人IL-10 cDNA克隆、表达及其生物学活性

目的 在大肠杆菌中表达重组人白细胞介素10(Human Interleukin,hIL-10)。方法 用RT-PCR自白血病细胞株K562 RNA扩增hIL-10 cDNA片段(约500bp),克隆至质粒载体pSK(+),并对克隆的DNA片段进行序列分析。用限制酶EcoRI和 BamHI消化pSK/IL-10重组质粒,分离hIL-10片段,并插入原核表达载体pBV220的相应限制酶位点,酶谱分析鉴定重组表达载体pBV/IL-10。转化菌株经42℃诱导,用SDS-PAGE和Westem blot鉴定表达的重组蛋白。表达的重组蛋白经谷胱甘肽复性缓冲液复性,用RT-PCR检测其对外周血单核细胞(PBMC)合成IFN-γ的抑制作用。结果 RT-PCR扩增的DNA片段与hIL-10 cDNA大小一致。重组质粒pSK/IL-10的DNA序列分析显示,克隆的DNA序列与文献报道的hIL-10 cDNA序列一致。SDS-PAGE表明,重组蛋白相对分子质量为18000,其表达量达菌体总蛋白的20％左右。Westem blot分析显示,重组蛋白能特异性地与抗hIL-10抗体结合,复性的重组蛋白能明显抑制PBMC合成IFN-γ。结论 已成功地构建了表达具有生物学活性的重组hIL-10(Recombinant hIL-10,rhIL-10)的工程菌株。

Cloning and Expression of Human IL-10 cDNA and Biological Activity of Expressed Product

Objective To express recombinant human IL - 10 in E. coli. Methods A hIL - 10 DNA fragment, with a length of about 500bp, was amplified from the RNA of leukemia cell strain K562 by RT - PCR and cloned to plasmid pSK( + ) ,and the cloned DNA fragment was sequenced. The recombinant plas-mid pSK/IL- 10 was digested with EcoRI and BamHI, then hIL- 10 fragment was isolated and inserted to the corresponding restriction site on procaryotic expression vector pBV220.The recombinant plasmid PBV/IL - 10 was identified by enzymogram and transformed to E. coli, then expressed by induction at 42℃ .The expressed product was identified by SDS-PAGE and Western blot and renaturalized with glutathione buffer, and the inhibitory effect of it on the production of IFN - γ in PBMC was detected by RT - PCR. Results The length of DNA fragment amplified by RT - PCR was consistent with that of hIL - 10 cDNA. DNA sequencing of pSK/ IL- 10 revealed that the cloned DNA sequence was identical to that of reported hIL- 10 cDNA.SDS - PAGE proved that the expressed product, with a relative molecular weight of 18000,contained about 20% of total somatic protein. Western blot showed that the recombinant protein could specifically bind to anti-hIL-10 antibody and,after being renaturalized, could inhibit the production of IFN-γ in PBMC significantly. Conclusion A recombinant bacterial strain for expressing hIL - 10 with biological activity was successfully constructed.