抗HBsAg单链抗体与白细胞介素-2融合蛋白的构建与序列测定

目的:构建以人抗乙肝表面抗原（HBsAg）单链抗体（ScFv）为导向作用,白细胞介素-2（IL-2）为治疗作用的融合蛋白原核表达载体pQE 40/ScFv-IL-2。方法:应用PCR技术将抗HBsAg单链抗体与白细胞介素-2在分子水平上进行基因重组,构建中间载体pGEM7Zf（+）/ScFv-IL-2,经酶切鉴定及序列测定正确后,将目的基因片段克隆到pQE 40表达载体中,转化大肠杆菌,经IPTG诱导表达。结果:SDS-PAGE结果显示在相对分子质量约4.3×104Da处,可见蛋白表达带。以鼠抗人IL-2单克隆抗体经免疫印迹验证,该表达蛋白为所设计的融合蛋白。结论:融合蛋白ScFv-IL-2的成功构建及表达,为融合蛋白的纯化和研究开发新型的乙肝导向治疗的基因工程药物打下良好的基础。

The Construction and Expression of A Fusion Protein Consisting of Anti-HBsAg Single Chain Fv and Interleukin-2 E. coli

Objective: To construct an expression vector for anti-HBsAg ScFv and interleukin-2 fusion protein in E. coli-2. Methods: Using DNA recombination and overlap PCR technology, a cloned vector PGEM7Zf( + )/ScFv for anti-HBsAg ScFvand interleukin-2 fusion protein was constructed. Enzyme digestion and DNA sequence measurement confirmed that the ScFv-IL-2 gene had been correctly cloned to the vector, and then the target gene was cloned to the expression vector PQE40, choosing the passive clone to transform into E. coli and induced by IPTG to expression protein. Results: A recombinant expression plasmid PQE40/ ScFv-IL-2 was constructed. 4. 3 kU expression protein was observed by SDS-PAGE, and Western blot with mouse anti-human IL-2 McAb showed that the expressed products were designed fusion protein. Conclusion: The success in construction and expression of fusion protein makes it possible to carry out further studies on its purification and the targeted therapy of gene-engineering pharmaceutic to HB virus.