hUNC93-B1基因的克隆、表达纯化及抗体制备

Expression and purification of fusion protein of cloned hUNC93-B1 gene and antibody preparation

目的:构建人hUNC93-B1基因的原核表达载体,诱导其表达并纯化该蛋白,制备特异性高效价抗体.方法:设计针对hUNC93-B1基因的特异性引物,通过PCR的方法扩增目的基因,克隆入pMD-18T载体,测序全部正确后亚克隆入原核表达载体pRSET-A.将构建的载体导入大肠杆菌BL21中,用IPTG诱导表达.经SDS-PAGE电泳鉴定后用镍柱纯化融合蛋白.与免疫佐剂联用,免疫家兔制备特异性抗体.结果:PCR扩增得到长度为1808bp的片断,结果与GenBank中人hUNC93-B1的基因序列完全一致,成功构建了原核表达载体hUNC93-B1-pRSET-A.导入大肠杆菌后经诱导得到一条约Mr72000的新蛋白条带.免疫家兔后获得了1∶64000的高效价特异性抗人hUNC93-B1血清.结论:成功地克隆了人hUNC93-B1基因,使其编码的蛋白质在原核细胞中顺利表达,并纯化出该种蛋白.制备了特异性抗体,为研究hUNC93-B1基因与人类心血管疾病的关系提供依据.

AIM： To construct the prokaryotic expression vector containing hUNC93-B1 gene, then induce the expression of this gene and purify the fusion protein, and at last using target protein to make the specific and high titer antibody. METHODS： The specific primers of hUNC93 gene were designed and the gene was amplified by PCR. The PCR product was then ligated into pMD- 1ST vector. After sequence analysis, it was sub-cloned into pR- SET-A which could express the target protein as a （His）6-tagged fusion. The bacterial strain BL21 （DE3） was used as the host cell to induce the expression of the fusion protein by IPTG. The purified hUNC93-B1 fusion protein was easily achieved on a Ni2- NTA affinity column by virtue of its （His） 6 tag. Rabbits were im- muned with the expressed protein and immunological adjuvant for specific antibody preparation. RESULTS： An 1808bp length fragment was amplified by PCR. The sequence was completely matched with hUNC93-B1 gene sequence registered in Genbank; hUNC93-Bl-pRSET-A vector was successfully constructed. After the fusion protein was transfected into E. coil, a Mr 72 000 new protein was got by IPTG induction. A specific and high titer anti- hUNC93-B1 serum in 1：64 000 was gained at last. CONCLUSION： hUNC93-B1 gene has been successfully cloned and hUNC93-B1 fusion protein has been correctly expressed in E. coil and purified by Ni2-NTA agarose beads. A specific and high titer antibody has been prepared. All this makes it possible to do further investigation on the relationship between hUNC93-B1 and heart failure.