猪囊尾蚴单链抗体PE40重组载体构建
发布时间：2018-08-29 12:54
【摘要】： 猪囊尾蚴病是全球性寄生性蠕虫病,发展中国家更为常见,尤其在我国,该病的流行给人民生产和生活带来诸多危害,给人类的健康带来严重的影响。我国至少已有31个省(市、自治区)有过该病的报道,囊尾蚴病患者约1000万,每年屠宰的囊尾蚴病猪约1200万头,直接经济损失约20亿元。脑囊尾蚴病对人类的健康危害极大,在某些发展中国家,脑囊尾蚴病是引起人死亡的主要原因之一。由于抗药性的不断出现以及药物残留等问题的严重性,使得利用免疫预防技术来取代药物成为控制猪囊尾蚴病的必然。免疫毒素是一种导向药物,它将毒素基因和靶向基因融合,表达融合蛋白,具有识别和选择性地破坏受体靶细胞的功能,它由导向部分和毒性部分组成。绿脓杆菌外毒素通常作为重组毒素的毒性成分,其毒性本质是它能够催化真核细胞延伸因子的ADP核糖基化,阻止肽链延长并中断细胞蛋白质的合成而行使细胞毒作用。作为重组毒素的导向部分,除能够与特定靶细胞表面受体结合外,同时能够与毒素基因相融合,表达出融合蛋白,且二者不影响各自独立结构和功能。将猪囊尾蚴头节单链抗体基因与绿脓杆菌外毒素PE40基因连接,装入表达载体pET-28(a)中,以获得重组毒素,研究其对猪囊尾蚴的杀灭作用。 从绿脓杆菌ATCC27853菌株中提取基因组,根据GenBank发表的绿脓杆菌外毒素基因全序列,自行设计引物,分别引入XbaⅠ和HindⅢ酶切位点。采用PCR方法扩增PE40基因,在反应体系中加入10%总体积的甘油,电泳检测,纯化的PCR产物与pMD18-T载体连接构建质粒pMD18-T-PE40,转入大肠杆菌感受态JM109中,蓝白斑筛选阳性克隆菌,提取质粒进行酶切鉴定及PCR鉴定,并测序。质粒pMD18-T-PE40经XbaⅠ和HindⅢ双酶切后电泳显示2692bp和1083bp两条带,测序结果显示扩增的PE40基因共有10个碱基发生改变,变异的碱基引起7个氨基酸发生变化,但PE40肽段的重要活性位点与绿脓杆菌标准产毒株PA103相比均未发生变化。含有ScFv基因的质粒pMD18-T-ScFv已经构建,且ScFv基因两端的酶切位点分别为EcoRⅠ和XbaⅠ,用XbaⅠ和HindⅢ分别对质粒pMD18-T-PE40和pMD18-T-ScFv进行双酶切,利用XbaⅠ和HindⅢ酶切位点将PE40基因与质粒pMD18-T-ScFv相连接,转入大肠杆菌感受态JM109中,挑取阳性菌酶切鉴定。经酶切电泳分析,得到大小约为1800bp的目的片段,与预期结果相同,说明ScFv与PE40已连接成功。用EcoRⅠ和HindⅢ分别对重组质粒pMD18-T-ScFv-PE40和表达载体pET-28(a)进行酶切,将ScFv-PE40转入pET-28(a)表达载体中构建融合毒素pET28-ScFv-PE40,转入大肠杆菌感受态细胞DH5α中,挑取阳性菌落提取质粒,酶切电泳检测,得到目的条带大小约为1800bp,保存菌种,为进一步研究重组免疫毒素对猪囊尾蚴的杀灭作用奠定基础。
[Abstract]:Cysticercosis is a global parasitic worm disease, which is more common in developing countries. Especially in China, the epidemic of cysticercosis has brought a lot of harm to people's production and life, and brought serious impact to human health. At least 31 provinces (municipalities and autonomous regions) have reported the disease. About 10 million cysticercosis patients and 12 million cysticercosis pigs slaughtered each year with direct economic loss of 2 billion yuan. Cerebral cysticercosis is harmful to human health. In some developing countries, cerebral cysticercosis is one of the main causes of death. Due to the continuous emergence of drug resistance and the severity of drug residues, the use of immunoprophylaxis to replace drugs has become a necessity for the control of cysticercosis. Immunotoxin is a kind of targeted drug, which fuses toxin gene and target gene, expresses fusion protein, has the function of recognizing and selectively destroying receptor target cells, and it consists of directed and toxic parts. Pseudomonas aeruginosa exotoxin is usually used as the toxic component of recombinant toxin. Its toxic essence is that it can catalyze the ADP ribosylation of eukaryotic cell extension factor, prevent peptide chain from prolonging and interrupt the synthesis of cellular protein. In addition to binding to the surface receptors of specific target cells, the recombinant toxin can be fused with the toxin gene to express the fusion protein, and they do not affect their independent structures and functions. The single chain antibody gene of cysticercus cephalosporium was linked with the PE40 gene of Pseudomonas aeruginosa and inserted into the expression vector pET-28 (a) to obtain the recombinant toxin and to study its killing effect on cysticercus cellulosae. The genome was extracted from Pseudomonas aeruginosa ATCC27853 strain. According to the whole sequence of exotoxin gene of Pseudomonas aeruginosa published by GenBank, primers were designed to introduce Xba 鈪，
本文编号：2211274
[Abstract]:Cysticercosis is a global parasitic worm disease, which is more common in developing countries. Especially in China, the epidemic of cysticercosis has brought a lot of harm to people's production and life, and brought serious impact to human health. At least 31 provinces (municipalities and autonomous regions) have reported the disease. About 10 million cysticercosis patients and 12 million cysticercosis pigs slaughtered each year with direct economic loss of 2 billion yuan. Cerebral cysticercosis is harmful to human health. In some developing countries, cerebral cysticercosis is one of the main causes of death. Due to the continuous emergence of drug resistance and the severity of drug residues, the use of immunoprophylaxis to replace drugs has become a necessity for the control of cysticercosis. Immunotoxin is a kind of targeted drug, which fuses toxin gene and target gene, expresses fusion protein, has the function of recognizing and selectively destroying receptor target cells, and it consists of directed and toxic parts. Pseudomonas aeruginosa exotoxin is usually used as the toxic component of recombinant toxin. Its toxic essence is that it can catalyze the ADP ribosylation of eukaryotic cell extension factor, prevent peptide chain from prolonging and interrupt the synthesis of cellular protein. In addition to binding to the surface receptors of specific target cells, the recombinant toxin can be fused with the toxin gene to express the fusion protein, and they do not affect their independent structures and functions. The single chain antibody gene of cysticercus cephalosporium was linked with the PE40 gene of Pseudomonas aeruginosa and inserted into the expression vector pET-28 (a) to obtain the recombinant toxin and to study its killing effect on cysticercus cellulosae. The genome was extracted from Pseudomonas aeruginosa ATCC27853 strain. According to the whole sequence of exotoxin gene of Pseudomonas aeruginosa published by GenBank, primers were designed to introduce Xba 鈪，
本文编号：2211274
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