人清道夫受体A胞外段的原核表达

目的获得具有生物学活性的人清道夫受体A（SRA）胞外段蛋白。方法从人外周血单个核细胞（PBMC）中提取RNA，逆转录为eDNA，采用PCR技术从PBMC．eDNA中扩增出目的基因片段，将其克隆至原核表达载体pET41a（＋），经PCR、限制性酶切和测序确证后，以IPTG诱导其在大肠杆菌中的表达。包涵体经变性、复性后以Ni—NTA亲和层析柱纯化融合蛋白，以SDS-PAGE、Western blowing进行分析鉴定。FCM及EHSA方法检测SRA胞外段对ConA诱导PBMC细胞增殖及分泌细胞因子IL-2的影响。结果PCR扩增得到长约1100bp的目的基因片段，插入pET41a（＋）载体所获重组表达载体pET41a—SRAECD的酶切图谱和序列与预期的一致。重组子经诱导表达。表达产物主要以包涵体形式存在。以Ni-NTA亲和层析柱纯化获得约75000Mr的SRA胞外段融合蛋白。纯化的人SRAECD蛋白能与抗人SRA单克隆抗体特异性结合并具有活性，可抑制T细胞增殖及细胞因子IL-2的分泌。结论获得了具有生物学活性的人SRA胞外段蛋白．为进一步探索SRA的效应功能提供了实验材料。

Prokaryotic expression of human scavenger receptor A extra-cellular domain

Objective To express the extracellular domain of human scavenger receptor A （SRA） in E. coli. Methods The gene fragment of the SRA was amplified by PCR from the human PBMC cDNA, and then inserted into the prokaryotic expression vector pET41a（＋） and identified by PCR, restriction mapping, and sequencing. The recombinant expression vector was transformed into E. coli strain BL21 （DE3） and induced to express the recombinant protein by IPTG. The fusion protein was purified by Ni-NTA gel affinity chromatography from denatured and renatured products of inclusion bodies and analyzed by SDS-PAGE and western blotting. ELISA and FCM determine the effect of expressed protein on ConA-induced PBMC proliferation and IL-2 production. Results A gene fragment of about 1 100 bp was amplified by PCR and the recombinant expression vector pET41a-SRAECD was constructed, and was confirmed by restriction maping and sequencing. The expressed products in BL21 （DE3） were existed as inclusion body, from which the His-SRAECD fusion protein of Mr 75 000 were obtained by Ni-NTA gel affinity chromatography. The purified human SRAECD protein could react with anti-SRA monoclonal antibody and inhibit ConA activated T cell proliferation and the secretion of cytokines IL-2. Conclusion The recombinant human His-SRAECD protein with bioactivities is obtained, which provides the necessary material for studying the functions of SRA.