人IL-32-IgG4(Fc)融合蛋白在CHO/DG44细胞中的表达

The expression of human IL-32-IgG4(Fc) fusion gene in CHO/DG44 cell line

【目的】构建人白细胞介素32(IL-32)和IgG4 Fc段的融合基因真核表达载体IL-32-IgG4(Fc)-pOp-tiVEC,建立稳定转染的中华仓鼠卵巢细胞(CHO/DG44)克隆,并检测其重组蛋白的表达。【方法】用PCR方法从脂多糖(LPS)激活的人CD4+T细胞cDNA中扩增出IL-32蛋白基因,并克隆到IgG4(Fc)-pOptiVEC载体上,构建重组质粒IL-32-IgG4(Fc)-pOptiVEC,并进行酶切和测序鉴定。采用脂质体法,将重组质粒线性化后转染CHO/DG44细胞,进行RT-PCR检测,筛选阳性克隆,并对筛选的阳性克隆进行氨甲喋呤(MTX)加压筛选。利用ProteinG-Agarose亲和层析纯化转染CHO/DG44细胞培养上清液中的融合蛋白IL-32-IgG4(Fc),通过SDS-PAGE、Western-blot对融合蛋白IL-32-IgG4(Fc)的表达和生物学活性进行检测。【结果】PCR扩增获得了长度为564 bp的人IL-32蛋白基因cDNA,经测序与GenBank报道的序列一致。真核表达载体IL-32-IgG4(Fc)-pOptiVEC经EcoRⅠ和XhoⅠ双酶切,获得了IL-32蛋白基因片段,其长度为564 bp。筛选出了能稳定表达IL-32-IgG4(Fc)融合蛋白的CHO/DG44细胞克隆。亲和纯化后的IL-32-IgG4(Fc)融合蛋白经SDS-PAGE和Western-blot鉴定,其分子质量约为50.0 ku,与预期结果一致。MTX加压后,IL-32-IgG4(Fc)融合蛋白的表达量明显升高。【结论】成功构建了人IL-32蛋白融合基因的真核表达载体IL-32-IgG4(Fc)-pOptiVEC,获得了能够表达具有生物学活性的IL-32-IgG4(Fc)融合蛋白的CHO/DG44细胞克隆。

【Objective】The study constructed eukaryotic expression vector IL-32-IgG4（Fc）-pOptiVEC for expression of IL-32-IgG4（Fc） fusion protein in CHO cells.【Method】IL-32 gene was amplified by PCR from the cDNA library of human CD4＋ T cell after LPS induction and inserted into IgG4（Fc）-pOptiVEC resulting in an eukaryotic expression vector IL-32-IgG4（Fc）-pOptiVEC.Then the eukaryotic expression vector was identified by enzyme digestion and sequencing.The CHO/DG44 cells were successfully transfected with the lined plasmid IL-32-IgG4（Fc）-pOptiVEC by Lipofectamine TM2000 and confirmed by detecting mRNA of the IL-32 in the transfected cells by RT-PCR.The positive clones were further screened in the presence of MTX at different concentrations.The IL-32-IgG4（Fc） fusion protein was purified from medium from the transfected CHO/DG44 clones by protein G affinity chromatography and then its expression and bioactivity were identified by SDS-PAGE and Western-blot.【Result】A 564 bp long cDNA sequence of human IL-32 gene was cloned by PCR.The sequence of IL-32 was consistent with that reported in GenBank.Eukaryotic expression vector IL-32-IgG4（Fc）-pOptiVEC was confirmed constructed successfully by restriction enzyme digestion,and was transfected into the CHO/DG44 cells which were further selected in presence of MTX.The immune activity of the fusion protein was verified by Western-blot,and its relative molecular weight was about 50 ku,which was very close to its expected value.The production of IL-32-IgG4（Fc） fusion protein was increased obviously by MTX selection.【Conclusion】An eukaryotic expression vector IL-32-IgG4（Fc）-pOptiVEC has been constructed successfully and the recombinant CHO/DG44 cell clone that can steadily express IL-32-IgG4（Fc） fusion protein with bioactivity has been obtained.