摘要
为了在乳酸乳球菌中分泌表达具有生物活性的猪IL-18蛋白,并检测其生物活性,故通过分离猪外周血单核淋巴细胞(PBMC),以其为模板,采用RT-PCR方法扩增猪白细胞介素18(pIL-18)基因,将目的基因与乳酸乳球菌表达载体pAMJ399进行连接,并电转化至乳酸乳球菌MG1363中,通过SDS-PAGE和Western blotting分析检测目的蛋白的表达,并通过脾淋巴细胞增殖试验和细胞病变抑制法对pIL-18的生物活性进行检测。Western blotting分析检测结果与生物活性检测结果显示,在重组菌pAMJ399-pIL18/MG1363的上清和菌体沉淀中19 kDa处均出现pIL-18的特异蛋白反应带,且分泌表达的pIL-18蛋白能明显促进猪脾淋巴细胞的增殖,并对病毒增殖有明显的抑制作用。以上结果表明pIL-18可在乳酸乳球菌分泌表达,且表达产物具有良好的生物活性。
To obtain active protein of plL-18 expression in Lactococcus lactis, and to observe its biological activity, the total RNA was extracted as template from peripheral blood mononuclear cells. Porcine interleukin 18 (plL-18) was amplified by RT-PCR. The resulting fragment was cloned into pAMJ399 L. lactis vector, and then transformed to L. lactis MG1363 cells by electroporation. Expression of plL-18 protein was detected by SDS-PAGE and Western-blotting. Bioactivity of the product was tested by pig spleen lymphocyte proliferation test and cytopathogenic effect inhibition assay. The result of Western blotting and bioactivity test shows that the molecular weight of plL-18 protein was 19 kDa. The react line was observed in both supernatant and precipitated of the recombinant bacteria pAMJ399-plL18/MG1363. The expressed plL-18 can promote the proliferation of pig spleen lymphocyte, and significantly inhibit virus multiplication. As conclusion, porcine interleukin-18 was successfully expressed in L. Lactis, and the product was biologically active.
出处
《生物工程学报》
CAS
CSCD
北大核心
2014年第10期1541-1548,共8页
Chinese Journal of Biotechnology
基金
"十二五"农村领域国家科技计划课题(No.2011AA10A213)
东北农业大学博士启动基金(No.2009RC59)资助~~
关键词
猪IL-18
乳酸乳球菌
分泌表达
生物活性
porcine IL-18, Lactococcus lactis, secretory expression, biological activity