猪白细胞介素18成熟蛋白基因克隆及在不同原核表达系统中的表达

Cloning of Procine Interleukin-18 Mature Gene and Its Expression in Different Prokaryotic Expression Vectors

RT-PCR方法直接从猪脾脏淋巴细胞中扩增出猪白细胞介素18成熟蛋白基因的cDNA,克隆到pGEM-T载体,构建重组质粒pGEM-TpIL18,转化E.coliJM109感受态细胞,取PCR和酶切鉴定为阳性的重组质粒进行序列测定.测序结果表明,pIL-18成熟蛋白基因核苷酸长度为474 bp,编码157个氨基酸.将其分别克隆到表达载体pQE30、pET-28a、pGEX6P-1中,构建重组表达质粒pQE-mpIL18、pET-mpIL18、pGEX-mpIL18.用IPTG诱导表达,重组菌菌体裂解物SDS-PAGE可检测到相对分子质量分别为19 000、21 000、45 000的重组蛋白.经薄层扫描分析,pQE-mpIL18、pET-mpIL18、pGEX-mpIL18菌体裂解物分别占菌体总蛋白的17%、28%、27%,均以包涵体形式存在.

Porcine interleukin-18 mature protein gene was amplified from porcine spleen cells by RT-PCR. PCR product was cloned into the T vector pGEM-T for sequencing. The result showed that the nucleotide sequence of this gene was 474 bp, which encodes 157 amino acids. It was subcloned into the prokaryotic expressing plasmid vectors pQE30, pET-28a, pGEX6P-1, and transformed into host E. coli strain JM109 or BL21 for expression. The expression of pIL-18 mature protein gene was identified by 5DS-PAGE. The result revealed that the expression products were fusion protein with relative molecular mass of 19 000,21 000,45 000, respectively, and the percentage of expression protein in E. coil protein was 17% ,28% ,27% respectively.