中文摘要：

目的克隆表达出能够抑制补体活化的双功能域小分子补体受体1型（complement receptor type 1，CR1）衍生物。方法从外周血提取总RNA，进行RT—PCR扩增出功能性片段I（CR1-SCR1—3）；以本室构建的pET-32a—CR1-SCR15—18为模板扩增功能性片段Ⅱ（CRl-SCR15—17）；利用重叠延伸PCR（SOE—PCR）法扩增出包含双功能域的融合基因。将融合基因重组于pET-32a（＋）载体，转化大肠杆菌Rosetta，经酶切及DNA序列测定鉴定后，IPTG诱导表达，SDS—PAGE和Western blot对表达蛋白质进行分析鉴定。蛋白质经Ni柱亲和层析及透析复性后，采用50％补体溶血抑制试验（CH50）进行功能鉴定。结果酶切及DNA序列测定证实成功构建出了pET-32a—CR1-SCR（1-3＋15-17）重组表达载体，IPTG诱导表达后在相对分子质量（Mr）为63×10^3处有一明显的条带，经Western blot鉴定为目的蛋白，Ni柱亲和层析获得纯度约为92％的目的蛋白，功能试验证实，在0～200μg／ml的融合蛋白剂量范围内，随着浓度的增加，补体抑制活性增强。结论成功构建并在大肠杆菌内表达了具有较高生物活性的重组双功能域小分子CR1衍生物，为体内保护实验研究提供实验数据。

英文摘要：

Objective To construct and express a small complement receptor type 1 （CR1） derivative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional fragment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already constructed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with splicing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a （ ＋ ） and transformed into E. coli Rosetta （DE3）. The inserted gene was verified by enzyme digestion and DNA sequencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The complement inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a（ ＋ ）. The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass（ Mr ） of the expressed protein was 63 × 10^3. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully constructed and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.