CAJ | 학술논문

目的探讨原核表达稀释复性的可溶性人FcγR Ⅱ a(shuFcγRⅡa)的胞外区蛋白在体外与配体的结合活性.方法应用PCR技术从重组质粒pc3huR Ⅱ中扩增huFcγR Ⅱ a的胞外区基因,将其克隆于原核表达载体pET-28a中.所构建的重组质粒经PCR和酶切鉴定后转化大肠杆菌B121(DE3),IPTG诱导表达,制备融合蛋白包涵体.并采用多次洗涤法分离和纯化包涵体,用稀释法对纯化后的目的蛋白进行复性,ELISA和流式细胞术测定重组shuRⅡ在体外与配体的结合活性.结果扩增到了huFcγRⅡa的胞外区基因,所构建的pETshuRⅡ重组质粒经PCR和酶切鉴定与设计序列一致.转化BL21(DE3)后,IPTG诱导目的蛋白表达率约为30%.SDS-PAGE初步测定目的蛋白的相对分子质量(Mr)约为24.8×103,与理论预期值一致.破菌后电泳证实目的蛋白主要以包涵体形式表达,经多次洗涤后蛋白纯度大于90%.ELISA法测得重组shuRⅡ与人IgG在体外有良好的结合活性,流式细胞术测得重组shuRⅡ对配体与受体结合有抑制作用.结论本复性方法可得到和天然蛋白类似生物学活性的目的蛋白.
목적 탐토원핵표체희석복성적가용성인FcγR Ⅱ a(shuFcγRⅡa)적포외구단백재체외여배체적결합활성.방법 응용PCR기술종중조질립pc3huR Ⅱ중확증huFcγR Ⅱ a적포외구기인,장기극륭우원핵표체재체pET-28a중.소구건적중조질립경PCR화매절감정후전화대장간균B121(DE3),IPTG유도표체,제비융합단백포함체.병채용다차세조법분리화순화포함체,용희석법대순화후적목적 단백진행복성,ELISA화류식세포술측정중조shuRⅡ재체외여배체적결합활성.결과 확증도료huFcγRⅡa적포외구기인,소구건적pETshuRⅡ중조질립경PCR화매절감정여설계서렬일치.전화BL21(DE3)후,IPTG유도목적 단백표체솔약위30%.SDS-PAGE초보측정목적 단백적상대분자질량(Mr)약위24.8×103,여이론예기치일치.파균후전영증실목적 단백주요이포함체형식표체,경다차세조후단백순도대우90%.ELISA법측득중조shuRⅡ여인IgG재체외유량호적결합활성,류식세포술측득중조shuRⅡ대배체여수체결합유억제작용.결론 본복성방법가득도화천연단백유사생물학활성적목적 단백.
Objective To study the effect of soluble, refolded, recombinant extracellular domain of the human Fc gamma receptor Ⅱ a (huFcγRⅡa) on the binding of human IgG to cells. Methods Extra-cellular domain of the huFcγRⅡ a gene was amplified from recombinant plasmid pe3huR Ⅱ by PCR and then cloned into pET-28a vector. The recombinant plasmid pETshuR Ⅱ was transformed into E. coli BL21 (DE3) after identified by PCR and doubly digested. The inclusion bodies of fusion protein were extracted and purified by washing, dissolved in 6 mol/L guanidine buffer, and refolded by rapid dilution technique. The refolding protein activity was tested by ELISA and flow cytometry. Results Extraceilular domain of the huFcγRⅡa gene was successfully cloned into pET-28a. The results of SDS-PAGE showed that the molecular mass (Mr) of the expressed protein was 24.8 × 103, and the expression rate was 30%. The purity of recom-binant shuR Ⅱ was up to 90% after washing. ELISA showed that the recombinant shuR Ⅱ was able to bind human IgG in a dose dependent manner, shuRⅡ could competitively inhibit the binding of human IgG to huFcγRⅡa expressed on the surface of COS-7 cells by flow cytometry. Conclusion The results demon-strate that it is possible to obtain large quantities of recombinant shuR Ⅱ which has comparable binding prep-erties to those of the whole membrane bound huFcγR Ⅱ a.