發(fā)布時間：2018-04-27 23:01

  本文選題：血吸蟲病 + 信號蛋白14-3-3��； 參考：《安徽醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:誘導(dǎo)表達(dá)本室保種的含有日本血吸蟲信號蛋白14-3-3(Sj14-3-3)編碼基因的重組表達(dá)載體pET28a(+)-Sj14-3-3,制備并純化出重組蛋白Sj14-3-3(rSj 14-3-3),建立重組抗原間接ELISA方法(rSj-ELISA)診斷日本血吸蟲病,并與日本血吸蟲可溶性蟲卵抗原(SjSEA)的間接ELISA、間接血凝試驗(IHA)和環(huán)卵沉淀試驗(COPT)平行檢測,比較幾種方法的敏感性和特異性之間的差異,以探討重組抗原用于診斷血吸蟲病的實用性和可靠性。方法:誘導(dǎo)含Sj14-3-3編碼基因的重組質(zhì)粒pET28a(+)-Sj14-3-3表達(dá)出rSj14-3-3。SDS-PAGE和Western blotting驗證表達(dá)量和免疫活性,使用親和層析法純化重組蛋白,并用Bradford法檢測純化蛋白的濃度。將rSj14-3-3和SEA包被反應(yīng)板,棋盤滴定分別確定最適抗原包被量和血清稀釋度,建立rSj-ELISA和SEA-ELISA診斷日本血吸蟲病方法。用rSj-ELISA,SEA-ELISA,SEA-IHA和COPT四種方法平行檢測急性日本血吸蟲病患者64例,慢性日本血吸蟲病患者56例,華支睪吸蟲患者28例,腸道線蟲患者以及正常人血清各32例,比較不同方法之間敏感性,特異性以及交叉反應(yīng)性。結(jié)果:誘導(dǎo)表達(dá)重組質(zhì)粒pET28a(+)-Sj14-3-3,Ni2+親和層析法純化獲得rSj14-3-3,SDS-PAGE檢測其分子量與理論值一致;Western blotting顯示rSj14-3-3能被其特異性單克隆抗體識別。以rSj14-3-3作為診斷抗原分子,經(jīng)過條件優(yōu)化建立了rSj-ELISA診斷日本血吸蟲病的方法。用rSj-ELISA,SEA-ELISA,SEA-IHA和COPT四種方法平行檢測急、慢性血吸蟲病患者,華支睪吸蟲患者,腸道線蟲患者以及正常人血清,結(jié)果示rSj14-3-3用于日本血吸蟲病免疫診斷與SEA-ELISA以及SEA-IHA具有相似的敏感性和特異性; rSj-ELISA的敏感性顯著優(yōu)于COPT,但特異性(假陽性以及與其他寄生蟲病的交叉反應(yīng)性)遜于COPT。結(jié)論:本研究成功表達(dá)并純化了rSj14-3-3抗原,以此抗原單獨(dú)包被建立rSj-ELISA方法診斷日本血吸蟲病。重組抗原制備簡便,成本低廉,制備周期短,方法易于標(biāo)準(zhǔn)化,更適合商品化生產(chǎn)和流行區(qū)血吸蟲病的血清學(xué)輔助診斷。 目的:構(gòu)建日本血吸蟲蟲卵cDNA文庫并用日本血吸蟲急性感染患者血清對其進(jìn)行免疫學(xué)篩選,以尋找有效的早期診斷分子和疫苗候選分子。方法:收集日本血吸蟲感染6周蟲卵,提取總RNA,逆轉(zhuǎn)錄生成cDNA,PCR合成cDNA雙鏈。經(jīng)過純化、sfi I酶切、過柱除去小片段后,與λTriplEx2噬菌體載體兩臂連接,體外包裝后形成日本血吸蟲蟲卵cDNA文庫。用大腸桿菌預(yù)吸收的急性血吸蟲病患者血清對文庫進(jìn)行免疫學(xué)篩選,對復(fù)篩得到的14個陽性克隆進(jìn)行插入片段的核苷酸序列測定,結(jié)果呈送GenBank進(jìn)行同源性分析,根據(jù)其理論氨基酸組成,初步確定該cDNA插入片段所編碼蛋白的特性并利用生物信息學(xué)軟件分析和預(yù)測其結(jié)構(gòu)和用途。結(jié)果:所構(gòu)建的日本血吸蟲蟲卵cDNA初始文庫的容量為2.7x106個重組子,經(jīng)感染XL1-Blue菌后擴(kuò)增文庫的滴度達(dá)1010pfu/ml。任意挑取克隆經(jīng)自身環(huán)化后提取質(zhì)粒,酶切后片段大小范圍為400- 1500bp,重組率85%以上。用急性日本血吸蟲患者血清初篩得到28個陽性克隆,復(fù)篩得到14個持續(xù)陽性的克隆,測序后送GenBank進(jìn)行BLAST比對,其中9號為空載序列,2號無同源基因匹配為可能的新基因,其余序列均為匹配的日本血吸蟲相關(guān)基因。經(jīng)生物信息學(xué)分析示2號克隆基因具有一個432bp的完整開放閱讀框架,Antheprot軟件和多種基于網(wǎng)頁的在線軟件分析其結(jié)構(gòu)和功能并預(yù)測可能為一跨膜蛋白。結(jié)論:本試驗成功地構(gòu)建了日本血吸蟲蟲卵cDNA文庫,篩選所得到的陽性克隆有望成為早期診斷抗原或保護(hù)性疫苗分子,值得進(jìn)一步研究。
[Abstract]:Objective: to induce the recombinant expression vector pET28a (+) -Sj14-3-3 containing the encoding gene of Schistosoma japonicum signal protein 14-3-3 (Sj14-3-3), to prepare and purify the recombinant protein Sj14-3-3 (rSj 14-3-3), to establish a recombinant antigen indirect ELISA method (rSj-ELISA) for the diagnosis of schistosomiasis japonicum and to the soluble egg antigen of Schistosoma japonicum. (SjSEA) indirect ELISA, indirect hemagglutination test (IHA) and cyclic egg precipitation test (COPT) parallel detection, compare the differences between the sensitivity and specificity of several methods, in order to explore the practicability and reliability of recombinant antigen used in the diagnosis of schistosomiasis. Method: the recombinant plasmid pET28a (+) -Sj14-3-3 containing Sj14-3-3 encoding gene expressed rSj14-3- 3.SDS-PAGE and Western blotting were used to verify the expression and immune activity. The recombinant protein was purified by affinity chromatography, and the concentration of purified protein was detected by Bradford. RSj14-3-3 and SEA were coated with the reaction plate, and the optimum antigen envelope and serum dilution were determined by chessboard titration, and rSj-ELISA and SEA-ELISA were built to diagnose the Japanese schistosomiasis side. Methods: four methods of rSj-ELISA, SEA-ELISA, SEA-IHA and COPT were used to detect 64 cases of acute schistosomiasis, 56 cases of chronic schistosomiasis, 28 cases of Clonorchis sinensis, intestinal nematode and 32 normal human serum. The sensitivity, specificity and cross reactivity between different methods were compared. The plasmid pET28a (+) -Sj14-3-3, Ni2+ affinity chromatography was purified to obtain rSj14-3-3, and the molecular weight of SDS-PAGE was consistent with the theoretical value; Western blotting showed that rSj14-3-3 could be identified by its specific monoclonal antibody. RSj14-3-3 was used as a diagnostic antigen, and the method was optimized to establish rSj-ELISA for the diagnosis of schistosomiasis japonica. Four methods of ELISA, SEA-ELISA, SEA-IHA and COPT were used to detect acute, chronic schistosomiasis, Clonorchis sinensis, intestinal nematode and normal human serum. The results showed that rSj14-3-3 was similar in sensitivity and specificity to SEA-ELISA and SEA-IHA in the immunodiagnosis of schistosomiasis japonica, and the sensitivity of rSj-ELISA was significantly better than that of COPT. But the specificity (false positive and cross reactivity with other parasitic diseases) is inferior to the COPT. conclusion: This study successfully expressed and purified the rSj14-3-3 antigen, and the antigen was successfully established by the rSj-ELISA method for the diagnosis of schistosomiasis japonicum. The preparation of recombinant antigen is simple, low cost, short preparation period, easy to standardize and more suitable for commodity. Serological diagnosis of schistosomiasis in chemical production and endemic areas.
Objective: to construct the cDNA Library of Schistosoma japonicum eggs and to screen it with sera from acute infected patients with Schistosoma japonicum to find effective early diagnostic molecules and vaccine candidates. Methods: to collect the eggs of 6 weeks infected by Schistosoma japonicum, extract total RNA, reverse transcriptase cDNA and PCR to synthesize cDNA double strands. After purification, SFI I enzyme digestion, After the small fragments were removed, the cDNA Library of Schistosoma japonicum eggs was formed after packing with the two arms of lambda TriplEx2 phage carrier. The sera of patients with acute schistosomiasis preabsorbed by Escherichia coli were immunologically screened, and the nucleotide sequence of the 14 positive clones was detected by the insertion fragment. The result was Ge NBank homology analysis, based on its theoretical amino acid composition, preliminarily determined the characteristics of the cDNA inserted fragment and analyzed and predicted its structure and use by bioinformatics software. Results: the capacity of the initial library of the egg cDNA of Schistosoma japonicum was 2.7x106 recombinant, and after infection of XL1-Blue bacteria, Kuo Zengwen The titer of the library was 1010pfu/ml. randomly selected to extract the plasmid after its own cyclization. The size of the fragment was 400- 1500bp and the recombination rate was more than 85%. 28 positive clones were screened from the sera of acute Schistosoma japonicum patients and 14 continuous positive clones were screened and then sent to GenBank for BLAST comparison, of which 9 was empty load. Sequence, No. 2 homologous genes are matched as possible new genes, and the rest of the sequence are matched genes of Schistosoma japonicum. By bioinformatics analysis, the 2 clone gene has a complete open reading frame of 432bp, Antheprot software and a variety of web based online software to analyze its structure and function and predict that it may be a transmembrane Conclusion: this experiment successfully constructed the cDNA Library of Schistosoma japonicum eggs. The screening of the positive clones is expected to be an early diagnostic or protective vaccine molecule. It is worth further study.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R383

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