摘要
目的 :克隆及表达Fas死亡信号激发域 (FasActivationDomain ,FasAD)片段 ,获得具有生物学活性的FasAD多肽。方法 :应用半巢式逆转录 多聚酶链反应 (RT PCR)扩增Fas死亡信号激发域cDNA片段 ,构建Intein表达型原核表达载体FasAD pTYB12 ,应用IMPACTTM CN系统表达及进行一步法亲合层析分离、纯化FasAD多肽。结果 :DNA序列测定显示克隆的FasADcDNA碱基排列顺序与Genebank(M6 74 5 4 )所示完全一致。重组表达载体FasAD pTYB12经IPTG诱导 ,成功表达可溶性融合蛋白 ,进一步分离、纯化获得分子量约 5 0 0 0的FasAD多肽 ,Westemblot显示FasAD多肽能被兔抗人Fas多抗识别。初步的生物学活性鉴定显示 ,该FasAD多肽抑制rhFasL诱导的细胞凋亡的生物学活性最高可达 70 %。结论 :可利用IMPACTTM CN系统制备Fas死亡信号激发域的小分子量多肽 ,为深入研究Fas结构与功能的关系及与配体的相互作用提供了相关的实验基础 ,为进一步开发相关的生物免疫调节剂提供了实验依据。
Objective:To clone and express human Fas activation domain(FasAD)with the bioactivity.Methods:The FasAD cDNA was amplified by seminested RT PCR,and then inserted into the prokaryote vector of pTYB12 expressed intein protein to construct the recombinant plasmid of FasAD pTYB12.The FasAD peptide was expressed and purified in IMPACT TM CN system by the method of one step affinity purification.Results:Sequence analysis revealed that cloned FasAD cDNA sequence was completely same as that of Genebank record(M67454).Soluble fusion protein was successfully expressed by induction of IPTG.The FasAD peptide with a molecular weight of 5 000 was obtained by purification and was recognized by the rabbit anti human Fas polyclonal antibody in Western blot analysis.It’s activity of inhibition of apoptosis induced by rhFasL can each to 70% in primary biological detection.Conclusion:The above results indicated that FasAD peptide could be prepared by using the IMPACT TM CN system,thus laid a relatively experimental foundation for further research of relationship between structure, function and interaction with it’s ligands,and for further development of biological immune modulator. [
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2003年第9期599-601,610,共4页
Chinese Journal of Immunology
基金
广东省自然科学基金课题资助 (No .980 2 2 2 )
