IL-13融合蛋白表达载体与非融合蛋白表达载体的构建与比较

Construction and Comparison of IL-13 Fusion-protein and Non-fusion-pro-tein Expression Vector

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.