猪白细胞介素18成熟蛋白基因的克隆与表达

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。

The cloning of pig interleukin 18 mature protein gene and its expression in Lactic acid bacteria

Objective To clone porcine interleukin 18 mature protein gene and express it in Lactobacillus plantarum NC8(Lb.plantarum NC8).Methods The porcine interleukin 18 mature protein gene(pIL-18) was amplified from its spleen cells by RT-polymerase chain reaction(PCR) method,then subcloned to pMD18-T and sequenced.The positive gene fragment was subcloned to lactic acid bacteria expression vectors PSIP-409 to construct recombinant expression vector PSIP-409-IL-18 and identified by enzyme digestion and PCR.The recombinant expression vector PSIP-409-IL-18 was transformed to Lb.plantarum NC8 through electric perforation techniques.After the recombinant strains were induced to express,the expression product was analysed by SDS-PAGE and Western-blot.Results Sequencing results showed that the pIL-18 mature protein gene contained 579 bp of bases and coded 193 nucleotides.The recombinant expression vector pSIP-409-IL-18 was proved to have been successfully constructed by enzyme digestion and PCR.SDS-PAGE and Western-blot analysis indicated that 18 kD fusion protein was expressed by these recombinant strains and it can react with the IL-18 polyclonal antibody of mouse anti-porcine.Conclusion The pIL-18 mature protein gene was amplified.And the pIL-18 recombinant Lactic acid bacteria with boligical activity was successfully constructed,which lay the foundation for the development of IL-18 recombinant Lactobacillus.