基因重组人血清淀粉样蛋白A1的制备及检测方法学的建立

发布时间：2019-04-29 15:34

【摘要】：人血清淀粉样蛋白A1是人血清淀粉样蛋白A(SAA)的家庭的一员,目前,细菌、病毒感染、冠心病、动脉粥样硬化、肿瘤等疾病中均检测到血清SAA增加。作为新型炎症标志物,SAA对于临床疾病中的监测具有重要的引导和应用意义。本文采用大肠杆菌BL21(DE3)原核系统表达人血清淀粉样蛋白A1(SAA1),根据BL21(DE3)表达偏好性设计并合成SAA1蛋白基因片段,构建表达载体pET-28a-SAA1,转化至表达宿主菌BL21(DE3)中,经表达条件优化,获得重组目的蛋白的最佳诱导表达条件:诱导剂IPTG终浓度为0.6 mM,34℃,诱导8 h,培养基pH7,诱导转速180rpm。过Ni柱纯化浓缩蛋白,得到纯度高达95%以上,最终浓度为8.09 mg/mL的SAA1蛋白。本文利用抗原与抗体的特异性结合,建立了SAA1的酶联免疫(ELISA)检测方法和比浊检测方法。通过试验对反应条件以及实验用耗材和试剂来进行优化,达到最佳检测效果。对检测试剂的敏感性、重复性和特异性进行评估,建立了ELISA检测方法,确定了阳性样品的临界值OD450 nm=0.222,线性范围在100-2400 ng/mL之间,最低检出限为50 ng/mL。性能评估显示出了本检测试剂特异性良好,批内以及批间变异系数均小于7%,回收率均在允许范围内且平均回收率为99.91%,精密度高。同时,成功建立了比浊检测方法。在紫外分光光度计560 nm下检测其吸光值与加入SAA1蛋白含量呈线性相关,其标准方程是Y=0.0698ln(X)+0.255,线性范围是0.075-2.4μg/mL,其相关系数R2=0.99287,其回收率在93.6%-106.4%之间,均在允许范围内。
[Abstract]:Human serum amyloid A1 is a member of the family of human serum amyloid protein A (SAA). At present, the increase of serum SAA has been detected in bacterial, viral infection, coronary heart disease, atherosclerosis, tumor and other diseases. As a new inflammatory marker, SAA plays an important role in the monitoring of clinical diseases. In this paper, human serum amyloid A1 (SAA1) was expressed in E. coli BL21 (DE3) prokaryotic system, and SAA1 gene fragment was designed and synthesized according to BL21 (DE3) expression preference. The expression vector pET-28a-SAA1, was constructed. The recombinant protein was transformed into the host strain BL21 (DE3) and the optimal expression conditions were obtained: the final concentration of IPTG was 0.6mM,34 鈩，

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