【作者】 朱建国；

【导师】 蔡幼民；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2001， 博士

【摘要】 目的及意义 血吸虫病（Schistosomiasis）为世界性分布、危害严重的人畜共患的寄生虫病。血吸虫（Schistosoma）在吸虫中独特的雌雄异体形式，使得雌雄虫在生殖生理方面表现出复杂的功能差异。雌雄合抱是血吸虫雌虫性成熟的前提，而雌虫性成熟产卵又是主要致病因素。因此，控制血吸虫性成熟生殖是防制血吸虫病的重要关键，其中对血吸虫性别特异性基因及其功能的研究，已引起了研究者广泛重视。这些研究不仅对阐明血吸虫发育及生殖机理具有重要理论意义，而且对抗血吸虫生殖免疫及研制有效防治新药也具有重要的实践意义。但总体来讲，该领域的研究积累尚少，所获得的基因数量有限，对其功能的研究则有待深入。尤其是对国内流行的日本血吸虫中国大陆株的研究更需要展开。本研究以研究日本血吸虫性别特异性基因及其功能为主要目的，从分析比较日本血吸虫中国大陆株雌雄虫之间蛋白质组成的差异性入手，鉴定雌雄虫特异性蛋白质；以此为线索克隆日本血吸虫性别特异性基因并对其免疫保护功能进行较为系统的研究，为从分子水平上揭示血吸虫性别发育、成熟及其生殖机制和探索防制新途径莫定一定的基础。 方法及结果 首先分别利用SDS-PAGE和双向电泳技术对日本血吸虫中国大陆株雌雄成虫虫体蛋白质进行分析研究。软件分析SDS-PAGE结果显示，有2处（79kDa、43kDa）雄虫蛋白质相对含量明显高于雌虫，另有4处（35kDa、28kDa、25kDa、22kDa）雌虫蛋白质的相对含量明显高于雄虫；双向电泳分析进一步查明了血吸虫雌雄虫蛋白质组成的诸多差异。在43kDa，pI5.60-5.90处雄虫有一条长度和宽度均超过雌虫，由多个斑点连在一起组成的条带；雌虫则有7个特有斑点（38kDa，pI5.71；38kDa，pI5.65；35kDa，pI5.7O；35kDa，pI5.58；32kDa，pI5.72；22kDa，pI5.45；18kDa，pI5.40处）；在28kDa，pI6.90-7.35处，雌雄虫各有3个斑点，但雄 虫较雌虫的斑点大；在28kD左右，pl4．80乃．50处，有许多斑点组成的着 色区，但雌虫的着色程度较雄虫深得多。该项研究揭示了日本血吸虫雌 雄虫之间虫体组成蛋白质明显的差异性，为开展血吸虫性别特异性基因的 研究积累了有益基础。 SDS－PAGE及双向电泳结果在43kD处蛋白质显示雌雄差异。以此为． 线索，检索查得编码曼氏血吸虫肌动蛋白的 CDNA（ShactZ夕，据此设 计一对引物，以日本血吸虫中国大陆株成虫InRNA为模板，用RT．PCR 扩增出一 11 31 hP的 CDNA片段。测序结果表明该片段为编码日本血吸虫 肌动蛋白基因的完整阅读框 CDNA，与 ShaCtZ cDNA序列同源性为 92％，命名为SjAct。将其克隆到表达载体pET28atO，在大肠杆菌中获 得了高效表达，SDSIAGE分析估计表达产物（伽ct）分子量约43kDa。用 兔抗日本血吸虫成虫粗抗原血清进行Western印迹分析，在预测位置出现 了明显的识别条带，说明咖Ct具有良好的抗原性。用巾Ct免疫昆明系 小鼠，攻击血吸虫尾抱，7周后剖杀小鼠，进行成虫和虫卵计数，其减虫一 率为28．40，肝脏减卵率为29．2O，和对照组比较，P刃．05差异显著，显示 了血吸虫肌动蛋白一定的免疫保护功能。 SDS．PAGE结果还显示在约79all处雄虫蛋白质的相对含量高于雌 虫，检索查得曼氏血吸虫 CDNA片段（SmGC），该片段编码曼氏血吸虫 抱雌沟分泌蛋白质79kDa肽段。根据SmGCP保守区片段设计一对引物， 以日本血吸虫中国大陆株成虫mRNA为模板，用RT．PCR法扩增出一大 小为868hp的基因片段。经测序证明该片段为编码日本血吸虫抱雌沟蛋 白基因保守区的。DNA，与SmGCP相应片段碱基同源性86石％，命名为 SjGCPj，编码的氨基酸序列同源性为 84．l％。将其克隆到表达载体． pET28归中，在大肠杆菌中获得了较高量的表达，SDS｛AGE分析估计表 达产物（均GCP八分子量约 29kD。利用兔抗日本血吸虫成虫粗抗原血 清进行 Western检测，在预测位置出现了明显的识别条带，说明 TSjGCPI 2 具有良好的抗原性。用TSjGCP］免疫昆明系小鼠，攻击尾蛐，7周后剖杀 小鼠，进行成虫和虫卵计数，其减虫率为35．2％，肝脏减卵率为37．8％，与 对照组比较，Pm刀5差异显著，表明血吸虫抱雌沟蛋白具有一定的免疫保 护功能。 为进一步研究 SjGCP］的核酸免疫特性，将其克隆到真核表达载体’ PCDNA3中，得到的重组质粒直接免疫昆明系小鼠，设不含 SjGCP］的 pCDNA3质粒为对照以进行效果比较。自免疫开?更多还原

【Abstract】 [Objective] Schistosome parasites are unique among the trematodes that the sex are separate. The development and fertility of the female Schistosome is completely dependent on continuous pairing with male .Unpaired female remain stunted and sexually immature .lnterested in this phenomenon, the study attention in this field have been focused on interaction between the male and female worm. In an effort to shed more light on the molecular mechanism of development and sexually mature of Schistosoma and the effective measure for the prevention of Schistosomiasis , our research aim is to study the sexspecific genes adult worm of Schistosoma japonicum (Chinese strain) and further to investigate their protective immunity.[Methods and Resultsl The study on sex-differentiation of proteins between male and female adult worm of Schistosoma was carried out firstly SDSPAGE -and Two-electrophoresis were used to analyse the sex difference of protein. The SDS-PAGE revealed that female has more proteins than male, but the amount of two molecular(79kDa~. 43kDa) are much more in male than those in female .The female,by comparison, has more amount of four protein polypeptides(35kDa 28kDa ~. 25kDa 22kDa) .The significant sexdifference of proteins were further detected by two-dimensional electrophoresis .A smear consisting of several dots with an MW of 43kDa and a isoelectric point (p1) of 5.60 ?5.90 is longer in the male than that in the female. The female has at least 7 specific molecular(38kDa,pI 5.71; 38kDa,pI 5.65; 35kDa,pI 5.70; 35kDa,pI 5.58; 32kDa,p15.72; 22kDa, p1 5.45; l8kDa,pI5.40)which have not been find in male worms .It shown that sex-differentiation of proteins between male and female adult worm of Schistosoma japonicum is significant.109Based on the result of the above, a pair of primers were designed according to the published SmAct2 to amplify a 1131 bp eDNA fragment by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNASequence analysis indicated that this fragment, named SjAct, with 92% homology to SmAct2, was a complete ORF of eDNA encoding actin of Schistosomajaponicum (Chinese strain). SjAct was cloned into the expression vector pET28a(+) and subsequently expressed in Escerichia co/i. SDS-PAGE, revealing that the expression of the product, named rSiA ci, was very effective. Probed with the rabbit serum immunized against Sj worm antigen preparation, Western Blotting showed that rSiAct has good antigencity. In order to observe the immunity protection induced by the recombinant protein, further study was performed by vaccinating Kuming mice three times with rSjAct. The mice was then challenged with 40 cercariae of Sj I week after the last injection and perfused 7 weeks post-challenge. The reduction rate of worm and liver egg were 28.4% and 29.2% respectively , indicating the significantly protection in animal immunized with rSjA ci.We also designed the primers on the basis of published SmGCP encoding Gynecophoral canal protein, and a 868 bp eDNA fragment was amplified by RT-PCR from adult Schistosomajaponicum (Chinese strain) mRNA .Sequence analysis indicated that this fragment, named SjGCPJ, was a conserved region of the gene encoding Gynecophoral canal protein of Schistosoma japonicum (Chinese strain), and the sequence was 86.6% homology to SmGCP, SjGCP1 was also cloned into the expression vector pET28a(+) and subsequently expressed in Escerichia co/i. . SDS-PAGE revealed that the molecular weight of this expressed product ,named rSjGCP1, was around 29kD. Western Blotting shown that S]GCP1 was recognized by the rabbit serum immunized against SI worm antigen preparation The recombinant protein was used to vaccinate Kuming mice .The vaccinated mice were then challenged with 40 cercariae of 5] and perfused 7 weeks after the challenge. 3 5.2% worm reduction rate and11037.8% liver Egg reduction rate were obtained in the vaccinated mice comparing with the control group. In order to study the immunity effect of Nuclie acid vaccination of S1GCP1, SIGC更多还原