细粒棘球绦虫六钩蚴M26基因的克

发布时间：2018-05-30 04:34

本文选题：六钩蚴 + M26　；参考：《石河子大学》2017年硕士论文

【摘要】：细粒棘球绦虫(Echinococcus granulosus)主要寄生于犬、狼、狐狸等犬科动物终末宿主体内,其虫卵和孕节随粪便排出体外,污染草料和饮水,牛、羊以及人等中间宿主吞食虫卵后感染此病,进入消化道的六钩蚴钻入肠壁经血流或淋巴散布到体内各处,主要在肝和肺脏引起病变导致生长缓慢,严重危害人的健康和畜牧业的快速发展,给世界经济造成严重损失。因此,防治此病在人类健康和畜牧业发展方面非常重要。长期以来,各国研究者都在探索快速诊断此病的方法,但是并未在早期诊断方面取得突破性成果。绵羊是该寄生虫的中间宿主,若在绵羊感染早期,即在六钩蚴进入血液,早期分泌蛋白时诊断出此病,并采取有效治疗措施,将对控制此病流行具有重要意义。目前,包虫病的诊断方式主要通过影像检测(X线射片,CT,超声,MR,DSA/SCA),皮内试验及血象检测等。但这些方法多为后期诊断(即形成包囊后)。而本试验的方法是采用六钩蚴分泌蛋白为抗原通过ELISA法对包虫病进行早期诊断,目前针对包虫病早期诊断的相关研究国内外尚未有过报道。因此选择特异性高的抗原,建立敏感性高以及特异性强的早期诊断方法十分必要。本研究为了探索在感染早期能够检测出此病的抗原,在实验室前期研究基础上选择了六钩蚴差异表达蛋白——副肌球蛋白(M26)作为研究对象,经原核表达制备多克隆抗体和筛选阳性杂交瘤细胞,建立ELISA方法检测感染早期宿主血清中的抗原,即六钩蚴分泌的蛋白。为寻找有效的诊断抗原和建立高效、特异的诊断方法进行了如下试验。1.细粒棘球六钩蚴抗原基因的筛选及原核表达:在本实验室以细粒棘球绦虫的成虫,六钩蚴,原头蚴为材料,通过i TRAQ方法分离检测差异蛋白,其中六钩蚴的差异表达蛋白可作为早期诊断抗原的前期研究基础上,本研究在这些差异表达蛋白中筛选出具有高效免疫保护性的副肌球蛋白(M26),扩增其基因并成功构建克隆载体和表达载体,经原核表达得到大小约为41k Da的重组蛋白,蛋白浓度为0.5 mg/mL,纯度达85%。2.M26多克隆抗体的制备与纯化:以M26为抗原免疫新西兰大白兔,成功制备了M26多克隆抗体,抗体效价为1:12800。经过Western blot验证,有且只有一条带,表明该抗体能够特异性识别M26。采用间接ELISA法利用多克隆抗体检测人工感染绵羊包虫病早期各时间点(5-10h,24 h和48 h)血清中副肌球蛋白,结果表明,当多抗的稀释倍数在1:3200-1:12800时,各时间点的样品孔与阴性孔的数据比值均大于或等于2.1,即为阳性,说明各时间点都有该蛋白。3.M26阳性杂交瘤细胞筛选:以M26作为抗原免疫Balb/c小鼠,经细胞融合、杂交瘤筛选和ELISA法,筛选出2株阳性杂交瘤细胞,通过间接ELISA法利用此细胞株检测人工感染绵羊细粒棘球绦虫早期各时间点(5-10 h,24 h和48 h)血清中副肌球蛋白,在10 h和24 h检测出该蛋白,结果说明此检测方法成功建立,可以表明利用该蛋白作为抗原采用间接ELISA法能够有效诊断出绵羊感染早期包虫病。
[Abstract]:Echinococcus granulosus (Echinococcus granulosus) mainly parasitized in the end host of canine, wolves, foxes and other canine terminal hosts. Its eggs and pregnancy were discharged from the feces, contaminated grass and drinking water, cattle, sheep and human intermediate hosts swallowed the eggs and infected the disease. The six hooks entered the intestinal wall and were drilled into the intestinal wall through blood flow or lymph distribution into the body. All places, mainly caused by the liver and lung disease causing slow growth, serious harm to human health and rapid development of animal husbandry, cause serious loss to the world economy. Therefore, prevention and control of the disease is very important in human health and animal husbandry development. For a long time, researchers in various countries have been exploring the method of rapid diagnosis of this disease, but it has not been found Breakthroughs have been achieved in early diagnosis. Sheep are the intermediate host of the parasite. If six cercariae enter the blood in the early stage of the sheep infection, the disease is diagnosed when the early secretory protein is secreted, and the effective treatment measures will be of great significance to control the epidemic. Shoot, CT, ultrasound, MR, DSA/SCA), intradermal tests and Hemogram detection, but these methods are mostly late diagnosis (that is, after the formation of the capsule). The method of this experiment is to use the six hook secretory protein as the antigen to diagnose hydatid disease by ELISA method, and the related research on the early diagnosis of hydatid disease has not been reported at home and abroad. This study is necessary to select high specific antigen and to establish an early diagnostic method with high sensitivity and specificity. In order to explore the antigen of this disease in the early stage of infection, we chose the differential expression protein of six hooks, the accessory myosin (M26), as the research object and the prokaryotic expression on the basis of the early laboratory study. To prepare the polyclonal antibody and screen positive hybridoma cells, the ELISA method was established to detect the antigen in the serum of the early infected host, that is, the protein secreted by the six cercariae. In order to find an effective diagnostic antigen and establish an efficient and specific diagnostic method, the following experiments were carried out to screen and express the antigen gene of the.1. fine spinous Echinococcus spinous Echinococcus. In the laboratory, the differentially expressed proteins were detected by I TRAQ method with the adult of Echinococcus granulosus, six hook and Echinococcus, and the differential expression protein of six Echinococcus could be used as a preliminary study on the early diagnosis of antigen. In this study, the effective and protective paromyosin (M26) was screened in these differentially expressed proteins. The gene was added and the cloning vector and expression vector were successfully constructed. The recombinant protein of 41K Da was obtained by prokaryotic expression. The protein concentration was 0.5 mg/mL and the purity of 85%.2.M26 polyclonal antibody was prepared and purified. The New Zealand white rabbit was immunized with M26 as antigen, and the M26 polyclonal antibody was successfully prepared. The antibody titer was 1:12800. through Western B. Lot verification, with only one band, shows that the antibody can specifically identify M26. by indirect ELISA method using polyclonal antibody to detect the paromyosin in the serum of artificial infected sheep hydatid disease (5-10h, 24 h and 48 h). The result shows that when the dilution multiple of polyclonal antibody is in 1:3200-1:12800, the sample holes and negative of each time point are negative. The ratio of the data of the holes was greater than or equal to 2.1, that is, the positive, indicating that the protein.3.M26 positive hybridoma cells were screened at all time points: Balb/c mice were immunized with M26 as antigen, and 2 positive hybridoma cells were screened by cell fusion, hybridoma screening and ELISA method, and the artificial infected sheep were detected by the indirect ELISA method. In the early stages of Echinococcus granulosus (5-10 h, 24 h and 48 h), the para myosin was detected in the serum of 10 h and 24 h. The results showed that this detection method was successfully established. It can be shown that the use of this protein as an antigen by indirect ELISA can effectively diagnose early echinococcosis in sheep.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.734

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