【摘要】： 日本血吸蟲病是中國嚴重的寄生蟲病之一,政府每年投入大量資金用于血吸蟲的防治工作,然而由于血吸蟲生活史的復雜性,血吸蟲病的仍然沒有得到很好的控制。血吸蟲病控制的重要措施之一在于建立準確而又靈敏的診斷方法。本研究即以尋找新的診斷抗原為目的,用感染兔血清免疫學方法篩選日本血吸蟲童蟲cDNA文庫,對獲得的24個持續(xù)陽性克隆進行測序分析,其中13個是日本血吸蟲22.6kDa膜相關蛋白(Sj22.6)基因,這表明Sj22.6基因在日本血吸蟲童蟲表膜高豐度表達,故推測其具有一定診斷作用。為驗證該作用,我們設計引物體外成功擴增該基因,將其克隆到原核表達載體pET28a中,轉(zhuǎn)化大腸桿菌BL21并進行誘導表達。在37℃下,OD600為0.6時,加入IPTG(異丙基硫代-β-半乳糖苷,最終濃度1mmol/L)進行誘導,通過SDS-PAGE電泳發(fā)現(xiàn)誘導4小時后表達量最大且為可溶蛋白。重組蛋白通過鎳柱親和層析純化。純化后的蛋白免疫4只BABL/c小鼠,多抗效價皆高于1:16 000,說明所表達的重組蛋白具有較好的免疫原性。再通過Western blotting方法發(fā)現(xiàn)可以被日本血吸蟲感染兔血清強烈識別而不被正常兔血清識別,說明本實驗表達蛋白具有免疫反應性。同時,該蛋白也可被急性日本血吸蟲病人血清以及慢性日本血吸蟲病人血清識別而不與正常人反應,說明其可能具有一定診斷潛力。然而在檢測日本血吸蟲感染兔血清時,我們只獲得77.8%(14/18)陽性率,以及高達22.2%(4/18)假陽性率。我們還對兩批人血清進行檢測,經(jīng)反復檢測,第一批血清(18份急性日本血吸蟲病人血清和18份正常人血清)rSj22.6陽性率僅為5.6%(1/18),假陽性率為5.6%(1/18);而日本血吸蟲成蟲粗抗原(AWA)陽性率為61.1%(11/18),假陽性率為5.6%(1/18)。而第二批人血清(15份急性日本血吸蟲病人血清和15份正常人血清)rSj22.6陽性率僅為26.7%(4/15),假陽性率為6.7%(1/15);而AWA陽性率為93.3%(14/15),假陽性率為6.7%(1/15)。檢測結果初步否定了rSj22.6蛋白作為診斷抗原的作用。另外,本課題組還對rSj22.6蛋白抗凝血功能作初步研究,當rSj22.6蛋白達到200μg/ml時,PT與APTT時間開始出現(xiàn)延長現(xiàn)象,并在本實驗所容許的最大蛋白濃度800μg/ml下,PT延長4.5s而APTT延長12s。在本實驗條件下,PT與APTT時間與rSj22.6濃度成劑量相關,但延長效果較弱。我們還將rSj22.6蛋白與牛凝血酶按照不同的物質(zhì)的量比混合并分別于23℃孵育,然后12 000g 4℃離心15min,留取上清,以rSj22.6蛋白免疫的小鼠血清作抗體,Western Blotting發(fā)現(xiàn)與牛凝血酶作用的rSj22.6蛋白與抗體的反應強度比單純的rSj22.6蛋白要弱,而且與牛凝血酶作用的rSj22.6蛋白出現(xiàn)比rSj22.6蛋白稍小的條帶,這說明牛凝血酶能結合并能水解rSj22.6蛋白。本實驗初步證實了rSj22.6蛋白具有良好的免疫原性以及免疫反應性,但不適合作為診斷抗原。該蛋白具有一定的抗凝血功能,可以與牛凝血酶結合,并能被水解。rSj22.6蛋白的抗凝功能可能是通過與凝血酶相互作用而發(fā)揮抗凝血作用。
[Abstract]:Schistosomiasis japonica is one of the most serious parasitic diseases in China. The government invests a lot of money every year in the prevention and treatment of schistosomiasis. However, due to the complexity of the life cycle of schistosomiasis, schistosomiasis has not been well controlled. One of the important measures to control schistosomiasis is to establish accurate and sensitive diagnostic methods. In order to find a new diagnostic antigen, the cDNA Library of Schistosoma japonicum was screened by rabbit serological immunoassay. 24 clones were sequenced and analyzed. Among them, 13 were the genes of Schistosoma japonicum 22.6 kDa membrane-associated protein (Sj22.6). This indicated that Sj22.6 gene was a high abundance surface marker of Schistosoma japonicum. To verify this effect, we designed primers to amplify the gene in vitro, cloned it into prokaryotic expression vector pET28a, transformed Escherichia coli BL21 and induced its expression. The recombinant protein was purified by nickel column affinity chromatography. The purified protein was immunized to 4 BABL/c mice with multiple antibody titers higher than 1:16 000, indicating that the recombinant protein had good immunogenicity. The strong recognition of rabbit serum infected with Schistosoma japonicum but not that of normal rabbit serum indicates that the expressed protein has immunoreactivity. At the same time, the protein can also be recognized by serum of acute and chronic schistosomiasis japonicum patients without reacting with normal human serum, indicating that it may have certain diagnostic potential. The positive rate of rSj22.6 was only 5.6% (1/18) and the false positive rate was 5.6% (1/18) in the first batch of sera (18 sera from acute schistosomiasis japonica patients and 18 sera from normal persons). The positive rate of rSj 22.6 was 26.7% (4/15) and 6.7% (1/15) in the second group of human sera (15 sera from acute schistosomiasis japonica patients and 15 sera from normal persons), while the positive rate of AWA was 93.3% (14/15) and 6.7% (1/15) respectively. Results The role of rSj22.6 protein as a diagnostic antigen was preliminarily negated. In addition, the anticoagulant function of rSj22.6 protein was preliminarily studied. When rSj22.6 protein reached 200 ug/ml, PT and APTT time began to prolong, and PT prolonged 4.5 s and APTT prolonged 12 s at the maximum protein concentration of 800 ug/ml allowed in this experiment. Under the experimental conditions, PT and APTT time were dose-dependent with rSj22.6 concentration, but the prolongation effect was weak. We also mixed rSj22.6 protein and bovine thrombin at different mass ratios and incubated them at 23 C respectively, then centrifuged at 12 000 g 4 C for 15 min. The supernatant was taken from the serum of mice immunized with rSj22.6 protein as antibody. Western Blotting found that The reactivity of rSj22.6 protein with bovine thrombin with antibody was weaker than that of rSj22.6 protein, and the bands of rSj22.6 protein with bovine thrombin were slightly smaller than that of rSj22.6 protein, which indicated that bovine thrombin could bind to and hydrolyze rSj22.6 protein. It can bind to bovine thrombin and can be hydrolyzed. The anticoagulant function of rSj22.6 protein may be mediated by interaction with thrombin.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392;R532.2

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相關期刊論文 前8條

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本文編號：2201423