以乙肝病毒核心抗原HBcAg为载体的utrophin抗体的制备

Preparation of antibody against utrophin based on HBc particles as the expression vector

目的构建以乙肝病毒HBc为载体的带有utrophin的两个B细胞表位多肽的融合表达质粒pHBc-2UB,表达出融合蛋白并纯化,通过免疫新西兰大白兔获得抗utrophin的抗体。方法通过DNAstarⅡ模拟出utrophin的两个显著优势的B表位。用PCR法合成片段插入HBcAg序列的78位,将该合成片段克隆至pET28a载体中,构建重组表达质粒,表达出融合蛋白HBc-2UB,纯化后免疫新西兰白兔。再将该合成片段克隆至pGEX4T-2载体中,构建重组表达质粒,表达出融合蛋白GST-2UB,用于检测两个表位的抗体。结果测序结果表明重组质粒构建成功,SDS-PAGE电泳显示融合蛋白表达正确,ELISA检测到高滴度抗体。结论以HBc呈现utrophin的B表位被成功表达和纯化,获得高滴度的与两个B表位结合的抗体,为utrophin检测和DMD治疗药物研究打下了基础。

Objective A recombinant plasmid pHBc-2UB was constructed, in which 2 epitopes of utrophin were inserted into the trunicated HBc vector. The fusion protein expressed was purified and used to immunize the rabbits to induce antibody against utrophin. Methods Two B epitopes were selected by DNAstar analysis. By PCR, two epitopes were inserted into the sequence of amino acid residues 78 of HBc. The PCR product was then cloned into pET28a to construct recombinant expression plasmid, which was transformed to E. coli BL21 to express the fusion protein through induction. After purification,this protein termed as HBe-2UB was used to immunize the rabbits to induce antibody against utrophin. The PCR product was also cloned into pGEX4T-2 to express fusion protein GST- 2UB as the antigen for detecting the antibody against utrophin. Results The recombinant plasmid was successfully construtcted as demonstraned by DNA sequencing analysis. A fusion protein with correct molecular mass was expressed and confirmed by SDS-PAGE. High titre antibody was elicited in the rabbits＇immunized with HBe-2UB, as demonstrated by ELISA. Conclusion The HBc particle vector containing 2B epitopes of utrophin has been successfully prepared and purified. High titre antibody against utrophin has been prepared, which will be very useful for utrophin detection and research of DMD.