Zhao Qing-tao, Ma Long-yang, Xue Guo-zhu, Zhao Ai-zhi, Dou Ke-feng
Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Jul;23(7):649-51.
To express and characterize an active form of a single-chain antibody (scFv) from the gene of human phage antibodies which is specific for hepatocellular cancer.
The complementary DNAs encoding the variable regions of the light chain (V(L)) and heavy chain (V(H)) were connected by a (Gly(4)Ser)(3) linker using a splicing by overlap extension polymerase chain reaction. The resultant scFv gene was cloned to the pET28a(+) vector and expressed in E.coli as inclusion bodies. Then the inclusion bodies were solubilized, denatured and renatured. Finally, the affinity constant of scFv was determined by noncompetitive enzyme immunoassay.
The target protein amounted 26% of the total protein in the condition of A(600) at 0.8 for 6 hours. After renatured, the purity of target protein was 95% and the affinity constant of the scFv was 3.6x10(7) mol/L.
An active form of scFv which is specific for hepatocellular cancer can bind selectively with hepatocellular cancer cells, which provides a theoretical basis for immunological detection and clinical use of scFv.
从人噬菌体抗体基因中表达并鉴定一种对肝细胞癌具有特异性的单链抗体（scFv）活性形式。
使用重叠延伸聚合酶链反应通过（Gly(4)Ser）(3)接头连接编码轻链可变区（V(L)）和重链可变区（V(H)）的互补DNA。将所得的scFv基因克隆到pET28a(+)载体中，并在大肠杆菌中以包涵体形式表达。然后将包涵体溶解、变性和复性。最后，通过非竞争性酶免疫测定法测定scFv的亲和常数。
在A(600)为0.8的条件下培养6小时，目标蛋白占总蛋白的26%。复性后，目标蛋白纯度为95%，scFv的亲和常数为3.6×10(7) mol/L。
对肝细胞癌具有特异性的scFv活性形式可与肝癌细胞选择性结合，为scFv的免疫检测及临床应用提供了理论依据。
