抗Ⅳ型胶原酶单链抗体的表达及其对肿瘤细胞侵袭的抑制作用

Expression of Anti Type Ⅳ Collagenase scFv Fragment and Inhibition of Tumor Cells Invasion

目的:制备抑制肿瘤细胞侵袭转移的单链抗体片段,用于构建小型化的免疫偶联物。方法:应用噬菌体呈现技术,以产生抗Ⅳ型胶原酶单抗的杂交瘤细胞为基础克隆构建单链抗体(scFv)基因。以表达质粒pET30a+为载体在大肠杆菌中进行诱导表达。ELISA法测定表达产物的免疫反应性,明胶酶谱法测定表达产物对靶酶活性的影响。BoydenChamber测定表达产物对肿瘤细胞侵袭的影响。结果:表达的单链抗体以包涵体形式存在,经变性和复性后,检测表明表达产物保留了对抗原的免疫反应性,对高转移性人肺癌PG细胞分泌的Ⅳ型胶原酶有抑制作用。同时在体外能抑制肿瘤细胞的侵袭,抑制率与表达产物的剂量浓度相关,1mg/ml浓度的片段具有68%的抑制率。结论:运用基因工程手段获得的单链抗体片段保留了亲本抗体的若干特性,对Ⅳ型胶原酶有抑制作用,并能抑制肿瘤细胞的侵袭。

Objective: To obtain the single chain antibody fragment (scFv) of a monoclonal antibody directed against type Ⅳ collagenase. Methods: The gene encoding scFv was cloned and constructed using phage display technique. The DNA fragment of scFv was cloned into expression vector pET 30a (+) and inducible expressed in E.coli BL21 (DE3). The immunoreactivity of expression product was detected with ELISA. The inhibition of targeted enzyme and invasion of tumor cells in vitro was investigated using gelatin zymography analysis and Boyden chamber assay, respectively. Results: The expressed protein mainly lies in inclusion body. After denaturation and renaturation, the expression product retains the affinity to the antigen type Ⅳ collagenase, and it partially inhibits the activity of type Ⅳ collagenase that was secreted by highly metastatic human lung carcinoma cell line PG cells. In addition, it inhibits the invasion of tumor cells in vitro in a dose dependent manner. The fragment of 1 mg/ml inhibits the invasion by 68%. Conclusion: The engineered scFv fragment retains the properties of the parent antibody. It shows the inhibition of type Ⅳ collagenase and invasion of tumor cells.