【摘要】：人血清淀粉樣蛋白A1是人血清淀粉樣蛋白A(SAA)的家庭的一員,目前,細(xì)菌、病毒感染、冠心病、動(dòng)脈粥樣硬化、腫瘤等疾病中均檢測(cè)到血清SAA增加。作為新型炎癥標(biāo)志物,SAA對(duì)于臨床疾病中的監(jiān)測(cè)具有重要的引導(dǎo)和應(yīng)用意義。本文采用大腸桿菌BL21(DE3)原核系統(tǒng)表達(dá)人血清淀粉樣蛋白A1(SAA1),根據(jù)BL21(DE3)表達(dá)偏好性設(shè)計(jì)并合成SAA1蛋白基因片段,構(gòu)建表達(dá)載體pET-28a-SAA1,轉(zhuǎn)化至表達(dá)宿主菌BL21(DE3)中,經(jīng)表達(dá)條件優(yōu)化,獲得重組目的蛋白的最佳誘導(dǎo)表達(dá)條件:誘導(dǎo)劑IPTG終濃度為0.6 mM,34℃,誘導(dǎo)8 h,培養(yǎng)基pH7,誘導(dǎo)轉(zhuǎn)速180rpm。過(guò)Ni柱純化濃縮蛋白,得到純度高達(dá)95%以上,最終濃度為8.09 mg/mL的SAA1蛋白。本文利用抗原與抗體的特異性結(jié)合,建立了SAA1的酶聯(lián)免疫(ELISA)檢測(cè)方法和比濁檢測(cè)方法。通過(guò)試驗(yàn)對(duì)反應(yīng)條件以及實(shí)驗(yàn)用耗材和試劑來(lái)進(jìn)行優(yōu)化,達(dá)到最佳檢測(cè)效果。對(duì)檢測(cè)試劑的敏感性、重復(fù)性和特異性進(jìn)行評(píng)估,建立了ELISA檢測(cè)方法,確定了陽(yáng)性樣品的臨界值OD450 nm=0.222,線性范圍在100-2400 ng/mL之間,最低檢出限為50 ng/mL。性能評(píng)估顯示出了本檢測(cè)試劑特異性良好,批內(nèi)以及批間變異系數(shù)均小于7%,回收率均在允許范圍內(nèi)且平均回收率為99.91%,精密度高。同時(shí),成功建立了比濁檢測(cè)方法。在紫外分光光度計(jì)560 nm下檢測(cè)其吸光值與加入SAA1蛋白含量呈線性相關(guān),其標(biāo)準(zhǔn)方程是Y=0.0698ln(X)+0.255,線性范圍是0.075-2.4μg/mL,其相關(guān)系數(shù)R2=0.99287,其回收率在93.6%-106.4%之間,均在允許范圍內(nèi)。
[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，

本文編號(hào)：2468326