异常凝血酶原N端F1片段及其抗血清的制备

Preparation of Abnormal Prothrombin Fl Fragment and Its Antiserum

正常人血浆经钡盐吸附、分段盐析、DE-52离子交换层析分离纯化到正常凝血酶原,然后经牛凝血酶酶切、DE-52离子交换层析分离到凝血酶原片段1,再经加热脱羧后得到异常凝血酶原的N端F1片段.将该片段免疫家兔,获得的抗血清与肝细胞性肝癌(HCC)患者血浆有抗原抗体反应,而与正常人血浆无反应,为研制新型的HCC检测试剂盒奠定了基础.

Hepatocellular carcinoma ( HCC) are incidenced highly in China. Alpha-fetoprotein (AFP) has been used as the tumor marker of this malignancy since the 1960s but falseness often occurred. Abnormal prothrombin (APT) is a new marker of HCC. Compared with prothrombin, APT lackes γ-carboxyglutamic acid (GLA) at N-terminus, which results from the abnormalities of posttranslational modification. The mechanism responsible for the high APT level of patients with HCC remains a speculation. Plasma APT level could be used in diagnosis of HCC and did not correlate with AFP levels. When APT and AFP were used together, the sensitivity increased. Thus APT was useful in early HCC diagnosis, predictable prognosis and indicating recurrence. We purified prothrombin from fresh human plasma by barium salt absorbtion、 (NH4)2SO4 step-precipitation and DE-52 ion exchange chromatography . Then the purified prothrombin was cleaved by bovine thrombin to release prothrombin fragment 1 (F1) which was isolated with DE-52 ion exchange chromatography. The F1 fragment was further decarboxylated by heat, and immunized the rabbit. Finally we got the anti-abnormal prothrombin F1-fragment antiserum, which interacted with the plasma of HCC patients, but did not interact with plasma of healthy human beings. Thus a new HCC test kit was developed.