【摘要】：目的 肝細(xì)胞性肝癌(Hepatocellular carcinoma, HCC)是最常見的惡性腫瘤之一,其發(fā)病率和死亡率呈逐年上升的趨勢,早期診斷和早期治療對肝癌病人的預(yù)后起著決定性的作用。目前HCC的常規(guī)篩查和診斷方法包括體格檢查、影像學(xué)檢查及血清甲胎蛋白(Alpha fetoprotein, AFP)檢測。AFP雖為診斷HCC的首選指標(biāo),但其敏感性只有60%-70%,特別是早期HCC, AFP敏感性僅40%左右。最近的研究發(fā)現(xiàn),血清異常凝血酶DCP (des-γ-carboxy prothrombin, DCP)在診斷HCC中其敏感性和特異性是可以與AFP相媲美的一個(gè)標(biāo)志物。因此,本試驗(yàn)擬制備抗DCP單克隆抗體(mAb),初步鑒定其生物學(xué)功能,并為進(jìn)一步將所制備的單克隆抗體用于建立定量檢測DCP的方法奠定基礎(chǔ)。 方法 1.構(gòu)建DCP原核表達(dá)載體pCold Ⅱ-DCP,導(dǎo)入E.coli. BL21(DE3)菌株,IPTG誘導(dǎo)表達(dá)后進(jìn)行SDS-PAGE電泳鑒定,以His TrapFF親和層析柱純化His-DCP融合蛋白。 2.以該融合蛋白免疫BALB/c小鼠,取免疫后小鼠的脾細(xì)胞與骨髓瘤細(xì)胞Sp2/0融合,依次進(jìn)行陽性雜交瘤細(xì)胞株的篩選及亞克隆化,以獲得能穩(wěn)定分泌a-抗DCP mAb雜交瘤單克隆細(xì)胞株。 3.以間接ELISA法檢測抗體效價(jià)；以Sigma公司提供的mAb亞類檢測試劑盒進(jìn)行抗體亞類鑒定；以Western blot及免疫組織化學(xué)鑒定抗體特異性。 結(jié)果 1.成功構(gòu)建了表達(dá)質(zhì)粒pCold Ⅱ-DCP。 2.成功誘導(dǎo)表達(dá)并純化了His-DCP融合蛋白,濃度為1.5mg/mL;純度達(dá)80%以上。 3.獲得了2株穩(wěn)定分泌抗人DCP mAb雜交瘤細(xì)胞株,分別命名為385和6H4；其免疫球蛋白亞類都為IgG2a型,每株抗體效價(jià)均達(dá)1：243,000以上。利用Western blot和免疫組織化學(xué)結(jié)果顯示抗人DCP單克隆抗體能與DCP發(fā)生特異性結(jié)合。 結(jié)論 本研究成功構(gòu)建了pCold Ⅱ-DCP表達(dá)質(zhì)粒,并表達(dá)了His-DCP融合蛋白；以該融合蛋白為免疫原,成功制備出了抗人DCP mAb,為進(jìn)一步將該抗體應(yīng)用于肝癌的早期診斷提供了重要工具。
[Abstract]:Aim: hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the most common malignant tumors. Its morbidity and mortality are increasing year by year. Early diagnosis and early treatment play a decisive role in the prognosis of HCC patients. At present, the routine screening and diagnosis methods of HCC include physical examination, imaging examination and serum alpha-fetoprotein (Alpha fetoprotein, AFP) detection. Although AFP is the first choice for the diagnosis of HCC, its sensitivity is only 60% ~ 70%, especially in the early stage of HCC,. The sensitivity of AFP was only about 40%. Recent studies have found that the sensitivity and specificity of serum abnormal thrombin DCP (des- 緯-carboxy prothrombin, DCP) in the diagnosis of HCC is comparable to that of AFP. Therefore, the anti-DCP monoclonal antibody (mAb), was prepared to identify its biological function and lay a foundation for the further application of the monoclonal antibody to establish a quantitative method for the detection of DCP. Method 1. The prokaryotic expression vector pCold 鈪，

本文編號：2472023