| 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.
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