摘要
采用RT-PCR技术直接从激活的健康人外周血单个核细胞中扩增得到hIL-13的基因片段,通过DNA重组技术分别将其插入到含有PRPL启动子的高效表达载体pBV220及含tac启动子、lac1阻遏蛋白基因及凝血酶识别位点的融合蛋白表达载体pGEX-4T-2中,成功地构建了hIL-13的原核非融含蛋白表达菌株hIL-13-PBV220/DHS5a及融合蛋白表达菌株hIL-13-PGEX-4T-2/TG1,分别经42℃热诱导及异丙基硫代半乳糖苷(IPTG)化学诱导后,SDS-PAGE结果表明IL-13仅以GST融合蛋白的形式在E.coli中获得了表达,表达量约占菌体蛋白总量的10%~30%。IL-13的蛋白单体在原核细胞中表达量很低。
A 0. 3 kb of hIL- 13 fragment, directly amplified from activated healthy human peripheral blood mononuclear cells (HPBMC) by RT-PCR method, was separately inserted into high expression vectors pBV220 and pGEX-4T-2 containing tac promotor, lac I repressor and thrombin recognition site so as to construct prokaryotic non-fusion-protein expression bacterial IL- 13-pBV220/DH5a and fusion protein expression bacterial IL- 13-pGEX-4T-2/TG1. After heat induction (42℃ ) for the former engi neering bacterial and chemical induction (IPTG) for the latter, IL- 13 got expressed in E.coli only in an GST fusion-protein form, and the GST-IL-13 expression quantities took up about 10% ~ 30% of the total bacterial proteins. The expression of IL- 13 monomers in prokaryocytic cells was quite low-lev el.
出处
《中国生化药物杂志》
CAS
CSCD
1997年第5期224-228,共5页
Chinese Journal of Biochemical Pharmaceutics