日本血吸虫硫氧还蛋白谷胱甘肽还原酶（SjTGR）的克

发布时间：2019-03-01 21:21

【摘要】：血吸虫病(schistosomiasis)是一种危害严重、分布广泛的人畜共患病。半个多世纪以来,我国血吸虫病防治取得了举世瞩目的成就,但由于重复感染严重,单靠治疗药物不能控制血吸虫病的流行,加强血吸虫病疫苗候选分子的筛选和疫苗的研制开发无疑是血吸虫病防治的重要举措。硫氧还蛋白谷胱甘肽还原酶是血吸虫体内一种特殊的氧化还原酶类,对保护血吸虫不受宿主免疫系统和自身新陈代谢而产生的游离氧的损害具有重要作用。本研究以SjTGR为研究对象,开展了该基因的克隆、表达及重组蛋白的免疫保护效果的研究,为深入探讨SjTGR的生物学功能,评估其作为疫苗候选分子和药靶的应用前景提供了基础。本实验室前期应用biotin-avidin分析日本血吸虫成虫表面蛋白质组时发现,SjTGR蛋白也出现在虫体表面,推测SjTGR在血吸虫中具有重要功能。本研究鉴于此发现,并参考SmTGR基因的扩增方法,获得SjTGR的全长cDNA序列。通过生物信息学分析,发现SjTGR基因的ORF为1791bp,编码596个氨基酸,理论分子量为64940.25Da,理论等电点为6.38, SjTGR和SmTGR的氨基酸序列相似性为82%。本研究成功地构建了重组表达质粒SjTGR-pET-28a和SjTGR-pET-32a,两种重组蛋白都在E.coli(DE3)中以可溶性蛋白形式表达,分子量分别69kD和72kD, Western blotting分析表明两种重组蛋白具有较好的抗原性和免疫原性。用纯化的SjTGR-pET-28a和SjTGR-pET-32a重组蛋白免疫BALB/c小鼠,结果与空白对照组相比,重组蛋白SjTGR-pET-28a在小鼠中分别诱导了91.24%的减虫率和93.37%的肝脏减卵率,差异极显著(p0.01)。重组蛋白SjTGR-pET-32a诱导了42.78%的减虫率和41.29%的肝脏减卵率,差异显著(p0.05)。用ELISA方法检测小鼠血清中特异性IgG抗体水平变化,结果表明,重组蛋白可诱导小鼠体内抗重组抗原的特异性IgG抗体迅速产生,并且维持在一个较高的水平。表明该重组蛋白具有发展为抗血吸虫病候选疫苗和新药靶的潜力及深入研究的价值。提取7d、14d、21d、28d、35d、42d和42d雌雄各个时期的虫体蛋白,并用新鲜虫体制作冰冻切片,以α-tublin为内参,通过Western blotting检测各个时期虫体中TGR蛋白的表达情况；经过免疫荧光检测方法,对该蛋白的表达部位进行了分析。同时,以看家基因α-tublin为内参,应用荧光定量PCR技术分析TGR基因在日本血吸虫各个时期虫体内的转录情况。结果显示,TGR蛋白在日本血吸虫各个虫体中均有表达,表达部位主要在体被表膜；该基因在日本血吸虫感染宿主后7d、14d、21d、28d、35d、42d虫体及42d雌虫和雄虫内均有转录,在35d-42d虫体的表达量较高,雌虫表达量高于雄虫。在35d到42d这个阶段,虫体发育成熟并开始产卵,新陈代谢比较旺盛,虫体产生的活性氧增多,TGR的高表达量可能与减少活性氧对虫体的损害、与血吸虫的生长发育和繁殖相关。本研究为深入研究SjTGR基因的生物学功能奠定了基础,为筛选新的血吸虫病疫苗候选分子和药物靶标提供了新思路
[Abstract]:Schistosomiasis is a serious and widely distributed man-and-animal disease. Since more than half a century, the prevention and control of schistosomiasis in China has made great achievements, but because of the serious repetition of the infection, the treatment of the medicine alone cannot control the epidemic of the schistosomiasis, The research and development of the screening and vaccine for strengthening the candidate molecule of the schistosomiasis vaccine is undoubtedly an important measure of the prevention and control of the schistosomiasis. The thioredoxin glutathione reductase is a special redox enzyme in the body of the schistosome, and has an important role in protecting the schistosome from the damage of the free oxygen produced by the host immune system and the metabolism of the host. In order to study the biological function of SjTGR, this study provided a basis for exploring the biological function of SjTGR, and to evaluate its application prospect as a candidate molecule and target. It was found that the SjTGR protein also appeared on the surface of the worm body, and it was suggested that the SjTGR had important work in the schistosome. The full-length cDNA sequence of SjTGR was obtained in view of this finding and with reference to the amplification method of the SmTGR gene. The amino acid sequence similarity of SjTGR gene was found to be 1791 bp, encoding 596 amino acids, the theoretical molecular weight was 64940.25 Da, the theoretical isoelectric point was 6.38, and the similarity of the amino acid sequence of SjTGR and SmTGR was 82. %. The recombinant expression plasmid SjTGR-pET-28a and SjTGR-pET-32a were successfully constructed in this study. Both of the two recombinant proteins were expressed in the form of soluble protein in E. coli (DE3). The molecular weight was 69 kD and 72 kD, respectively. Western blotting analysis indicated that the two recombinant proteins had better antigenicity and immunogen. The recombinant protein SjTGR-pET-28a and SjTGR-pET-32a recombinant protein were used to immunize the BALB/ c mice. The recombinant protein SjTGR-pET-32a induced 42.78% of the reduction rate and 41.29% of the liver egg reduction rate, and the difference was significant (p0.05). The results showed that the recombinant protein could induce the rapid production of the specific IgG antibody against the recombinant antigen in the mouse, and it was maintained at a higher water level. It shows that the recombinant protein has the potential to develop the candidate vaccine for anti-schistosomiasis and the target of new drug, and the price of the in-depth study. The expression of TGR was detected by Western blotting, and the expression of TGR was detected by Western blotting. the measuring method is carried out on the expression part of the protein, The effects of TGR gene on the body of Schistosoma japonicum in various stages of Schistosoma japonicum were analyzed by using fluorescence quantitative PCR (PCR) as an internal reference at the same time. The results showed that the TGR protein was expressed in the various insect bodies of Schistosoma japonicum, and the expression site was mainly expressed in the surface of the body. The gene was transcribed in 7 days,14 days,21 days,28 days,35 days,42 days, and 42 days after the infection of the host of Schistosoma japonicum. the amount of the female worm is high, the expression amount of the female worm is high, in this stage of 35d to 42d, the insect body is mature and the oviposition is started, the metabolism is more vigorous, the active oxygen generated by the insect body is increased, the high expression quantity of the TGR may be related to the reduction of the damage of the active oxygen to the worm body, the development and the propagation of the schistosome, The study provides a basis for studying the biological function of the SjTGR gene and provides the candidate molecular and drug target for screening new schistosomiasis vaccine.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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