日本血吸虫22.6kDa表膜蛋白功能的初步研究
发布时间：2018-08-24 16:49
【摘要】： 日本血吸虫病是中国严重的寄生虫病之一,政府每年投入大量资金用于血吸虫的防治工作,然而由于血吸虫生活史的复杂性,血吸虫病的仍然没有得到很好的控制。血吸虫病控制的重要措施之一在于建立准确而又灵敏的诊断方法。本研究即以寻找新的诊断抗原为目的,用感染兔血清免疫学方法筛选日本血吸虫童虫cDNA文库,对获得的24个持续阳性克隆进行测序分析,其中13个是日本血吸虫22.6kDa膜相关蛋白(Sj22.6)基因,这表明Sj22.6基因在日本血吸虫童虫表膜高丰度表达,故推测其具有一定诊断作用。为验证该作用,我们设计引物体外成功扩增该基因,将其克隆到原核表达载体pET28a中,转化大肠杆菌BL21并进行诱导表达。在37℃下,OD600为0.6时,加入IPTG(异丙基硫代-β-半乳糖苷,最终浓度1mmol/L)进行诱导,通过SDS-PAGE电泳发现诱导4小时后表达量最大且为可溶蛋白。重组蛋白通过镍柱亲和层析纯化。纯化后的蛋白免疫4只BABL/c小鼠,多抗效价皆高于1:16 000,说明所表达的重组蛋白具有较好的免疫原性。再通过Western blotting方法发现可以被日本血吸虫感染兔血清强烈识别而不被正常兔血清识别,说明本实验表达蛋白具有免疫反应性。同时,该蛋白也可被急性日本血吸虫病人血清以及慢性日本血吸虫病人血清识别而不与正常人反应,说明其可能具有一定诊断潜力。然而在检测日本血吸虫感染兔血清时,我们只获得77.8%(14/18)阳性率,以及高达22.2%(4/18)假阳性率。我们还对两批人血清进行检测,经反复检测,第一批血清(18份急性日本血吸虫病人血清和18份正常人血清)rSj22.6阳性率仅为5.6%(1/18),假阳性率为5.6%(1/18);而日本血吸虫成虫粗抗原(AWA)阳性率为61.1%(11/18),假阳性率为5.6%(1/18)。而第二批人血清(15份急性日本血吸虫病人血清和15份正常人血清)rSj22.6阳性率仅为26.7%(4/15),假阳性率为6.7%(1/15);而AWA阳性率为93.3%(14/15),假阳性率为6.7%(1/15)。检测结果初步否定了rSj22.6蛋白作为诊断抗原的作用。另外,本课题组还对rSj22.6蛋白抗凝血功能作初步研究,当rSj22.6蛋白达到200μg/ml时,PT与APTT时间开始出现延长现象,并在本实验所容许的最大蛋白浓度800μg/ml下,PT延长4.5s而APTT延长12s。在本实验条件下,PT与APTT时间与rSj22.6浓度成剂量相关,但延长效果较弱。我们还将rSj22.6蛋白与牛凝血酶按照不同的物质的量比混合并分别于23℃孵育,然后12 000g 4℃离心15min,留取上清,以rSj22.6蛋白免疫的小鼠血清作抗体,Western Blotting发现与牛凝血酶作用的rSj22.6蛋白与抗体的反应强度比单纯的rSj22.6蛋白要弱,而且与牛凝血酶作用的rSj22.6蛋白出现比rSj22.6蛋白稍小的条带,这说明牛凝血酶能结合并能水解rSj22.6蛋白。本实验初步证实了rSj22.6蛋白具有良好的免疫原性以及免疫反应性,但不适合作为诊断抗原。该蛋白具有一定的抗凝血功能,可以与牛凝血酶结合,并能被水解。rSj22.6蛋白的抗凝功能可能是通过与凝血酶相互作用而发挥抗凝血作用。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R532.2
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R532.2
【参考文献】
相关期刊论文 前8条
1 于复东;康斌;李园园;李亦学;;预测和分析血吸虫含有EF-hand结构域的表膜蛋白的功能[J];科学通报;2007年13期
2 胡雪梅,张兆松,李春林,吴海玮,苏川,季e鹲，
本文编号：2201423
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