【作者】 韩冬梅；

【导师】 仲飞；

【作者基本信息】 河北农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 大肠杆菌不耐热肠毒素(heat-labile enterotoxin, LT)是肠产毒性大肠杆(Enterotoxigenic Escherichia coli,ETEC)分泌的一种热不稳定肠毒素,LT是由一个具有毒性的A亚单位(LTA)和形成环状的五个能够与神经节苷脂GM1结合而粘附于真核细胞膜上的B亚单位(LTB)组成。能引起人和其他哺乳动物的水样腹泻。LT除具有毒性外,还能够有效的启动局部及全身的体液和细胞免疫,具有很强的粘膜免疫原性和粘膜佐剂活性,在粘膜免疫研究中以及粘膜免疫疫苗开发中具有重要医学价值和经济价值。为制备大量具有免疫活性的LT蛋白,本实验通过提取产肠毒素大肠杆菌44815菌株的基因组,采用PCR方法分别扩增不耐热肠毒素A亚基(LTA)和B亚基(LTB)的编码基因,并将其连接在pMD18-T载体上,构建成LTA和LTB基因的重组载体pMD18-T-LTA和pMD18-T-LTB。阳性克隆筛选采用蓝白斑选择和限制性内切酶XhoⅠ和NcoⅠ消化,最后进行序列测定。然后采用定点突变方法将LTA的第63位的丝氨酸改变为赖氨酸,构建成pMD18-T-LTAK63突变体。再分别将pMD18-T-LTAK63和pMD18-T-LTB通过XhoⅠ和NcoⅠ位点插入到pET-20b(+)原核表达载体,分别构建成LTA和LTB的原核表达载体pET-20b-LTAK63和pET-20b-LTB。将重组LTA和LTB表达载体分别转化宿主菌Bl21,制备工程菌进行表达。表达产物采用SDS-PAGE方法检测,目的蛋白分离纯化采用镍琼脂糖胶粒吸附法。为鉴定带有人CD5信号肽的LTAK63基因能否在真核细胞中表达,以pMD18-T-LTAK63为模板,通过PCR扩增LTAK63基因,将LTAK63基因连接在pcDNACD5sp真核表达载体上,构建成LTAK63重组质粒pcDNACD5sp-LTAK63。利用磷酸钙方法将pcDNACD5sp-LTAK63转染人胚胎肾细胞HEK 293T细胞使其瞬时表达。本研究构建的重组质粒pMD18-T-LTA和pMD18-T-LTB的测序结果表明:所克隆的LTA基因与GenBank中大肠杆菌标准产毒株LTA基因的大小完全一致,有4个碱基发生了改变,同源性为99%。在这4个突变中有3个是无义突变,不造成氨基酸的改变,但有1个碱基的突变使谷氨酸变为赖氨酸。可见LTA基因存在着多态性;所克隆的LTB基因与GenBank中大肠杆菌标准产毒株LTB基因的完全一致,同源性为100%。利用定点突变方法构建的pMD18-T-LTAK63突变体的测序结果表明LTA的第63位的丝氨酸成功的改变为赖氨酸。构建的原核表达载体经XhoⅠ和NcoⅠ双酶切,得到了符合预期大小的特异性片段,结果证明pET-20b-LTAK63和pET-20b-LTB原核表达载体构建成功。SDS-PAGE检测宿主菌Bl21表达目的蛋白的结果显示:在细胞周质中有目的蛋白表达,分别在分子量约为28Kda(LTA)及14Kda(LTB)处有一条明显的带。而在细胞的培养基及包涵体中未见目的蛋白表达。同时通过镍吸附法纯化得到了单一目的蛋白。结果表明LTAK63和LTB在Pel信号肽的引导下进行了表达并分泌到细胞周质,而不是以包涵体形式存在,因此表达目的蛋白具有一定的活性,且易于分离纯化。在LTAK63基因进行真核表达的基础上构建的pcDNACD5sp-LTAK63真核表达载体转染HEK293T细胞,Western blot检测结果显示在分子量约为34.8KDa处有一条明显的杂交带,而空载体在相应位置未见杂交带。结果证明pcDNACD5sp-LTAK63真核表达载体构建成功,并实现了它在真核细胞中的分泌性表达。更多还原

【Abstract】 Escherichia coli heat-labile enterotoxin (heat-labile enterotoxin, LT) was the intestinal toxicity secreted by Enterotoxigenic Eshchirichia coli (ETEC). LT was composed of subunit A (LTA) and subunit B, the former has toxicity; the latter formed five rings which can bind GM1 ganglioside on the membrane of eukaryotic cell. LT can cause diarrhea in human and other mammals. In addition to toxicity, LT was also able to trigger humoral immunity and cellular immunity, so it has the strong immunogenicity and adjuvanticity in mucous membrane and has important medical value in the study of mucosal immunity and in vaccine development.In order to prepare LT proteins at large scale with immunocompetence, the genes encoding of heat-labile enterotoxin A subunit (LTA) and the B subunit (LTB) were amplified with PCR method from the genomic DNA of Enterotoxigenic Escherichia coli strain 44815. The amplified LTA and LTB genes were inserted into pMD18-T vector to construct pMD18-T-LTA and pMD18-T-LTB recombinant plasmids, respectively. The positive clones were identified through the blue-white screening system and double-enzyme digestion(Nco I and Xho I), vertified by sequencing. The LTA gene was modified by changing Ser63 to Lys63 with Site-directed mutagenesis on the pMD18-T-LTA recombinant plasmid. LTAK63 and LTB genes were transfered into the pET-20b (+) prokaryotic expression vectors respectively through Nco I and Xho I sites and construct the prokaryotic expression vectors of pET-20b-LTAK63 and pET-20b-LTB. Recombinant expression vectors of LTA and LTB were transformed into host bacteria Bl21 respectively for preparation of engineering bacteria. The expression products were detected by SDS-PAGE and the interest protein was separated and purified by nickel-agarose beads. In order to construct LTAK63 gene eukaryotic expression vector, the LTAK63 gene with NheI and ApaI sites was amplified by PCR using pMD18-T-LTAK63 plasmid as a template. The gene was ligased with pcDNACD5sp by NheI and ApaI sites to construct the recombinant plasmid, pcDNACD5sp-LTAK63. To identify whether LTAK63 gene with human CD5 signal peptide can express in eukaryotic cells, HEK 293T cells were applied to transiently express LTAK63 gene with the calcium phosphate transfection method.The experimental results showed that the LTA gene amplified by PCR was consistent with the Gene Bank published sequence and the homology was 99%. Four bases of LTA gene were changed, but only one base results amino acid substitution from glutamic acid to Lysine. Therefore LTA gene has polymorphism. LTB gene amplified was in coincidence with the published in GenBank. Sequence results showed that Ser63 of LTA was changed to Lys63 successfully. The constructed prokaryotic expression vectors, pET-20b-LTAK63 and pET-20b-LTB, were correct by identification with Xho I and NcoⅠdigestion. The two proteins (MW of 28 kDa (LTA) and 14kDa (LTB)) were detected in shock–release sulution by SDS-PAGE, which indicated that the two interest proteins were expressed as secretive form. Purified protein by nickel adsorption showed the single band by SDS-PAGE. All illustrated that the LTAK63 and LTB were mediated by signal peptide Pel , can make the LTAK63 and LTB expressed and secreted to the periplasm, rather than existing in form of inclusion body, so the expressed protein has activity. and separation and purification were easy. Western blot results showed that there was a clear hybrid band at about 34.8KDa, but at the corresponding location of empty vector there was no hybrid band. Therefore the eukaryotic expression vector pcDNACD5sp-LTAK63 was constructed successfully, the secretive expression for LTA63 gene was achieved in eukaryotic cells.更多还原