【作者】 江文才；

【导师】 高琪；

【作者基本信息】 江苏省血吸虫病防治研究所 ， 病原生物学， 2011， 硕士

【摘要】 华支睾吸虫病(clonorchiasis)是由华支睾吸虫(Clonorchis sinensis, Cs)寄生于人体肝内胆管所引起的寄生虫病。华支睾吸虫病是一种重要的食源性寄生虫病。广泛流行于东亚地区,包括中国、日本、朝鲜、韩国、越南,特别是中国的南部和东北部,感染者约占全球总感染人数的一半。根据2004年第二次全国人体重要寄生虫病现状调查资料推算,我国华支睾吸虫感染者约有1 249万人,与1989年第一次全国调查相比,感染率升高了74.85%。随着人口的流动,人们饮食习惯的改变,淡水养殖业和物流业的迅速发展,以及华支睾吸虫卫生检疫工作的相对滞后,华支睾吸虫感染率仍可能继续上升。华支睾吸虫的成虫和虫卵毒素及代谢产物可引起的肝脏和胆管系统疾病,长期、慢性感染者可导致肝组织纤维化、肝脏肿瘤,甚至胆管癌,严重威胁着病人的健康。华支睾吸虫的早期诊断和及时治疗是华支睾吸虫病防治工作的重要组成部分。目前,华支睾吸虫病的主要检测方法包括病原学诊断和免疫学诊断。病原学诊断因华支睾吸虫虫卵较小,容易漏检。同时,华支睾吸虫虫卵与灵芝孢子、猫后睾吸虫卵和异形类吸虫卵形态、大小极为相似,易造成误诊。免疫学诊断正成为华支睾吸虫病研究的主要方向之一,该方法包括抗原检测和抗体检测两类,其中抗体检测因可同时作为病原学检测的初筛和流行病学筛查工具,应用更为广泛。目前,已用于华支睾吸虫病血清学检测的抗原包括:成虫可溶性抗原、纯化抗原和重组抗原3类,其中成虫可溶性抗原制备简单,敏感性高(83.10%-100.00%),但特异性低,与日本血吸虫病、卫氏并殖吸虫病、肝片吸虫病、麝猫后睾吸虫病以及横川后睾吸虫病等存在交叉反应。纯化虫体抗原克服了成虫水溶性抗原交叉反应率高的缺点,但制备方法繁琐以及原材料限制,无法大量制备,很大程度上限制了其广泛应用。随着分子生物学的发展,重组抗原广泛用于寄生虫病的诊断,如日本血吸虫、疟疾等。华支睾吸虫重组蛋白的分子生物学研究虽起步较晚,但也已经发现了一些具有诊断价值的重组蛋白,如重组7kDa蛋白和26kDa谷胱甘肽S转移酶敏感性分别为81.30%和73.30%,特异性分别为92.60%和83.90%,显示出较高的敏感性和特异性。重组蛋白不仅具有较高的诊断效果,也便于大量制备,因此,在免疫诊断中具有广阔的应用前景。然而目前已发现的重组抗原的敏感性普遍低于成虫可溶性抗原,在筛查病人时存在一定漏诊的可能性,因此,寻找敏感性更高的重组抗原意义重大。半胱氨酸蛋白酶(Cysteine protease ,CP)是一类在酶的活性中心含有半胱氨酸残基的蛋白水解酶,该酶广泛地存在于人类、寄生虫等多种生物体内,参与多种生化反应。寄生虫半胱氨酸蛋白酶主要与虫体的毒力、对组织和细胞的侵袭力和免疫逃避等方面有关。该酶作为一种寄生虫特异性抗原也广泛应用于寄生虫病的免疫研究。根据文献报道,华支睾吸虫的半胱氨酸蛋白酶,主要分布于虫体的肠腔、子宫内虫卵和睾丸。本研究通过对GenBank中已公布的序列号为AF093242华支睾吸虫半胱氨酸蛋白酶基因克隆、表达、纯化,并对获得的重组蛋白的血清学诊断效果进行评估。研究内容主要包括:一华支睾吸虫半胱氨酸蛋白酶(Cscp)基因的克隆及原核表达根据已知的华支睾吸虫半胱氨酸蛋白酶基因序列,应用SignalP 3.0对其氨基酸序列进行分析,除去信号肽,设计引物。提取华支睾吸虫成虫mRNA,逆转录获得成虫cDNA,进一步PCR扩增目的片段。随后,通过TA克隆、菌落PCR和测序、相似性比较对获得目的基因片段进行鉴定。将CP片段和原核表达质粒pET28a(+)双酶切,酶切产物通过T4连接酶连接,构建pET28a-CP重组质粒,电转化入E. coli DH5α感受态细胞中,克隆、扩增重组质粒,抽提重组质粒双酶切鉴定和测序鉴定,随后电转化入BL21(DE3)中,在IPTG诱导剂的诱导下表达、电泳鉴定后纯化,得到以包涵体形式存在的该重组蛋白。二华支睾吸虫半胱氨酸蛋白酶的酵母表达为克服原核表达获得的华支睾吸虫半胱氨酸蛋白酶CsCP重组蛋白为包涵体,其变性、纯化过程复杂,且不易获得具有生物活性的天然状态蛋白的不足。因此本研究选用毕赤酵母GS115,对华支睾吸虫半胱氨酸蛋白酶的CsCP重组质粒进行酵母表达探索。将已获得CP基因与酵母表达质粒pPIC9K双酶切,T4连接酶连接后,电转入E .coli DH5α感受态细胞中,抽提质粒双酶切鉴定,阳性结果进一步测序鉴定。成功构建的酵母表达重组质粒pPIC9K-CP,SacⅠ单酶切线性化后,电转入感受态GS115酵母中。MD平板营养筛选,挑取单菌落PCR鉴定,阳性菌落转入BMGY培养液中30℃培养至OD600≈2-6,离心后将菌体重悬于BMMY中至OD600≈1.0,诱导表达,对表达产物纯化,获得可溶性的华支睾吸虫半胱氨酸蛋白酶重组蛋白。三华支睾吸虫半胱氨酸蛋白酶重组蛋白用于血清学诊断的初步研究纯化后的华支睾吸虫半胱氨酸蛋白酶重组蛋白,经弗氏完全佐剂乳化后(浓度250μg/ml)背部皮下多点注射免疫Balb/c小鼠,200μl/只,第3和第5周再用弗氏不完全佐剂乳化重组蛋白,浓度、方法均同第1次免疫。加强免疫2次,末次免疫后2周摘眼球取血。采用ELISA法检测血清中抗华支睾吸虫半胱氨酸蛋白酶特异性IgG效价,结果显示抗体滴度可达1:64 000。同时,用该重组蛋白分别与华支睾吸虫病人血清、正常人血清反应,以及用该重组蛋白免疫小鼠后血清与华支睾吸虫成虫水溶性抗原反应,实验结果表明所制备的重组蛋白具有较好的免疫原性及生物活性。通过western-blot法对该重组蛋白的特异性进行分析,结果显示该重组蛋白与日本血吸虫病人血清无交叉反应,与卫氏并殖吸虫病人血清反应率为20.00%(2/10)。随后应用Dot-ELISA法分别检测60份华支睾吸虫病人血清和84正常人血清,检测敏感性和特异性分别达到91.67%(55/60)和97.62%(82/84)。表明用该华支睾吸虫半胱氨酸蛋白酶重组蛋白检测血清中特异性IgG具有较高敏感性和特异性,是华支睾吸虫一个有潜在血清学诊断价值的重组蛋白抗原。更多还原

【Abstract】 Clonorchiasis is a food-borne parasitic disease caused by Clonorchis sinensis(Cs) , which is endemic to eastern Asian including China, Japan, Democratic People’s Republic of Korea, Republic of Korea and Vietnam, pardicular in the south and northeast China. It was estimated that China accounted for nearly half of total population infected by this paraiste in the world. Based on the data from the second national survey of important human parasitic diseases in 2001 to 2004, there were 12.49 million cases in China, increased 74.85% compared to the first national survey conducted in 1989. Even now, the risk of infection by Clonorchis sinensis still keeps increasing, due to mobile population, changing of eating habit, rapid development of fishing industry, and relative lagging behind of health and quarantine technique.The C. sinensis adult, egg toxin and metabolite contribute to chronic impairment to hepatic and biliary system, long-term chronic infections often lead to hepatic fibrosis, hepatic neoplasm and even cholangiocarcinoma. Early diagnosis and timely treatmentis is crucial for clonorchiasis control and prevention. At present, fecal examination is the standard diagnostic method, but tiny eggs are often missed by microscopic examination of stool samples, it is also difficult to collect feces because of indifference of the inhabitants. Meanwhile, it’s very difficult to be discriminated C. sinensi egg with Ganoderma spore, O. felineus and alien class Trematoda due to the similar morphologies and size, Sero-diagnosis is a potential technique to replace the light microscopy detection of the eggs in feces, which depends on high quality of antigens. The crude antigen is easy to produce and sensitive to detect patients with C. sinensis (83.10%-100.00%), but cross-reactions are known to occur with Schistosomiasis japonica, Paragonimiasis westermani, Fascioliasis, Opisthorchiasis viverrini and Metagonimiasis yokogawai. Purified antigens are better than the crude antigen, but it is not easy to produce then results in not able to be applied widely. Some of recombinant C. sinensis proteins are valuable on diagnosis of clonorchiasis such as 7 kDa and 26 kDa glutathione S-transferase, with high sensitivity and specificity Cysteine proteases (CP) is a class of enzymes with common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic dyad. Cysteine proteases are commonly encountered in human beings, parasites and other creatures and play multi-faceted roles, virtually in every aspect of physiology and development. Cysteine proteases play numerous indispensable roles in the biology of parasitic organisms. Besides previously known general catabolic functions and protein processing, cysteine proteases may be crucial to parasite immunoevasion, virulence, and cell and tissue invasion. Parasite cysteine proteases are unusually immunogenic and have been exploited as serodiagnostic markers and vaccine targets. It is reported that 9 kinds of cysteine proteases have been discovered in C. sinensis.They mainly locate in the adult worm intestines, intrauterine eggs and testis. In this study , one of C. sinensis Cysteine protease (Cscp),(access number: AF093242) was cloned and expressed in Pichias Pastoris GS115, and purified recombinant Cscp was evaluated for sero-diagnosis of clonorchiasis.Part I Cloning and prokaryotic expression of C. sinensis cysteine proteaseA pair of primers were designed, according to the analysis of gene sequence of C. sinensis cysteine protease using SignalP 3.0 .The total RNA was extracted from adult worm of C. sinensis, and cysteine protease gene was amplified by RT-PCR and inserted into pGEM-T vector by TA cloing. The insertion of fragment was confirmed through colony PCR and sequencing, then the gene was further cloned into the prokaryotic expression vector pET28a(+) and transformed into E.coli DH5α. The recombinant plasmid was identified by double digestion. Inducing by IPTG, the recombinant Cscp was expressed as inclusion bodies in E.coli BL21. The recombinant protein was purified by affinity chromatography.Part II Yeast expression of C. sinensis cysteine proteaseE. coli might produce a mis-folded protein, that is usually inactive or insoluble. Whereas P. pastoris is capable of producing disulfide bonds and glycosylations in proteins that is main advantage of Pichia over E. coli. Cscp gene would be re-amplified by primers with EcoR I and Not I. And the Cscp gene was cloned into eukaryotic vector pPIC9K. The recombinant plasmid was transformed into Pichia GS115 vector after linearized by Sac I digestion, and further screened through MD medium plates and colony PCR. And the recombinant plasmid was expressed in BMMY media for 4 days. The soluble recombinant protein was produced and was purified by ultrafiltration.Part III The preliminary evaluation of recombinant C.sinensis cysteine protease for sero-diagnosisSix weeks old BALB/c mice were immunized each with 50μg recombinant cysteine protease of C. sinensis (rCsCP), and after 3 times immunization, the anti- rCsCP antibody in immunized mice’s sera was tested with ELISA assay. The antibody titer was up to 1:64 000. Meanwhile, the mice’s sera immunized could be recognized by adult worm soluble antigen with Western-blot analysis. And rCsCP shows a strong reaction with human clonorchiasis sera, but it does not react with health people sera. The result demonstrated that the recombinant cysteine protease of C. sinensis protein was well expressed.Using western-blot, the rCsCP was probed with patients sera of Schistosomiasis japonicum and patients sera of Paragonimiasis westermani. There is no cross-reaction with human Schistosomiasis japonicum sera, and only 2 out of 10 human Paragonimiasis westermani sera react with rCsCP. Follwoing that, the value of rCsCP for sero-diagnosis was evaluated using Dot-ELISA, the sensitivity was 91.67% (55/60), and specificity was 97.62%(82/84). In conclusion, the rCsCP might be a potential candidate antigen for human clonorchiasis sero-diagnosis.更多还原