抗Sclerostin单链抗体基因的构建、表达及活性分析

目的 构建抗Sclerostin（SOST）单链抗体原核表达载体并对表达蛋白进行活性分析.方法 提取抗SOST单克隆抗体5H3D1杂交瘤细胞总RNA,用RT-PCR方法扩增抗体轻链可变区基因（VL）和重链可变区基因（VH）并通过重叠延伸拼接（SOE）PCR方法,在VL和VH基因之间引入Linker（Gly4Ser）3,连接成单链抗体SOST-scFv（VL-Linker VH）.将scFv基因克隆至表达载体PET-22b（＋）后转入HEK293细胞进行分泌表达,聚丙烯酰胺凝胶电泳SDS-PAGE鉴定表达产物,ELISA和West-blot检测表达蛋白的反应活性,茜素红结节染色法评估单链抗体对体外培养骨髓间充质干细胞成骨矿化的影响.结果 VL基因序列全长为339个碱基对,VH基因序列全长330个碱基对,均符合小鼠免疫球蛋白可变区基因特征,而SOST-scFv基因全长包括4个框架区（FR）、3个抗原互补决定区（CDR）以及具有抗体特征性的2个半胱氨酸残基;本研究构建的scFv全长687个碱基对,VL-Linker-VH为连接结构,SDS-PAGE分析表明大肠杆菌表达的scFv为可溶性蛋白,ELISA和West-blot检测证实表达scFv具有与SOST特异性结合的活性,细胞实验结果证实scFv能够促进骨髓间充质细胞成骨分化及矿化结节的形成.结论 成功构建抗SOST单链抗体表达载体,而且表达的scFv产物具有确切的免疫结合活性以及体外诱导成骨分化、矿化作用,为该抗体应用于骨质疏松疾病治疗奠定了实验基础.

Construction,expression and activity analysis of ScFv against SOST

Objective To construct the expression vector for ScFv against SOST and analyze antigen hinding activity of ScFv. Methods Total RNA was extracted from hybridoma cell line 5H3Dl secreting mAbs against SeFv and CDNA was amplified by retro- polymerase chain reaction （RT -PCR）. VL and VH were linked via a linker to construct SeFv （ VI, - Linker - VH） gene by SOE - PCR. The ScFv gene was clmled into vector and expressed in HEK293 cells. The expressed proteins were detected by SI）S - PAGE and antigen binding activity was analyzed by ELISA and Western blotting. The influences of ScFv on BMSCs differentiation were investiga- ted by alizarin red S （ARS） staining. Results The VL gene contained 339 base pairs and encoded 113 amine acid residues. The VH gene contained 330 base pairs and encoded 110 amine acid residues. There were four FRs, three CI）Rs and two characteristic cysteine residues in the YH gene and the VL gene, respectively. The ScFv gene contained 687 bases pairs and encode 229 amine acid residues with the structure of VL - linker - VH. ELISA analysis and Western blotting showed that the expression protein had specific antigen binding activity. The ScFv improve mineralized nodules formation. Conclusions The expression vector for ScFv against SOST has been successfully constructed to express protein having specific SOST binding activity. It has satisfactory effect on improving in vitro os- teogenic potential of BMSc and establishes the basis for the treatment of osteoporosis with antihody.