【摘要】 目的 克隆和表达HLA I类分子轻链(β2m)基因,为人工制备HLA I类分子提供基础。方法 利用逆转录PCR技术从Jeg-3细胞株中克隆β2m基因,与质粒pET22b(+)重组,构建原核表达载体pET22b(+)-β2m,转化宿主菌E.coli BL21(DE3),诱导β2m基因高效表达,并采用双抗体夹心ELISA法对表达产物进行抗原性和功能鉴定。结果 酶切和测序证实β2m基因克隆和载体构建成功,目的蛋白在宿主菌中高效表达于包涵体中;通过包涵体的分离和纯化获得初步纯化的β2m,所制备的β2m能够与特异性抗体结合,并能与HLA I类分子重链形成具有天然构象的HLA I类分子。结论 人β2m基因能够在原核宿主中高效表达,表达产物具有形成HLA I类分子的功能。更多还原

【Abstract】 Objective To study the cloning and expression of the light chain (β2m) gene of HLA I molecules to work out a protocol to prepare HLA class I /peptide complex. Methods The cDNA of β2m was cloned from the cell line Jeg-3 by RT-PCR and recombined into pET22b (+) to reconstruct prokaryotic expression vector pET22b (+) -β2m, which was used to transfer the host E. coli BL21 (DE3) and induce the high-level expression of β2m. The antigenicity and function of the target protein were determined by Sandwich ELISA. Results Restriction enzyme assay and DNA sequencing showed the β2 m gene was cloned and the vector was constructed successfully. The target protein was highly expressed in bacterial cells, and was primarily purified with the preparation of the inclusions. The target protein was able to bind to the β2m antibody specifically, and to form HLA I molecule associated with HLA-A2 heavy chain. Conclusion Human β2m gene can be highly expressed in the prokaryotic host, and the expressed product is able to form HLA I molecule when associated with the heavy chain and corresponding peptide.更多还原