摘要
通过PCR技术扩增出约0.5kb的IL-6基因片段,并且采用含有P<sub>R</sub>P<sub>L</sub>串联启动子及SD区的高效表达载体pBV<sub>2</sub>20,经DNA重组技术,成功地构建了rhIL-6的基因工程菌E.coli DH<sub>5</sub>a/pBV<sub>2</sub>20IL-6。所表达的IL-6经SDS-PAGE证明约17.5kD,表达量占菌体总蛋白的30%以上。以IL-6依赖细胞株MH<sub>6</sub>0·BSF<sub>2</sub>检测,表达产物粗提物1:100复性后效价可达到5×10<sup>8</sup>u/ml,每升发酵菌液可获10<sup>1</sup>2u IL-6粗品。
Human IL-6 cDNA fragment (0. 5kb) was amplified through PCR method, inserted into EcoR I /BamH I site of pBV220 expression vector which contains PRPL promoters and SD sequence. After transformation and screening the E. coli DH5α in Amp+/LB medium, rhIL-6/pBV220 positive clone ratio was as high as 80%. The expression level of rhIL-6 was more than 30% of the total bacterial protein in SDS-PAGE, molecular weight was 17. 5kD.The biological activity of renatured bacterial extracts(1 : 100 dilution) was 5×108 u/ml on MH60 ·BSF2 cell lines, that is, 1012u rhIL-6/L bacterial fermentation.
出处
《中国生化药物杂志》
CAS
CSCD
1994年第4期235-238,共4页
Chinese Journal of Biochemical Pharmaceutics
