疥螨丝切蛋白基因的原核表达、蛋白定位以及间接ELISA诊断方法的初步建立

发布时间：2018-04-15 15:45

  本文选题：疥螨 + 丝切蛋白　； 参考：《四川农业大学》2015年硕士论文

【摘要】：疥螨病作为一种重要的人兽共患寄生虫病,严重影响人类健康和生活质量,特别是在人口密度较大的区域内流行广泛,另外还能危害多种动物,给社会经济带来不可小觑的损失。目前该病已引起了世界许多国家和组织机构的重视,针对疥螨的分子学研究不断深入,但还没有有效的免疫学诊断方法和疫苗问世。本研究根据NCBI数据库中疥螨丝切蛋白基因的EST序列设计引物,通过扩增、克隆后获得447bp大小的目的基因片段,将测序结果在GenBamk数据库中比对后发现与已报道的尘螨主要变应原之一的Derf31基因(GenBank:KM010014.1)的序列相似性最高(90%)。根据软件预测该丝切蛋白分子量约为16.8kDa,等电点为pI=5.59,推导化学式为C751H1175N1910231S8,蛋白带负电荷,无信号肽切割位点,不存在跨膜结构域,是一种膜外蛋白。将目的基因片段连入表达载体pET32a(+)后转入表达菌大肠杆菌E.coliBL21(DE3),并优化诱导条件,通过聚丙烯凝胶电泳验证该蛋白表达后,利用Ni-亲和层析柱纯化疥螨丝切蛋白。在免疫印迹实验中,疥螨丝切蛋白重组蛋白与兔疥螨阳性血清反应后产生免疫印迹条带,说明其具有良好的抗原性。利用纯化后的疥螨丝切重组蛋白免疫制备疫苗免疫实验兔,将采集到的高免血清作为一抗用于荧光组织化学实验,结果发现该蛋白广泛分布于疥螨虫体组织中,但表皮中未检测到该蛋白存在。将纯化后的丝切重组蛋白作抗原,经过条件优化筛选和特异性、重复性试验,成功建立了间接ELISA检测方法。实验结果确定抗原最佳包被浓度为5μg/mL,血清的最佳工作浓度为1:100,酶标二抗最适使用浓度为1:3000。利用cut-off计算公式确定阴阳性临界值,最后得到cut-off值为0.188,即当血清OD450≥ 0.188判为阳性,反之为阴性。再根据公式计算出特异性87.9%,敏感性83.33%。另外该方法检测出,该重组蛋白与患痒螨的和患豆状囊尾蚴的兔阳性血清均不发生交叉反应;批内变异系数在1.28%～4.08%之间,批间变异系数为1.81%～5.30%之间。本试验证明疥螨丝切重组蛋白具有潜在的诊断价值。
[Abstract]:As an important zoonotic parasitic disease, scabies seriously affects human health and the quality of life, especially in areas with high population density.To the social economy brings the loss which cannot be underestimated.At present, the disease has attracted the attention of many countries and organizations in the world. Molecular studies on scabies mite have been deepened, but there is no effective immunological diagnosis method and vaccine.In this study, primers were designed according to the EST sequence of the filamentous protein gene of scabies mite in NCBI database, and the target gene fragment of 447bp size was obtained by amplification and cloning.The sequencing results were compared in GenBamk database and the sequence similarity was the highest with the reported Derf31 gene GenBank: KM010014.1).According to the software prediction, the molecular weight of the protein is about 16.8 kDa and the isoelectric point is Pi 5.59.The deduced chemical formula is C751H1175N1910231S8. The protein has negative charge, no signal peptide cleavage site, no transmembrane domain, and is an extramembrane protein.The target gene fragment was inserted into the expression vector pET32a () and then transferred into E. coli BL21DE3, and the induction conditions were optimized. The protein was identified by polyacrylamide gel electrophoresis and purified by Ni-affinity chromatography.In the Western blotting experiment, the recombinant protein of scabies mites silk cut protein reacted with the positive serum of the rabbit scabies mite to produce the immunoblotting bands, which indicated that the recombinant protein had good antigenicity.The immunized rabbits were immunized with purified recombinant protein of scabies mites. The high immune serum was used as a first antibody for fluorescent histochemical experiments. It was found that the protein was widely distributed in the tissue of scabies mite.However, the protein was not detected in the epidermis.The purified recombinant protein was used as antigen, and the indirect ELISA detection method was successfully established by optimized screening, specificity and repeatability test.The results showed that the best coating concentration of antigen was 5 渭 g / mL, the optimal working concentration of serum was 1: 100, and the optimum concentration of enzyme labeled second antibody was 1: 3000.The critical value of yin and yang was determined by using the cut-off formula, and the final cut-off value was 0.1888, that is, when serum OD450 鈮，

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