本文選題：幽門螺桿菌 + cagT�。� 參考：《江蘇大學(xué)》2008年碩士論文

【摘要】： 目的:探討cagT基因在幽門螺桿菌(Helicobacter pylori,H.pylori)cag致病島(cag pathogenicity island,cag PAI)編碼的Ⅳ型分泌系統(tǒng)(typeⅣsecretion system,TFSS)裝置中的作用;并通過與胃癌細胞相互作用,研究其重組蛋白對胃癌細胞分泌炎癥因子的能力和增殖的影響,為進一步闡明H.pylori的致病機制奠定基礎(chǔ)。 方法:根據(jù)GenBank收錄的H.pylori 22695全基因組序列,利用生物學(xué)軟件Primer Premier5.0設(shè)計擴增cagT基因的引物,并引入限制性核酸內(nèi)切酶酶切位點;應(yīng)用PCR技術(shù)從H.pylori NCTC 11637基因組DNA中擴增cagT編碼基因片段后,克隆至pGEM-T載體,轉(zhuǎn)化大腸埃希菌(Escherichia coli,E.coli)DH5α,雙酶切鑒定篩選陽性克隆,進行序列測定。應(yīng)用生物信息學(xué)技術(shù),對cagT基因編碼的氨基酸序列與已知的根癌農(nóng)桿菌(Agrobacterium tumefaciens,A.tumefaciens)TFSS結(jié)構(gòu)基因virB7進行同源性比對,并對其編碼的蛋白質(zhì)做理化特性的預(yù)測分析。再將其定向插入原核表達載體pQE30中,轉(zhuǎn)化至表達宿主菌E.coli M15,通過氨芐青霉素抗性篩選和雙酶切鑒定陽性克隆。IPTG誘導(dǎo)其表達,表達的蛋白經(jīng)Western blot鑒定,并以Ni~(2+)-NTA樹脂進行純化。純化后的重組蛋白免疫家兔,制備抗CagT多克隆抗體,酶聯(lián)免疫吸附試驗(enzyme-linked immunosorbentassay,ELISA)檢測血清抗體效價。重組蛋白與SGC-7901細胞作用一定時間,提取細胞總RNA,采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reversetranscriptase polymerase chain reaction,RT-PCR)檢測IL-8 mRNA的表達;并用四甲基偶氮唑藍(methyl thiazoly tetrazolium,MTT)法檢測蛋白對細胞增殖的影響。 結(jié)果: 1.H.pylori NCTC 11637 cagT基因全長843bp(GenBank登錄號為EF114758),編碼280個氨基酸,與H.pylori其它菌株基因序列的核苷酸同源性為96%～99%;與A.tumefaciens的VirB7具有同源性;DNAStar軟件預(yù)測其編碼的蛋白質(zhì)的相對分子量為32.33kDa,具有較強的免疫原性。 2.成功構(gòu)建pQE30-cagT原核表達重組質(zhì)粒,確定IPTG終濃度0.8mmol/L,溫度30℃,誘導(dǎo)時間4h為最佳誘導(dǎo)條件,Western blot檢測結(jié)果顯示該重組蛋白可與抗His標(biāo)簽抗體結(jié)合反應(yīng),經(jīng)Ni~(2+)-NTA純化后可獲得純度約為98%的CagT重組蛋白,相對分子質(zhì)量(Mr)約為32kDa,與預(yù)測結(jié)果一致。CagT重組蛋白免疫家兔,獲得抗CagT多克隆抗體,其效價為1:1.6×10~4。 3.終濃度10μg/mL的CagT重組蛋白作用于SGC-7901細胞4h后,可擴增出IL-8片段。 4.不同濃度的CagT重組蛋白與SGC-7901細胞作用6、24及48h后,細胞增殖的程度隨著CagT重組蛋白濃度的增加和作用時間的延長而逐漸降低。 結(jié)論: 1.成功克隆了cagT基因,其與已知的A.tumefaciens的TFSS結(jié)構(gòu)基因virB7具有同源性,是H.pylori cag PAI編碼的TFSS的結(jié)構(gòu)基因之一。 2.成功構(gòu)建了原核表達載體,獲得了CagT重組蛋白,并制備了兔源性抗CagT多克隆抗體。 3.CagT重組蛋白可誘導(dǎo)SGC-7901細胞表達IL-8 mRNA。 4.CagT重組蛋白對SGC-7901細胞的增殖有一定抑制作用,其抑制作用隨著蛋白濃度增加和作用時間延長而逐漸增強。
[Abstract]:Objective: To investigate the cagT gene of Helicobacter pylori (Helicobacter pylori, H.pylori (cag pathogenicity) cag pathogenicity island island, CAG PAI) encoding the type IV secretion system (type IV secretion system, TFSS) in the device; and with the gastric cancer cell interactions, affect the ability of proliferation and secretion of inflammatory factors on gastric cancer cells the recombinant protein, lay the foundation for elucidating the molecular pathogenesis of H.pylori.
Methods: according to the GenBank included H.pylori 22695 genome sequence, primers designed by biology software Primer Premier5.0 gene was amplified by cagT, and the introduction of restriction endonuclease sites; the application of PCR technology from H.pylori NCTC 11637 genome DNA gene fragment encoding cagT was amplified, cloned into pGEM-T vector and transformed into Escherichia coli Escherichia (coli E.coli) DH5 alpha, double enzyme digestion and positive clones were screened and sequenced. Using bioinformatics, amino acid sequence and known to the cagT gene encoding Agrobacterium tumefaciens (Agrobacterium tumefaciens, A.tumefaciens TFSS) virB7 gene homology comparison, analysis and prediction of its encoding protein physicochemical properties. Then inserted into the prokaryotic expression vector pQE30 and transformed to E.coli E.coli M15 by ampicillin resistance screening and double enzyme Cut the identification of positive clones induced by.IPTG expression, protein expression by Western blot identification, and Ni~ (2+) was purified by -NTA resin. The recombinant protein was used to immunize rabbits after purification, preparation of anti CagT polyclonal antibody, enzyme-linked immunosorbent assay (enzyme-linked Immunosorbentassay, ELISA) detection of serum antibody titer of recombinant protein and SGC-7901. The cells for a certain period of time, extraction of total cellular RNA by reverse transcriptase polymerase chain reaction (reversetranscriptase polymerase chain reaction, RT-PCR) to detect the expression of IL-8 and mRNA; four with methyl thiazolyl tetrazolium (methyl thiazoly tetrazolium MTT) method was used to detect the protein effect on cell proliferation.
Result:
The full-length 843bp 1.H.pylori NCTC 11637 cagT gene (GenBank accession number EF114758), encoding 280 amino acids, 96% homology with other H.pylori strains to 99% nucleotide sequences; homologous with A.tumefaciens VirB7; the relative molecular weight of the protein DNAStar software to predict the encoding of the 32.33kDa, has a strong immunogenicity..
2. successfully constructed pQE30-cagT prokaryotic expression plasmid, to determine the final concentration of IPTG 0.8mmol/L, a temperature of 30 DEG C, the induction time of 4H induced condition is the best, Western blot results showed that the recombinant protein could react with the antibody against His label, the Ni~ (2+) -NTA was obtained after about 98% purity of CagT recombinant protein the relative molecular mass (Mr) was about 32kDa, consistent with the prediction results of.CagT recombinant protein to immunize rabbits obtained anti CagT polyclonal antibody, and its titer was 1:1.6 * 10~4.
The CagT recombinant protein of 3. terminal concentration of 10 mu g/mL acts on the 4H of SGC-7901 cells, and the IL-8 fragment can be amplified.
4., after different concentrations of CagT recombinant protein interact with SGC-7901 cells for 6,24 and 48h, the degree of cell proliferation decreases with the increase of CagT recombinant protein concentration and the prolongation of action time.
Conclusion:
1., cagT gene has been cloned successfully. It is homologous with known A.tumefaciens TFSS structural gene virB7, and is one of the structural genes of TFSS encoded by H.pylori CAG PAI.
2. the prokaryotic expression vector was successfully constructed, the recombinant protein of CagT was obtained, and the Rabbit anti CagT polyclonal antibody was prepared.
3.CagT recombinant protein can induce the expression of IL-8 mRNA. in SGC-7901 cells
The recombinant protein of 4.CagT has a certain inhibitory effect on the proliferation of SGC-7901 cells, and its inhibitory effect gradually increases with the increase of protein concentration and the prolongation of action time.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378.3

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本文編號：1760084