎo)較強(qiáng)的特異性體液免疫和細(xì)胞免疫應(yīng)答。 (4) pcDNA3.1(+)/Lpp20核酸疫苗接種C57BL/6小鼠后能在肌細(xì)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- 緯 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- 緯 in the culture supernatant of mice immunized with nucleic acid vaccine was significantly increased (410.36 鹵56.23 PG / mL), which was significantly higher than that in the blank plasmid group (25.26 鹵10.85 PG / mL) (P0.01). The proliferation of spleen lymphocytes in the nucleic acid vaccine group (2.37 鹵0.22) was significantly higher than that in the blank plasmid group (1.53 鹵0.47) and PBS group (1.20 鹵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.
【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】：2007
【分類號】：R392

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