论文部分内容阅读
目的 :利用基因工程抗体技术构建抗人整合素ανβ3 单链抗体(scFv)。方法 :从分泌抗人整合素ανβ3 单抗 (mAb)的杂交瘤细胞E10总RNA中 ,用RT PCR扩增VH 和VL 基因 ,对其核苷酸序列分析后 ,通过PCR在VH 和VL 基因间插入柔性连接子(Gly4Ser) 3,组装成scFv基因 ,并克隆至原核表达载体pTIG TRX中。以重组子转化大肠杆菌BL2 1(DE3 )诱导目的基因表达。结果 :转化菌可表达相对分子质量 (Mr)为 3 10 0 0的scFv。Westernblot证实 ,具有His6标签蛋白的表达。经低剂量的IPTG诱导和较低温度培养 ,scFv获得了可溶性形式表达。表达产物经Ni NTA琼脂糖层析纯化后纯度达 91%以上。ELISA鉴定证实 ,scFv具有良好的抗原结合活性。结论 :成功地构建并表达抗人整合素ανβ3 的scFv ,为进一步临床研究奠定了基础。
OBJECTIVE: To construct an anti-human integrin ανβ3 single chain antibody (scFv) by using gene engineering antibody technology. Methods: VH and VL genes were amplified by RT-PCR from total E10 total RNA of hybridoma cells secreting anti-human integrin ανβ3 monoclonal antibody (mAb). After nucleotide sequence analysis, the VH and VL genes were amplified by PCR The flexible linker (Gly4Ser) 3 was inserted into the scFv gene and cloned into prokaryotic expression vector pTIG TRX. The recombinant plasmid was transformed into E. coli BL21 (DE3) to induce the expression of the target gene. Results: Transformants could express scFv with relative molecular mass (Mr) of 310,000. Westernblot confirmed His6 tagged protein expression. After low-dose IPTG induction and lower temperature incubation, scFv was obtained in soluble form. The purity of the expressed product was over 91% after purification by Ni NTA agarose chromatography. ELISA identification confirmed that scFv has good antigen binding activity. Conclusion: The successful construction and expression of anti-human integrin αvβ3 scFv laid the foundation for further clinical research.