本文選題：日本血吸蟲 + 童蟲　； 參考：《安徽醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的篩選新的可能具有診斷意義的血吸蟲蛋白分子,并對(duì)其進(jìn)行生物信息學(xué)分析。 方法以血吸蟲病人血清篩選日本血吸蟲15d肝期童蟲cDNA文庫(kù),對(duì)陽(yáng)性克隆插入片段的核苷酸進(jìn)行序列分析,將結(jié)果通過(guò)Internet送入NCBI GenBank進(jìn)行同源性比較并利用蛋白質(zhì)分析軟件進(jìn)行生物信息學(xué)分析。 結(jié)果經(jīng)3輪篩選,共獲15個(gè)陽(yáng)性克隆,對(duì)其進(jìn)行測(cè)序,獲得11個(gè)基因,其中5個(gè)為編碼日本血吸蟲線粒體的基因,1個(gè)為編碼日本血吸蟲肌球蛋白的基因,其他5個(gè)分別為編碼SjCHGC05315、SjCHGC01371、SjCHGC04782、SjCHGC05166、SjCHGC09769的基因。 結(jié)論從日本血吸蟲肝期童蟲cDNA文庫(kù)中篩選到一批日本血吸蟲基因,為血吸蟲病新的診斷候選分子的研究提供了材料。 目的日本血吸蟲病是中國(guó)嚴(yán)重的寄生蟲病之一,政府每年投入大量資金用于血吸蟲病的防治工作,但由于血吸蟲生活史的復(fù)雜性,血吸蟲病仍然沒(méi)有得到很好的控制。血吸蟲病控制的重要措施之一在于建立準(zhǔn)確而又靈敏的診斷方法。本研究以尋找新的血吸蟲病免疫診斷候選抗原分子為目的,對(duì)日本血吸蟲表膜蛋白Tetraspanin2(SjTsp2)進(jìn)行體外重組表達(dá)、純化,建立重組抗原間接ELISA方法(rSj-ELISA)檢測(cè)日本血吸蟲重組表膜蛋白rSjTsp2用于日本血吸蟲病免疫診斷的潛能,并與成蟲粗抗原(SjAWA)平行檢測(cè),比較兩種方法的敏感性和特異性之間的差異。并用純化的重組蛋白rSjTsp2免疫小鼠,制備單克隆抗體,以進(jìn)一步探討rSjTsp2用于血吸蟲病診斷的應(yīng)用價(jià)值。 方法誘導(dǎo)含SjTsp2編碼基因的重組質(zhì)粒pET28a(+)-SjTsp2表達(dá)出rSjTsp2,SDS-PAGE驗(yàn)證表達(dá)量,使用親和層析法純化重組蛋白,并用Lowry法檢測(cè)純化蛋白的濃度。將rSjTsp2和成蟲粗抗原(SjAWA)包被反應(yīng)板,棋盤滴定法分別確定最適抗原包被量和血清稀釋度,建立rSjTsp2-ELISA和AWA -ELISA檢測(cè)特異性抗體的方法并進(jìn)行比較。用rSj Tsp2-ELISA和AWA-ELISA兩種方法平行檢測(cè)正常家兔血清,感染日本血吸蟲后2周、4周和6周家兔血清,比較兩種方法之間敏感性和特異性。繼之,確定最佳試驗(yàn)條件后建立rSj Tsp2-ELISA檢測(cè)34例急性、31例慢性和25例肝吸蟲染者、9例線蟲感染者和49例正常對(duì)照者血清標(biāo)本。以rSjTsp2免疫小鼠6周后將其脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行雜交融合,HAT篩選出雜交瘤細(xì)胞,經(jīng)亞克隆及擴(kuò)大培養(yǎng),獲得5株單克隆抗體。用Antibody Isotype Kit鑒定單抗Ig類別及亞型,用間接ELISA法檢測(cè)雜交瘤上清和小鼠腹水效價(jià),免疫熒光實(shí)驗(yàn)鑒定其特異性。 結(jié)果成功誘導(dǎo)表達(dá)重組質(zhì)粒pET28a(+)-SjTsp2,6×His親和層析柱純化獲得rSjTsp2,SDS-PAGE檢測(cè)其分子量與理論值一致。以rSjTsp2作為診斷抗原分子,經(jīng)過(guò)條件優(yōu)化建立了rSjTsp2-ELISA診斷兔日本血吸蟲病的方法。用rSjTsp2-ELISA,AWA-ELISA兩種方法平行檢測(cè)正常兔血清,感染日本血吸蟲后2周、4周和6周兔血清,結(jié)果示rSjTsp2用于日本血吸蟲病免疫診斷與AWA-ELISA具有相似的敏感性和特異性, rSjTsp2檢測(cè)感染日本血吸蟲后2周兔血清陽(yáng)性率為75.4%,高于后者的68.9%。rSjTsp2檢測(cè)血吸蟲感染病人、其他寄生蟲感染病人及正常人血清特異性抗體,初步結(jié)果顯示該方法具有一定的敏感性和特異性。同時(shí)獲得5株特異性抗rSjTsp2表膜蛋白的單克隆抗體的雜交瘤細(xì)胞株。單抗Ig類別及亞型,雜交瘤上清和小鼠腹水效價(jià),免疫熒光鑒定其特異性,結(jié)果顯示rSjTsp2為日本血吸蟲成蟲表膜蛋白。 結(jié)論體外成功表達(dá)、純化了日本血吸蟲表膜蛋白Tetraspanin2(SjTsp2),建立了間接rSj-ELISA方法檢測(cè)感染家兔血清IgG抗體,其原核表達(dá)重組蛋白具有一定的診斷應(yīng)用潛能。本研究還獲得了5株特異性抗rSjTsp2表膜蛋白的單克隆抗體的雜交瘤細(xì)胞株。
[Abstract]:Objective to screen new schistosomiasis protein molecules that may have diagnostic significance and to carry out bioinformatics analysis.
Methods schistosomiasis screening of Schistosoma japonicum 15d hepatic schistosomula cDNA library, the positive clone fragment of nucleotide sequence analysis results through the Internet into the NCBI GenBank homology and protein analysis software by bioinformatic analysis.
Results after 3 rounds of screening, 15 positive clones were obtained and sequenced to obtain 11 genes, 5 of which encoding Schistosoma japonicum mitochondrial genes, 1 genes encoding for Schistosoma japonicum tropomyosin, the other 5 were SjCHGC01371, encoding SjCHGC05315, SjCHGC04782, SjCHGC05166, SjCHGC09769 genes.
Conclusion a number of Schistosoma japonicum genes were screened from Schistosoma japonicum liver worm cDNA library, providing materials for the study of new diagnostic candidates for schistosomiasis.
Objective schistosomiasis is one of the most serious parasitic disease China, the government invested a lot of money every year for schistosomiasis prevention and control work, but because of the complexity of the life history of schistosomiasis japonicum, still has not been well controlled. One of the important measures for schistosomiasis control is to establish accurate and sensitive diagnostic methods. This study to find the candidate antigen for immunodiagnosis of schistosomiasis new molecules for the purpose of Schistosoma japonicum membrane protein Tetraspanin2 (SjTsp2). The recombinant expression, purification and establishment of indirect ELISA recombinant antigen (rSj-ELISA) detection of Schistosoma japonicum recombinant membrane protein rSjTsp2 for immunodiagnosis of schistosomiasis japonica potential, and adult worm antigen (SjAWA) parallel detection, the difference between the two the sensitivity and specificity of the methods. And immunized with rSjTsp2 recombinant protein were purified, the preparation of single To clone antibody to further explore the application value of rSjTsp2 in the diagnosis of schistosomiasis.
The recombinant plasmid pET28a containing SjTsp2 gene encoding method to induce the expression of -SjTsp2 (+) rSjTsp2, expression of SDS-PAGE authentication, using the recombinant protein was purified by affinity chromatography, and the concentration of purified protein was detected by Lowry. RSjTsp2 and adult worm antigen (SjAWA) coated reaction plate, checkerboard titration method to determine the optimal antigen and the amount of serum dilution, and compared the method of establishing rSjTsp2-ELISA and AWA -ELISA detection of specific antibody. The parallel detection of normal rabbit serum by rSj Tsp2-ELISA and AWA-ELISA two methods, 2 weeks of infection of Schistosoma japonicum after 4 weeks and 6 weeks of rabbit serum, sensitivity and specificity of the comparison between the two methods. Then, the establishment of rSj Tsp2-ELISA was detected in 34 cases of acute to determine the optimal experimental conditions, 31 cases and 25 cases of chronic liver fluke infections, serum samples from 9 cases of nematode infections and 49 cases of normal controls. In 6 weeks after immunization of mice will be rSjTsp2 The spleen cells and myeloma cell SP2/0 were screened by HAT fusion, hybridoma cells, by subcloning andexpansion of training, obtained 5 monoclonal antibodies. Using Antibody Isotype Kit identification of monoclonal antibody Ig type and subtype detection, hybridoma supernatant and ascites titer by indirect ELISA, immunofluorescence assay. The opposite sex.
The expression of recombinant plasmid pET28a (+) rSjTsp2 affinity chromatography purification was -SjTsp2,6 * His, SDS-PAGE was consistent with the molecular weight and theoretical value. Using rSjTsp2 as diagnostic antigen molecules, after optimizing the condition to establish a diagnosis of rSjTsp2-ELISA rabbit schistosomiasis method. RSjTsp2-ELISA AWA-ELISA two parallel detection method of normal rabbit serum. The infection of Schistosoma japonicum after 2 weeks, 4 weeks and 6 weeks of rabbit serum, the results showed that rSjTsp2 in Japan for the immunodiagnosis of schistosomiasis and AWA-ELISA have similar sensitivity and specificity of rSjTsp2 detection on Schistosoma japonicum infection after 2 weeks of rabbit serum positive rate was 75.4%, higher than that in patients with 68.9%.rSjTsp2 infection the detection of schistosome, patients and normal human serum. The specific antibody infected with other parasites, preliminary results show that this method has certain sensitivity and specificity. At the same time obtain 5 strains of specific anti rSj The hybridoma cell lines with monoclonal antibodies against Tsp2 membrane proteins. Ig and its subtypes, the hybridoma supernatants and the ascites of mice were identified by immunofluorescence. The results showed that rSjTsp2 is the membrane protein of Schistosoma japonicum adult.
Conclusion in vitro successfully expressed Schistosoma japonicum membrane purified protein Tetraspanin2 (SjTsp2), was developed for the detection of serum IgG antibody in rabbits infected with indirect rSj-ELISA and prokaryotic expression of the recombinant protein has certain diagnostic potential. This study also obtained hybridoma cell lines of monoclonal antibody specific against 5 strains of the rSjTsp2 membrane protein.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R383.2

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