摘要
根据GenBank报道的人白细胞介素-2(hIL-2)基因分析设计并合成引物,以本室构建的pPIC9K/IL-2突变体(编码125位半胱氨酸→丙氨酸、18位亮氨酸→蛋氨酸、19位亮氨酸→丝氨酸)为模板,PCR扩增出突变型人白细胞介素-2基因.用EcoRI和BamHⅠ对目的基因进行双酶切后,插入大肠杆菌表达载体pBV220,转化宿主菌JM109,经菌落PCR筛选阳性重组子、酶切鉴定,DNA序列分析证实,重组质粒pBV220/IL-2含有突变型人IL-2编码序列及正确的开放阅读框.经42℃热诱导表达,SDS-PAGE电泳显示,诱导表达碎菌后的沉淀中有分子质量大小约为15kD的外源蛋白产生,经Western印迹证明为rhIL-2.成功建立了突变型人白细胞介素-2(rhIL-2)基因的大肠杆菌表达系统,为rhIL-2的生产及临床应用奠定基础.
One pair of primers were synthesized according to the hIL-2 gene reported in GenBank. RhlL-2 gene was amplified by PCR from pPIC9K/IL-2 triple mutant （C125A/ L18M/ L19S） we previously estab lished by using site-specific mutagenesis with a synthetic oligonucleotide as the template. RhlL-2 gene, after dige JM109. stion withEcoR Ⅰ and BamH Ⅰ, was inserted into pBV220, and then transferred into E. coli Colony PCR, restriction analysis and sequence ana pBV220/rhIL-2 contained the coding sequence of rhIL 2 and lysis showed that the recombinant plasmid the open reading frame was right. In SDSPAGE analysis after induction at 42 ℃, foreign protein with a molecular mass of 15 KDa was found in the precipitation of the disrupted strains and the protein was also confirmed by Western blotting. Thus, a recombinant bacterial strain expressing rhIL-2 was constructed successfully. This study provides a foundation for the production of hIL-2 mutant and clinical practice.
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第8期93-97,共5页
Journal of Southwest University(Natural Science Edition)
基金
重庆市自然科学基金资助项目(CSTC2007BB5353)
