成纤维细胞生长因子23表达纯化及其多克隆抗体的制备
发布时间：2018-10-05 20:29
【摘要】：背景： 慢性肾脏病（chronic kidney diease，CKD）的全球发病率呈逐年增高的趋势，其防治正面临严峻挑战，早期诊断和预防慢性肾脏病是目前全球急切需要解决的问题。据ISN和IFKF的估计，慢性肾脏病占世界人口的十分一，其中大部分慢性肾脏病患者没得到早期诊断和及时的治疗，结果是:由于肾功能下降，而导致肾功能衰竭，，患者生活质量下降，需要透析或肾移植，更重要的是在进入肾衰期之前就过早死于心脑血管疾病。 目前临床上仍然缺乏简单能够准确筛查慢性肾脏病的手段或方法。现在临床上以血肌酐，尿素氮和蛋白尿作为监测肾功能的指标，虽然对于慢性肾病和慢性肾衰竭的分期可以起到一定程度的作用,但其检测准确性低、特异性不高,常常需要同时结合血液及B超检查，综合判断才能确诊，而且一旦发现异常多数病程已经达到中晚期。 成纤维细胞生长因子23（fibroblast growth factor23,FGF23），作为肾功能早期衰退诊断的一个新型分子标志物，具有充分的临床资料与基础研究的文献支持，且与临床上现有的新的生物标物-肌氨酸酐相比，能更早地检测到病情，无须复杂的修正，仅需单独的测量。因此可作为早期慢性肾病的诊断方法，同时，它可通过检测体内治疗前后血浆水平，监测治疗的有效性。 目的： 诱导表达纯化具有生物学活性的可溶性重组人成纤维细胞生长因子23（FGF23）并制备FGF23多克隆抗体。 方法： 根据已构建的SUMO-FGF23菌株，通过亲和层析技术优化FGF23蛋白的分离纯化条件，SDS-PAGE电泳法检测纯化效果。用纯化的FGF23蛋白免疫兔子产生抗血清，得到FGF23蛋白的多克隆抗体，并以慢性肾病病人的血清中天然状态的FGF23蛋白作为阳性对照，用ELISA和Western blotting检测基因工程制备的FGF23多克隆抗体抗血清的效价及其生物学活性。 结果： FGF23蛋白在低温(16℃)诱导19h大部分以可溶形式表达,组氨酸亲和层析法分离纯化了蛋白,并用纯化的蛋白制备了兔抗FGF23多克隆抗体,结果显示制备的抗体特异性高，可进一步在免疫分析中使用。 结论: 本研究纯化了条带单一的并且具有生物学活性的FGF23蛋白,制备了具有良好特异性的兔抗FGF23多克隆抗体,为进一步研发FGF23试剂盒诊断早期CKD奠定了基础。
[Abstract]:Background: the global incidence of chronic kidney disease (chronic kidney diease,CKD) is increasing year by year. Its prevention and treatment are facing severe challenges. The early diagnosis and prevention of chronic kidney disease is an urgent problem to be solved in the world. According to ISN and IFKF estimates, chronic kidney disease accounts for one tenth of the world's population, and most of the patients with chronic kidney disease do not receive early diagnosis and timely treatment, resulting in renal failure due to a decline in renal function. Patients with reduced quality of life need dialysis or kidney transplantation and, more importantly, die prematurely from cardiovascular and cerebrovascular diseases before entering renal failure. Is there still a lack of simplicity in clinical practice? A method or method for accurate screening of chronic kidney disease. At present, serum creatinine, urea nitrogen and proteinuria are used as indicators to monitor renal function. Although they can play a certain role in the staging of chronic nephropathy and chronic renal failure, their accuracy and specificity are low. It is often necessary to combine blood and B-ultrasound examination to determine the diagnosis, and once it is found that most of the abnormal course of disease has reached the middle and late stage. Fibroblast growth factor (23 (fibroblast growth factor23,FGF23), as a novel molecular marker for the early diagnosis of renal function decline, has sufficient clinical data and basic literature support, and compared with the new biomarkers, creatinine, which is a new biomarker in clinic. The condition can be detected earlier, without complicated correction, but with individual measurements. Therefore, it can be used as a diagnostic method for early chronic nephropathy and can be used to monitor the effectiveness of treatment by detecting plasma levels before and after treatment in vivo. Aim: to induce the expression and purification of soluble human fibroblast growth factor 23 (FGF23) with biological activity and to prepare FGF23 polyclonal antibody. Methods: according to the constructed SUMO-FGF23 strain, the purification conditions of FGF23 protein were optimized by affinity chromatography. The purification effect was detected by SDS-PAGE. Rabbits were immunized with purified FGF23 protein to produce antiserum, and polyclonal antibody of FGF23 protein was obtained. The natural state of FGF23 protein in serum of patients with chronic nephropathy was used as positive control. ELISA and Western blotting were used to detect the titer and biological activity of FGF23 polyclonal antibody antiserum prepared by genetic engineering. Results: most of FGF23 protein was expressed in soluble form at low temperature (16 鈩
本文编号：2254772
[Abstract]:Background: the global incidence of chronic kidney disease (chronic kidney diease,CKD) is increasing year by year. Its prevention and treatment are facing severe challenges. The early diagnosis and prevention of chronic kidney disease is an urgent problem to be solved in the world. According to ISN and IFKF estimates, chronic kidney disease accounts for one tenth of the world's population, and most of the patients with chronic kidney disease do not receive early diagnosis and timely treatment, resulting in renal failure due to a decline in renal function. Patients with reduced quality of life need dialysis or kidney transplantation and, more importantly, die prematurely from cardiovascular and cerebrovascular diseases before entering renal failure. Is there still a lack of simplicity in clinical practice? A method or method for accurate screening of chronic kidney disease. At present, serum creatinine, urea nitrogen and proteinuria are used as indicators to monitor renal function. Although they can play a certain role in the staging of chronic nephropathy and chronic renal failure, their accuracy and specificity are low. It is often necessary to combine blood and B-ultrasound examination to determine the diagnosis, and once it is found that most of the abnormal course of disease has reached the middle and late stage. Fibroblast growth factor (23 (fibroblast growth factor23,FGF23), as a novel molecular marker for the early diagnosis of renal function decline, has sufficient clinical data and basic literature support, and compared with the new biomarkers, creatinine, which is a new biomarker in clinic. The condition can be detected earlier, without complicated correction, but with individual measurements. Therefore, it can be used as a diagnostic method for early chronic nephropathy and can be used to monitor the effectiveness of treatment by detecting plasma levels before and after treatment in vivo. Aim: to induce the expression and purification of soluble human fibroblast growth factor 23 (FGF23) with biological activity and to prepare FGF23 polyclonal antibody. Methods: according to the constructed SUMO-FGF23 strain, the purification conditions of FGF23 protein were optimized by affinity chromatography. The purification effect was detected by SDS-PAGE. Rabbits were immunized with purified FGF23 protein to produce antiserum, and polyclonal antibody of FGF23 protein was obtained. The natural state of FGF23 protein in serum of patients with chronic nephropathy was used as positive control. ELISA and Western blotting were used to detect the titer and biological activity of FGF23 polyclonal antibody antiserum prepared by genetic engineering. Results: most of FGF23 protein was expressed in soluble form at low temperature (16 鈩
本文编号：2254772
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