日本血吸虫表膜蛋白SjOST48的初步研究

发布时间：2018-03-27 23:09

本文选题：日本血吸虫　切入点：SjOST48基因　出处：《中国农业科学院》2015年硕士论文

【摘要】：日本血吸虫病是我国重点防控的人畜共患寄生虫病,对我国人民的身体健康和畜牧生产造成了重大损害。防治实践表明吡喹酮只针对成虫有效且不能避免重复感染,单靠吡喹酮药物治疗不能有效控制血吸虫病,因此,筛选、鉴定高效且安全的血吸虫病疫苗成为血防工作者们的努力方向。体被表膜蛋白被证明是血吸虫潜在的疫苗和药物靶标,具有很好的研究价值。本实验室前期在开展日本血吸虫体被蛋白组研究时鉴定到了SJCHGC01743蛋白,本文对该蛋白编码基因进行了克隆、表达,对其基本特性和生物学功能进行了初步的研究。为了鉴定日本血吸虫SJCHGC01743基因并评估其重组蛋白作为新的血吸虫病候选疫苗抗原的潜力,本研究利用PCR技术首次扩增了该基因,获得了1248 bp的cDNA序列。同源性分析结果显示,该基因为日本血吸虫寡糖转移酶OST48亚基,命名为SjOST48。实时定量PCR分析显示SjOST48在检测的童虫和成虫各个期别虫体中均有转录,其中在28 d虫体中的转录水平最高,在42 d雌虫中的转录水平显著高于雄虫。构建了重组表达质粒pET-28a(+)-SjOST48,并在大肠杆菌中成功表达,重组蛋白rSjOST48分子量约50 kDa。应用重组蛋白rSjOST48免疫BALB/c小鼠制备免疫血清,Western-blot分析表明rSjOST48具有较好的免疫原性。间接免疫荧光试验表明SjOST48蛋白主要分布于虫体体被,少量分布于实质。ELISA检测结果表明rSjOST48能诱导产生较高的特异性IgG、IgGl和IgG2a抗体。两次动物免疫保护试验结果表明SjOST48分别诱导小鼠产生了21.58%-32.62%(P0.05)的减虫率和50.94%-57.61%(P0.01)的肝脏虫卵减少率。为了探究SjOST48基因的生物学功能,本研究利用RNAi技术对该基因进行了初步研究。根据SjOST48核苷酸序列,设计了4对针对该基因的特异性siRNAs,通过浸泡法进行体外筛选,获得一体外干扰效果较好的siRNAs (siRNA-713),与无关对照组相比,siRNA-713浸泡组SjOST48的转录水平下降了40.00%。利用尾静脉注射法将siRNA-713导入感染血吸虫的小鼠体内进行体内干扰实验,结果显示,7次注射siRNA-713后,与无关对照组相比,siRNA-713干扰组小鼠体内的虫荷数减少了29.50%,肝脏虫卵数减少了30.08%,表明日本血吸虫表膜蛋白SjOST48可能对血吸虫的生长发育具有重要作用。本研究为深入探讨日本血吸虫SjOST48基因的生物学功能及筛选新的血吸虫病疫苗候选分子奠定了基础。
[Abstract]:Schistosomiasis japonicum is one of the most important zoonotic parasitic diseases in China, which has caused great harm to the health of our people and livestock production. The control practice shows that praziquantel is only effective against adults and can not avoid repeated infection. Praziquantel alone cannot control schistosomiasis effectively, so screening, Identification of highly efficient and safe schistosomiasis vaccine has become a major task for schistosomiasis control workers. Surface membrane proteins have been shown to be potential vaccine and drug targets for schistosomiasis. SJCHGC01743 protein was identified in the early stage of the study of Schistosoma japonicum, and the gene encoding the protein was cloned and expressed. In order to identify the SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of its recombinant protein as a new candidate vaccine antigen of schistosomiasis japonicum, the gene was amplified by PCR technique for the first time. A 1248 BP cDNA sequence was obtained. Homology analysis showed that the gene was a Schistosoma japonicum oligosaccharide transferase OST48 subunit named SjOST48.The real-time quantitative PCR analysis showed that SjOST48 was transcribed in all phases of the detected juvenile and adult worms. The transcriptional level of pET-28a (SjOST48) was the highest in 28 d and significantly higher in female than in male at 42 d. The recombinant expression plasmid pET-28a (SjOST48) was constructed and successfully expressed in Escherichia coli. The molecular weight of recombinant protein rSjOST48 was about 50 kDa.Western-blot analysis showed that rSjOST48 had good immunogenicity by immunizing BALB/c mice with recombinant protein rSjOST48. Indirect immunofluorescence assay showed that SjOST48 protein was mainly distributed in the body of the worm. The results of Elisa showed that rSjOST48 could induce the production of high specific IgGG l and IgG2a antibodies. The results of two animal immunoprotection tests showed that SjOST48 induced mice to produce the worm reduction rate of 21.58 -32.62% (P0.05) and the decrease of liver eggs of 50.94-57.61% (P0.01), respectively. To explore the biological function of the SjOST48 gene, In this study, RNAi technique was used to study the gene. According to the nucleotide sequence of SjOST48, four pairs of siRNAs-specific genes were designed and screened in vitro by immersion method. A siRNAs siRNA-713 was obtained with good interference effect in vitro. Compared with the unrelated control group, the transcription level of SjOST48 in the siRNA-713 immersion group was decreased by 40.00.The siRNA-713 was introduced into the mice infected with Schistosoma japonicum by caudal vein injection for in vivo interference experiment. The results showed that after 7 times of siRNA-713 injection, Compared with the unrelated control group, the number of worm load and the egg number of liver decreased by 29.50 and 30.08 respectively, indicating that SjOST48 may play an important role in the growth and development of Schistosoma japonicum. The biological function of SjOST48 gene of Schistosoma japonicum and the screening of new vaccine candidate molecules for schistosomiasis japonicum have been established.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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