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目的克隆猪白细胞介素18(pIL-18)成熟蛋白基因,并在植物乳杆菌(Lb.plantarum)NC8中进行表达。方法通过RT-PCR方法从猪脾脏细胞中扩增出pIL-18成熟蛋白基因,克隆到T载体pMD18-T后测序;将阳性基因片段克隆至大肠埃希菌-乳酸菌穿梭表达载体pSIP-409构建重组表达载体pSIP-409-IL-18,进行酶切和PCR鉴定;应用电穿孔技术将其转化至Lb.plantarum NC8中,经SppIP诱导表达后,进行SDS-PAGE及Western-blot分析。结果经测序,pIL-18成熟蛋白基因核苷酸长度为579 bp,编码193个核苷酸;酶切和PCR鉴定证明成功构建了重组表达载体pSIP-409-IL-18;SDS-PAGE及Western-blot分析表明重组菌表达了18 kD的融合蛋白,该重组蛋白可以与鼠抗猪IL-18多克隆抗体反应。结论成功克隆了pIL-18成熟蛋白基因,并获得有生物活性的pIL-18重组乳酸菌,为研制开发IL-18重组乳酸菌制剂奠定基础。
Objective To clone the mature protein gene of porcine interleukin 18 (pIL-18) and express it in Lb.plantarum NC8. Methods The pIL-18 mature protein gene was amplified from pig spleen cells by RT-PCR and cloned into T vector pMD18-T. The positive gene fragment was cloned into Escherichia coli-lactic acid bacteria shuttle expression vector pSIP-409 The recombinant expression vector pSIP-409-IL-18 was digested with restriction endonuclease and identified by PCR. The recombinant plasmid was transformed into Lactobacillus plantarum NC8 by electroporation and expressed by SppIP. SDS-PAGE and Western-blot analysis were performed. Results The nucleotide sequence of the mature protein of pIL-18 was 579 bp, encoding 193 nucleotides. The recombinant plasmid pSIP-409-IL-18 was successfully constructed by restriction enzyme digestion and PCR. SDS-PAGE and Western blot -blot analysis showed that the recombinant strain expressed a 18 kD fusion protein that can react with mouse anti-porcine IL-18 polyclonal antibody. Conclusion The pIL-18 mature protein gene was successfully cloned, and the pIL-18 recombinant lactic acid bacteria with biological activity was obtained. It laid the foundation for the development of IL-18 recombinant lactic acid bacteria.