CAJ | 학술논문

目的构建Fas胞外区(eFas)基因的表达载体,表达纯化重组蛋白,进行多克隆抗体的制备,为进一步功能研究奠定基础.方法通过重叠PCR获得eFas基因的鳊码序列,构建pET-22b(+)/eFas表达载体,转化大肠杆菌Rosetta-gami,IPTG诱导表达,Ni-NTA柱亲和纯化,SDS-PAGE鉴定重组蛋白的纯度.将纯化的eFas融合蛋白免疫新西兰白兔制备多克隆抗体,通过ELISA方法检测多克隆抗体的效价.结果获得了eFas的编码序列与表达裁体.目的蛋白主要在包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上.结论 eFas融合蛋白基因的构建、表达、纯化以及多克隆抗体的制备,为进一步研究Fas提供了材料.
목적 구건Fas포외구(eFas)기인적표체재체,표체순화중조단백,진행다극륭항체적제비,위진일보공능연구전정기출.방법 통과중첩PCR획득eFas기인적편마서렬,구건pET-22b(+)/eFas표체재체,전화대장간균Rosetta-gami,IPTG유도표체,Ni-NTA주친화순화,SDS-PAGE감정중조단백적순도.장순화적eFas융합단백면역신서란백토제비다극륭항체,통과ELISA방법검측다극륭항체적효개.결과 획득료eFas적편마서렬여표체재체.목적단백주요재포함체중표체,표체량점균체총단백적30%이상,순화적중조단백순도체95%이상.결론 eFas융합단백기인적구건、표체、순화이급다극륭항체적제비,위진일보연구Fas제공료재료.
Purpose To construct expression vector of Fas extracellular region gene(eFas) ,to express and purify recombination protein and to prepare polyclonal antibody, which have laid a foundation of studying its function. Methods The eFas gene encoding sequence was acquired through overlapping PCR, and pET-22b ( + )/eFas expression vector was constructed. Then this vector was transformed into E. coli Rosetta-gami. Re-combinant protein was expression being induced by IPTG,and was purified using Ni-NTA matrix of affinity chromatograph. The purity of recombination protein was identified by SDS-PAGE. Hereafter, the purified eFas recombinant protein was immunized to New Zealand white rabbit in order to prepare polyclonal antibody. The titer of polyclonal antibody was determined by ELISA. Results The encoding sequence and expression vector of eFas was obtained while the interest protein was mainly expressed in the inclusion body. The eFas fusion protein's expression quantity accounts for more than 30% proportion of total E. coli protein. The eFas protein we obtained was provided with the purity of at least 95 % . Conclusion The successful constrution, expression and purification of FasL fusion protein and preparation of polyclonal antibody will provide some material for further studies of Fas.