【摘要】：背景： 慢性腎臟病（chronic kidney diease，CKD）的全球發(fā)病率呈逐年增高的趨勢(shì)，其防治正面臨嚴(yán)峻挑戰(zhàn)，早期診斷和預(yù)防慢性腎臟病是目前全球急切需要解決的問(wèn)題。據(jù)ISN和IFKF的估計(jì)，慢性腎臟病占世界人口的十分一，其中大部分慢性腎臟病患者沒(méi)得到早期診斷和及時(shí)的治療，結(jié)果是:由于腎功能下降，而導(dǎo)致腎功能衰竭，，患者生活質(zhì)量下降，需要透析或腎移植，更重要的是在進(jìn)入腎衰期之前就過(guò)早死于心腦血管疾病。 目前臨床上仍然缺乏簡(jiǎn)單能夠準(zhǔn)確篩查慢性腎臟病的手段或方法�，F(xiàn)在臨床上以血肌酐，尿素氮和蛋白尿作為監(jiān)測(cè)腎功能的指標(biāo)，雖然對(duì)于慢性腎病和慢性腎衰竭的分期可以起到一定程度的作用,但其檢測(cè)準(zhǔn)確性低、特異性不高,常常需要同時(shí)結(jié)合血液及B超檢查，綜合判斷才能確診，而且一旦發(fā)現(xiàn)異常多數(shù)病程已經(jīng)達(dá)到中晚期。 成纖維細(xì)胞生長(zhǎng)因子23（fibroblast growth factor23,FGF23），作為腎功能早期衰退診斷的一個(gè)新型分子標(biāo)志物，具有充分的臨床資料與基礎(chǔ)研究的文獻(xiàn)支持，且與臨床上現(xiàn)有的新的生物標(biāo)物-肌氨酸酐相比，能更早地檢測(cè)到病情，無(wú)須復(fù)雜的修正，僅需單獨(dú)的測(cè)量。因此可作為早期慢性腎病的診斷方法，同時(shí)，它可通過(guò)檢測(cè)體內(nèi)治療前后血漿水平，監(jiān)測(cè)治療的有效性。 目的： 誘導(dǎo)表達(dá)純化具有生物學(xué)活性的可溶性重組人成纖維細(xì)胞生長(zhǎng)因子23（FGF23）并制備FGF23多克隆抗體。 方法： 根據(jù)已構(gòu)建的SUMO-FGF23菌株，通過(guò)親和層析技術(shù)優(yōu)化FGF23蛋白的分離純化條件，SDS-PAGE電泳法檢測(cè)純化效果。用純化的FGF23蛋白免疫兔子產(chǎn)生抗血清，得到FGF23蛋白的多克隆抗體，并以慢性腎病病人的血清中天然狀態(tài)的FGF23蛋白作為陽(yáng)性對(duì)照，用ELISA和Western blotting檢測(cè)基因工程制備的FGF23多克隆抗體抗血清的效價(jià)及其生物學(xué)活性。 結(jié)果： FGF23蛋白在低溫(16℃)誘導(dǎo)19h大部分以可溶形式表達(dá),組氨酸親和層析法分離純化了蛋白,并用純化的蛋白制備了兔抗FGF23多克隆抗體,結(jié)果顯示制備的抗體特異性高，可進(jìn)一步在免疫分析中使用。 結(jié)論: 本研究純化了條帶單一的并且具有生物學(xué)活性的FGF23蛋白,制備了具有良好特異性的兔抗FGF23多克隆抗體,為進(jìn)一步研發(fā)FGF23試劑盒診斷早期CKD奠定了基礎(chǔ)。
[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

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