幽门螺杆菌cagT基因的克隆表达及重组蛋白对诱导SGC-7901细胞炎症因子表达和细胞增殖的影响

发布时间：2018-04-16 18:26

  本文选题：幽门螺杆菌 + cagT　； 参考：《江苏大学》2008年硕士论文

【摘要】： 目的:探讨cagT基因在幽门螺杆菌(Helicobacter pylori,H.pylori)cag致病岛(cag pathogenicity island,cag PAI)编码的Ⅳ型分泌系统(typeⅣsecretion system,TFSS)装置中的作用;并通过与胃癌细胞相互作用,研究其重组蛋白对胃癌细胞分泌炎症因子的能力和增殖的影响,为进一步阐明H.pylori的致病机制奠定基础。 方法:根据GenBank收录的H.pylori 22695全基因组序列,利用生物学软件Primer Premier5.0设计扩增cagT基因的引物,并引入限制性核酸内切酶酶切位点;应用PCR技术从H.pylori NCTC 11637基因组DNA中扩增cagT编码基因片段后,克隆至pGEM-T载体,转化大肠埃希菌(Escherichia coli,E.coli)DH5α,双酶切鉴定筛选阳性克隆,进行序列测定。应用生物信息学技术,对cagT基因编码的氨基酸序列与已知的根癌农杆菌(Agrobacterium tumefaciens,A.tumefaciens)TFSS结构基因virB7进行同源性比对,并对其编码的蛋白质做理化特性的预测分析。再将其定向插入原核表达载体pQE30中,转化至表达宿主菌E.coli M15,通过氨苄青霉素抗性筛选和双酶切鉴定阳性克隆。IPTG诱导其表达,表达的蛋白经Western blot鉴定,并以Ni~(2+)-NTA树脂进行纯化。纯化后的重组蛋白免疫家兔,制备抗CagT多克隆抗体,酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)检测血清抗体效价。重组蛋白与SGC-7901细胞作用一定时间,提取细胞总RNA,采用逆转录聚合酶链反应(reversetranscriptase polymerase chain reaction,RT-PCR)检测IL-8 mRNA的表达;并用四甲基偶氮唑蓝(methyl thiazoly tetrazolium,MTT)法检测蛋白对细胞增殖的影响。 结果: 1.H.pylori NCTC 11637 cagT基因全长843bp(GenBank登录号为EF114758),编码280个氨基酸,与H.pylori其它菌株基因序列的核苷酸同源性为96%～99%;与A.tumefaciens的VirB7具有同源性;DNAStar软件预测其编码的蛋白质的相对分子量为32.33kDa,具有较强的免疫原性。 2.成功构建pQE30-cagT原核表达重组质粒,确定IPTG终浓度0.8mmol/L,温度30℃,诱导时间4h为最佳诱导条件,Western blot检测结果显示该重组蛋白可与抗His标签抗体结合反应,经Ni~(2+)-NTA纯化后可获得纯度约为98%的CagT重组蛋白,相对分子质量(Mr)约为32kDa,与预测结果一致。CagT重组蛋白免疫家兔,获得抗CagT多克隆抗体,其效价为1:1.6×10~4。 3.终浓度10μg/mL的CagT重组蛋白作用于SGC-7901细胞4h后,可扩增出IL-8片段。 4.不同浓度的CagT重组蛋白与SGC-7901细胞作用6、24及48h后,细胞增殖的程度随着CagT重组蛋白浓度的增加和作用时间的延长而逐渐降低。 结论: 1.成功克隆了cagT基因,其与已知的A.tumefaciens的TFSS结构基因virB7具有同源性,是H.pylori cag PAI编码的TFSS的结构基因之一。 2.成功构建了原核表达载体,获得了CagT重组蛋白,并制备了兔源性抗CagT多克隆抗体。 3.CagT重组蛋白可诱导SGC-7901细胞表达IL-8 mRNA。 4.CagT重组蛋白对SGC-7901细胞的增殖有一定抑制作用,其抑制作用随着蛋白浓度增加和作用时间延长而逐渐增强。
[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.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.3

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