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目的研究人类白细胞抗原(human leukocyte antigen,HLA)-B* 2705分子结构对其在原核细胞可溶性表达的影响。方法采用RT-PCR法将HLA-B* 2705基因的全长、胞外区和α1、α2功能区分别进行克隆并连接到pET-32a(+)表达载体。经IPTG诱导表达,并经超声破碎、层析和透析纯化蛋白,再经体外微量细胞淋巴毒反应检测蛋白质活性。结果成功构建3种pET-32a(+)表达载体,经IPTG诱导后,HLA-B* 2705基因全长和胞外区主要以包涵体的形式表达,可溶性蛋白的量较低;而α1和α2功能区则以高效可溶性形式表达,通过体外微量细胞淋巴毒实验证实,HLA-B* 2705的α1和α2功能区可溶性蛋白可与HLA-B27抗体特异性结合,并成功阻断补体介导的细胞毒作用。结论α1和α2功能区能够在原核细胞中高效表达可溶性蛋白,并具有蛋白功能,而HLA-B* 2705基因全长和胞外区主要以包涵体的形式表达,表明蛋白质的大小和空间结构对于其可溶性表达具有重要影响。
Objective To study the effect of molecular structure of human leukocyte antigen (HLA) -B * 2705 on its soluble expression in prokaryotic cells. Methods The full-length, extracellular domain and α1, α2 functional regions of HLA-B * 2705 gene were cloned by RT-PCR and ligated into pET-32a (+) expression vector respectively. After induced by IPTG, the protein was purified by ultrasonication, chromatography and dialysis. The protein activity was detected by in vitro microcytosis. Results Three pET-32a (+) expression vectors were successfully constructed. After induced by IPTG, the full-length and extracellular domains of HLA-B * 2705 were mainly expressed as inclusion bodies with low amounts of soluble proteins. The α1 and α2 The functional regions were expressed in highly efficient and soluble form. The in vitro cytotoxicity assay showed that α1 and α2 soluble proteins of HLA-B * 2705 could specifically bind to HLA-B27 antibody and successfully block the expression of complement-mediated cells Toxic effects. Conclusion The α1 and α2 functional domains can express soluble proteins in prokaryotes efficiently and have protein function. The full-length and extracellular domains of HLA-B * 2705 are mainly expressed as inclusion bodies, indicating that the size and spatial structure of proteins Its soluble expression has a significant impact.