CAJ | 학술논문

目的:克隆及可溶性表达 HLA-B*2704重链胞外功能区,并对其生物学功能进行初步鉴定.方法:以HLA-B 位点全长 cDNA 为模板,用 PCR-SSP 方法扩增 HLA-B*2704重链胞外区 cDNA,经测序鉴定后与 pET32a 可溶性核表达载体构建其重组核表达系统,并在大肠杆菌 BL21中表达,采用 Western 印迹及微量淋巴细胞毒阻断实验初步鉴定该蛋白的特异性及其生物学特性.结果:扩增出 HLA-B*2704重链胞外功能区 cDNA 片段,构建的HLA-B*2704 cDNA-pET32a 可溶性核表达载体可在大肠杆菌 BL21表达系统中得到较好表达,表达量约占菌体总蛋白的40%;通过 Western 印迹鉴定了表达蛋白的特异性;通过微量淋巴细胞毒阻断实验发现该重组蛋白具有生物活性并可特异性阻断 HLA-B27阳性细胞的微量淋巴细胞毒反应.结论:在大肠杆菌中表达了具有生物学活性的HLA-B*2704重链胞外功能区,为强直性脊柱炎的机理研究及特异性阻断药物的筛选提供了实验基础和新的靶标.
목적:극륭급가용성표체 HLA-B*2704중련포외공능구,병대기생물학공능진행초보감정.방법:이HLA-B 위점전장 cDNA 위모판,용 PCR-SSP 방법확증 HLA-B*2704중련포외구 cDNA,경측서감정후여 pET32a 가용성핵표체재체구건기중조핵표체계통,병재대장간균 BL21중표체,채용 Western 인적급미량림파세포독조단실험초보감정해단백적특이성급기생물학특성.결과:확증출 HLA-B*2704중련포외공능구 cDNA 편단,구건적HLA-B*2704 cDNA-pET32a 가용성핵표체재체가재대장간균 BL21표체계통중득도교호표체,표체량약점균체총단백적40%;통과 Western 인적감정료표체단백적특이성;통과미량림파세포독조단실험발현해중조단백구유생물활성병가특이성조단 HLA-B27양성세포적미량림파세포독반응.결론:재대장간균중표체료구유생물학활성적HLA-B*2704중련포외공능구,위강직성척주염적궤리연구급특이성조단약물적사선제공료실험기출화신적파표.
Objective: To investigate the feasibility of cloning and expression of soluble HLA-B*2704 extracellu?lar heavy chain and its preliminary biological functions. Methods: Full length cDNA of HLA-B locus was used as template to clone and amplify HLA-B*2704 extracellular heavy chain by PCR-SSP, and the correct sequence of HLA-B*2704 cDNA confirmed by DNA sequencing was inserted into pET32a soluble prokaryotic expression vec?tor. The expression of HLA-B*2704 extracellular heavy chain was tested by Western blot, and its biological func?tion was detected through micro-lymphocyte toxicity blockage experiment. Results: We successfully amplified the cDNA fragments of HLA-B*2704 extracellular heavy chain and constructed the fragment into soluble prokaryotic expression vector of pET32a. By using E.coli BL21 expression system, the total protein expression was around 40%. Western blot identified the HLA-B*2704 specificity of the expressed protein, and micro-lymphocytes block?ing experiment revealed that the protein possessed the biological activity to specifically block the B27 specific cyto?toxicity reaction. Conclusion: HLA-B*2704 extracellular heavy chain with biological activity was successfully ob?tained in E.coli, which will be of some help to the investigation of the mechanism of ankylosing spondylitis as well as for the selection of specific blockage drugs.