【作者】 刁薇；

【导师】 曹建平；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2008， 硕士

【摘要】 日本血吸虫病(schistosomiasis japonica)作为人兽共患寄生虫病,在我国仍然是一个重要的公共卫生问题。现阶段我国实行的是“以控制传染源”为主、综合治理的血吸虫病防治策略。其中,吡喹酮群体化疗是目前血吸虫病防治措施的基础,但血吸虫病仍在向新的地区蔓延,并且吡喹酮化疗也有一定的局限性,群体化疗并不能防止重复感染,还可能会产生吡喹酮抗性株。因此,在血吸虫病防治策略中,疫苗策略已被认为将是吡喹酮化疗措施的重要补充。目前已有酶性、肌性、膜相关性等多种疫苗候选抗原的基因得到了克隆和表达,并且开展了动物保护性实验,虽然取得了一定的预期效果,但继续寻找新的候选抗原分子以及提高候选疫苗分子的免疫原性仍是血吸虫病疫苗研究的重要方向,通过免疫筛选cDNA文库去发掘更有效的疫苗候选分子是获得新的候选抗原分子的有效途径。发展疫苗的主要目的是降低虫荷和虫卵在肝组织的沉积,因此疫苗的效应应多针对童虫阶段和成虫产卵。东方田鼠(Microtus fortis,Mf)是一种感染日本血吸虫后不致病的哺乳类动物。血吸虫童虫可能是宿主免疫系统较为适合的靶子,若能找到童虫阶段的特异性抗原分子,将血吸虫消灭在此阶段或阻止其生长、发育、成熟、产卵和致病,这不仅可以减轻血吸虫病所造成的病理损害,还可有效地阻止其传播。因此用东方田鼠血清免疫筛选日本血吸虫童虫cDNA文库中,以寻找相关的疫苗候选分子,可能会获得令人满意的结果,并为东方田鼠天然抗日本血吸虫机制的研究提供信息。精氨酸甲基化在血吸虫基因表达调节中起着重要的作用。这是一种翻译后修饰,它参与了多种细胞功能,包括RNA加工处理、细胞信号转导、蛋白亚细胞定位、转录后调节和DNA修复。高迁移率族蛋白B1(HMGB1)参与基因转录、复制、重组与修复。胞外HMGB1是一种重要的晚期炎症介质,它可以激活巨噬细胞释放TNF-α和IL-13等早期炎症因子。在血吸虫感染中,TNF-α和IL-13对虫卵周围肉芽肿的形成起着重要的免疫诱导作用,可能是宿主感染后免疫调节的关键分子。HMGB1与一些感染性疾病的发病密切相关。本研究从日本血吸虫童虫cDNA文库筛选得到的阳性克隆中选择了蛋白质精氨酸甲基转移酶1(PRMT1)和高迁移率族蛋白B1(HMGB1)编码基因进行研究。首先通过生物信息学分析获得其完整的开放阅读框,然后通过分子克隆技术对这2个基因进行了克隆表达。随后,对纯化重组蛋白reSjcHMGB1开展了动物免疫保护性试验,评价其作为疫苗候选抗原的价值。一、东方田鼠日本血吸虫天然抗性相关基因的免疫筛选用日本血吸虫天然抗性东方田鼠血清免疫筛选日本血吸虫肝期童虫cDNA文库,将3次复筛获得的阳性克隆转入大肠杆菌(E.coil)BM25.8环化成质粒,抽提质粒DNA,EcoRⅠ和HindⅢ双酶切琼脂糖凝胶电泳鉴定,插入片段进行核苷酸序列测序,并进行生物信息学分析。结果,经3次复筛后获得32个阳性克隆,插入片段为300 bp～1 100 bp之间,测序结果经同源性分析,共获得26个不同分子基因:高迁移率族蛋白B1(HMGB1)部分基因,蛋白质精氨酸甲基转移酶部分编码基因,细胞色素b部分编码基因,线粒体编码区基因,16个日本血吸虫未知蛋白编码基因,6个日本血吸虫未知新基因。本研究用东方田鼠血清筛选日本血吸虫童虫cDNA文库,获得一批新的日本血吸虫疫苗候选分子的编码基因,为研究血吸虫病疫苗和血吸虫病免疫诊断奠定了基础。二、日本血吸虫蛋白质精氨酸甲基转移酶(PRMT)1编码基因的克隆、表达和分析依据电子延伸得到的SjPRMT1基因序列设计一对引物,上游引物引入BamHⅠ酶切位点,下游引物引入XhoⅠ酶切位点。以日本血吸虫成虫总RNA为模板,经反转录PCR(RT-PCR)扩增目的编码基因。纯化PCR产物与pGEM-T载体连接后转化感受态大肠杆菌JM109,抽提重组质粒DNA用BamHⅠ和XhoⅠ双酶切及核苷酸序列测序进行鉴定。选择阅读框正确的克隆,纯化重组质粒中目的基因双酶切片段,亚克隆入pET28a原核表达载体,构建重组质粒pET28a-SjPRMT1,转化DH5α感受态菌,重组质粒经双酶切和核苷酸序列鉴定后,阳性克隆质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达并获得纯化的重组蛋白(简称为reSjPRMT1),采用SDS-PAGE和Western blotting分析和鉴定该重组蛋白。运用Gene Runner软件预测reSjPRMT1蛋白的二级结构、功能位点及表位特征。结果,RT-PCR扩增出一大小与预期一致的基因片段。TA克隆插入目的片段经核苷酸序列测定,cDNA全长1083 bp,编码360个氨基酸。序列分析表明该片段与SmPRMT1基因序列同源性为87%,推导的氨基酸序列同源性为95%。表达蛋白经SDS-PAGE和Western blotting分析显示,reSjPRMT1重组蛋白的分子质量约43 kDa(包括6个组氨酸),以可溶性方式表达,可被日本血吸虫感染小鼠血清和抗His-G HRP抗体识别。SjPRMT1基因的克隆、表达获得成功,并获得纯化的重组蛋白,为今后进一步研究其生物学特性以及免疫原性奠定了基础。三、日本血吸虫高迁移率族蛋白B1(HMGB1)编码基因的克隆、表达和免疫保护性研究依据公布的SmHMGB1基因序列设计一对简并引物,上游引物引入BamHⅠ酶切位点,下游引物引入SalⅠ酶切位点。以日本血吸虫成虫总RNA为模板,经反转录PCR(RT-PCR)扩增目的编码基因。纯化PCR产物与pGEM-T载体连接后转化感受态大肠杆菌JM109,抽提重组质粒DNA用BamHⅠ和SalⅠ双酶切及核苷酸序列测序进行鉴定。选择阅读框正确的克隆,纯化重组质粒中目的基因双酶切片段,亚克隆入pET28a原核表达载体,构建重组质粒pET28a-SjHMGB1,转化DH5α感受态菌,重组质粒经双酶切和核苷酸序列鉴定后,阳性克隆质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达并获得纯化的重组蛋白,采用SDS-PAGE和Western blotting分析和鉴定该重组蛋白。运用Gene Runner软件预测reSjHMGB1的二级结构、功能位点及表位特征。在免疫保护性实验中,雌性C57BL/6小鼠随机分为5组,分别为感染对照组、弗氏佐剂对照组、MontanideISA206佐剂对照组、reSjcHMGB1加弗氏佐剂免疫组、reSicHMGB1加MontanideISA 206佐剂免疫组。感染对照组不注射任何抗原和佐剂,两种佐剂对照组小鼠注射乳化的生理盐水加弗氏或Montanide ISA 206佐剂,两免疫组每只小鼠经背部皮下多点注射乳化的20μg reSjcHMGB1加弗氏或Montanide ISA 206佐剂,共免疫3次,间隔2周。末次免疫后2周,小鼠经腹部感染日本血吸虫尾蚴30±1条,攻击感染后6周剖杀小鼠,进行成虫和虫卵计数。并分别于免疫前、攻击感染前和小鼠剖杀前采血并分离血清,ELISA检测血清中特异性IgG抗体。结果,RT-PCR扩增的目的片段经琼脂糖凝胶电泳观察,与预计的一致。TA克隆插入目的片段经核苷酸序列测定,cDNA全长531 bp,编码176个氨基酸。序列分析表明该片段与SmHMGB1基因序列同源性为86%,推导的氨基酸序列同源性为93%。表达蛋白经SDS-PAGE分析显示,reSjHMG重组蛋白的分子质量约30 kDa(包括6个组氨酸),以可溶性方式表达。免疫印迹结果显示,日本血吸虫感染小鼠血清、重组抗原免疫小鼠血清和抗His-G HRP抗体均可识别该重组蛋白。生物信息学分析表明该蛋白包含两个保守的结构域(A盒和B盒)及含酸性氨基酸的C末端,同时存在多个潜在的抗原决定簇。在免疫保护性实验中,ELISA结果表明,免疫后重组抗原加两种佐剂免疫组小鼠的特异性IgG抗体水平均显著高于感染对照组和佐剂对照组(P＜0.05)。reSjcHMGB1加弗氏佐剂免疫组减虫率和肝组织减卵率分别为17.9%和17.6%,其虫荷数和每克肝组织虫卵数(EPG)与感染对照组相比均无统计学意义(P＞0.05)。reSjcHMGB1加Montanide ISA 206佐剂免疫组减虫率和肝组织减卵率分别为分别为33.2%和11.3%,其虫荷数与感染对照组相比有统计学意义(P＜0.05),但与ISA 206佐剂对照组相比无统计学意义(P＞0.05)。本研究成功克隆、表达SjHMGB1,并获得纯化的重组蛋白。在动物保护性实验中,重组抗原并未诱导小鼠产生明显的抗感染和抗生殖免疫保护作用。更多还原

【Abstract】 Schistosomiasis japonica, a zoonotic parasitic disease, is still recognized as a major public health problem in China. Current strategy to control schistosomiasis japonica in China is an integrated and comprehensive approach based on control of the source of infection. Those measures rely predominantly on mass chemotherapy with the effective antischistosome drug praziquantel (PZQ). But schistosomiasis is spreading to new areas, in addition, PZQ chemotherapy does have limitations including the inability with mass treatment to prevent reinfection and the increasing possibility to develop resistance to PZQ. Consequently, vaccine strategy represents an essential component for the future control of schistosomiasis as an adjunct to chemotherapy. Genes encoding antigens including enzymes, muscles and teguments of S. japonicum have been cloned and expressed, but in general they did not exhibite the expected protective efficacy. It is still main task to seek and search for new vaccine candidates. Immunoscreening of S. japonicum cDNA library seems to be an efficient way to identify new molecules as vaccine candidates with higher immunogenicity and antigenicity.The main aim to develop antischistosome vaccine is to reduce worm burden and egg depositon in the liver tissue. Thus the vaccine should effect on the schistosomula and fecundity of adult worms. Microtus fortis is a non-permissive mammal host for S. japonicum. S. japonicum infection does not lead to Schistosomiasis in Microtus fortis. It seems that schistosomula may be a suitable target of the immune system of the host. If some specific antigen molecules derived from the larvae could be found, it will be possible to kill it off in this stage or stop it from growing, developing, maturing, egg depositing and so on. In this case, not only the pathologic lesion caused by Schistosome can be alleviated, but also the transmission of schistosomiasis can be effectively interrupted. Therefore, encouraging results may be obtained by immunoscreening of S. japonicum schistosomula cDNA library with sera of Microtus fortis. Meanwhile, this study may provide related information on the mechanism of natural resistance to S. japonicum in Microtus fortis.Protein arginine methylation play important roles in the modulation mechanisms of gene expression in Schistosome. It is a posttranslational modification involved in various cellular functions including RNA processing, signal transduction, protein subcellular localization, transcriptional regulation and DNA repairing.High mobility group box-1 (HMGB1) involves in gene transcription, replication, recombination and DNA repair. Extracellularly released HMGB1 has been recently identified as a delayed mediator of inflammation and is shown to be a potent inducer of pro-inflammatory cytokines including TNF-αand IL-13. Since TNF-αand IL-13-mediated inflammation around eggs is a major pathology process associated with this infection, SjHMGB1 may have an important immunomodulatory role in the pathophysiology of this infection. HMGB1 has been implicated in the pathogenesis of several inflammatory disorders.In this study, the genes encoding protein arginine methyltransferases 1 (PRMT1) and high mobility group box-1 (HMGB1) selected from the positive clones of above Schistosome larvae library were further studied. Firstly the open reading frame was obtained respectively by tools of bio-informatics. Then gene encoding SjPRMT1 and SjHMGB1 were cloned and expressed using molecular cloning technique. Subsequently, recombinant SjcHMGB1 as vaccine candidate was carried out in mice experiments to evaluate its protective efficacy. 1. Immunoscreening of natural resistant molecules to Schistosoma japonicum in Microtus fortisS. japonicum schistosomula cDNA library was screened using pre-absorbing sera of Microtus fortis, which shows natural resistance to S.japonicum. The positive clones after three rounds of screening were transformed into E.coli BM25.8. E. coli BM25.8 clones containing the plasmid were cultured in LB, and then selected for plasmid extraction. the plasmid DNA was digested by EcoR I and HindIII, and analyzed by agarose gel electrophoresis. Then the positive clones were sequenced and analyzed through BLASTn software of NCBI. As a result, 32 positive clones were obtained after three rounds of screening and their sizes ranged from 300 bp to 1 100 bp. There were 26 different genes identified by BLASTn and BLASTp analysis that may code 26 proteins of S.japonicum including high mobility group box-1(HMGB1), cytochrome b5, mitochondrion coding region, 16 function-unknown S.japonicum proteins and 6 new proteins. Generally, in this study, the genes encoding some molecules which may have potencial efficacy against schistosomiasis by immunization were identified by immunoscreening of S. japonicum schistosomula cDNA library with sera of Microtus fortis, thus to be of assistance of the development of protective vaccine and diagnosis of schistosomiasis japonica.2. Cloning, expression and analysis of the gene encoding protein arginine methyltransferases 1 of Schistosoma japonicumA couple of primers were designed according to electronic elongation SjPRMT1 sequence, with BamH I restriction endonuclease site introduced in forward primer, and Xho I in reverse primer. The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) as template. The purified PCR product was ligated with pGEM-T easy vector and then transformed into JM109. After identified by endonucleases digestion and sequencing, the target DNA fragment was cut and purified, and then subcloned into the proper prokaryotic expressin vector and transformed into competent E.coli DH5α. After identification as above, plasmids isolated from positive recombinant clone were transformed into competent E.coli BL21 (DE3) and the recombinant protein was induced to express in the presence of Isopropyl-β-D-thio-galactopyranoside (IPTG). The purified recombinant protein was analyzed by SDS-PAGE, western blotting and tools of bio-informatics such as Gene Runner. As a result, a 1083 bp DNA fragment was acquired by RT-PCR. BLASTn results revealed that the identity of SjPRMT1 gene to SmPRMT1 gene is 87%, and their deduced amino acid sequence shows 95% identity. SDS-PAGE and Western blotting analysis revealed that the molecular weight of reSjPRMT1 is approximately 42 kDa (including 6 His) in the soluble form. The protein could be recognized by sera of mice immunized with cercariae of S. japonicum and anti-His G HRP antibodies. The bio-information analysis demonstrated that the protein has multiple potential antigenic determinants. The gene encoding high mobility group box-1 of S. japonicum was successfully cloned and expressed, and the result may facilitate for further investigation on its biological characteristics, and immunogenicity as well.3. Gene cloning, expression and immunological study on high mobility group box-1 of Schistosoma japonicumA couple of primers were designed according to published SmHMGB1, with BamH I restriction endonuclease site introduced in forward primer, as well as Sal I in reverse primer. The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) as template. The purified PCR product was ligated with pGEM-T easy vector and then transformed into JM109. After identified by endonucleases digestion and sequencing, the target DNA fragment was cut and purified, and was then ligated with pET28a and transformed into competent E.coli DH5α. After identification as above, plasmids isolated from positive recombinant clone were transformed into E.coli BL21 (DE3) and the recombinant protein was induced to express in the presence of IPTG. The purified recombinant protein was soluble and analyzed by SDS-PAGE, western blotting and tools of bio-informatics such as Gene Runner. In the immunological study, female C57BL/6 mice were randomly divided into five groups, challenge control group, Freund’s adjuvant control group, Montanide ISA 206 adjuvant control group, reSjcHMGB1 plus Freund’s adjuvant, reSjcHMGB1 plus Montanide ISA 206 adjuvant. As for challenge control group, mice were not treated with recombinant antigen or adjuvant. In two adjuvant control groups, each mouse was injected subcutaneously with sterile normal saline emulsified in FCA/FIA or Montanide ISA 206 respectively. Each mouse in two groups of reSjcHMGB1 plus Freund’s adjuvant or Montanide ISA 206 adjuvant was immunized subcutaneously with 20μg reSjcHMGB1 emulsified in FCA/FIA or Montanide ISA 206 respectively. All the mice were vaccinated three times at an interval of 2 weeks. Two weeks after final immunization, the mouse was challenged with cercariae of S. japonicum. At the sixth week after challenge, all mice were sacrificed and the worms and eggs in each mouse were counted. The sera collected from mice periodically before immunization, challenge infection and sacrifice were tested respectively using ELISA assay to detect specific anti-SjcHMGB1 IgG antibodies. As a result, a 531 bp in size of the DNA fragment was acquired by RT-PCR. The sequence analysis indicated that the fragment shows 86% in homology to that of SmHMGB1, and the deduced amino acid sequence shows to be 93% identical with that encoded by SmHMGB1. SDS-PAGE analysis revealed that the molecular weight of expressed protein re SjHMGB1 is approximately 26 kDa (including 6 His) in the soluble form. The protein could be recognized by sera of mice infected with S.japonucum, sera of mice immunized with recombinant antigen reSjcHMGB1 and anti-His G HRP antibodies. The bio-information analysis demonstrated that the protein had two conservative domains (A box and B box) and acidic C-terminal tails, as well as multiple potential antigenic determinants. In the immunological study, the results of ELISA showed that a higher level of specific IgG antibodies in two immunized groups with reSjcHMGB1 was detected in comparison with that of challenge group (P<0.05) . The worm reduction rate and egg reduction rate in mice immunized with reSjcHMGB1 plus Freund’s adjuvant were 17.9% and 17.6% respectively, but there was no significant difference compared with that in challenge control group (P>0.05) . Results also showed that reSjcHMGB1 plus Montanide ISA 206 adjuvant group showed 33.2% worm reduction rate and 11.3% egg reduction rate, and the difference in worm burden was significant when compared to that in challenge control group (P<0.05) . In this study, The gene encoding high mobility group box-1 of S. japonicum was successfully cloned and expressed. In the immunological study, no significant protective immunity and anti-fecundate immunity were found experimentally in mice immunized with recombinant antigen against challenge infection of S. japonicum.更多还原