重组人可溶性PDGFRβ/Fc在昆虫细胞Sf9中的表达

Expression of recombinant human soluble platelet-derived growth factor receptor Beta/Fc chimera in insect cell Sf9

【目的】利用昆虫细胞Bac-to-Bac杆状病毒表达系统表达血小板源性生长因子受体β(PDGFRβ)链膜外区与人IgGFc片段的可溶性受体融合蛋白sPDGFRβ/Fc,并检测重组蛋白的特异性和生物活性。【方法】采用Bac-to-Bac系统,构建重组转移质粒pFastbac-sPDGFRβ/Fc,转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,使目的基因与杆状病毒基因组DNA发生位点特异性重组,获得重组病毒DNA,将其通过脂质体转染昆虫细胞Sf9获得重组病毒。将该重组病毒感染Sf9无血清细胞系,在Sf9细胞中表达sPDGFRβ/Fc,对表达产物进行Western blotting检测和Protein A亲合层析纯化,并进一步通过MTT法检测获得的重组蛋白生物学活性。【结果】重组病毒感染Sf9细胞后,经Western blotting分析,能检测到一条分子量约为97kDa的特异性条带,与目的蛋白大小相符。通过ProteinA亲和层析,获得了纯度达75%以上,表达量为1μg/mL细胞培养上清的重组融合蛋白,MTT结果显示该重组融合蛋白sPDGFRβ/Fc具有抑制PDGF刺激的Balb/c3T3细胞增殖的能力。【结论】具有生物活性的重组可溶性受体融合蛋白sPDGFRβ/Fc可在昆虫细胞中成功地得到表达。

[ Aim] Bac-to-Bac baculovirus expression system was used to obtain a soluble fusion protein sPDGFRβ/Fc, combining the extracellular domain of PDGFR β chain and the Fc region of human IgG in insect cells sf9, and the specificity and bioactivity of the recombinant protein are detected. [ Methods ] The gene sPDGFRβ/Fc was cloned into a transfer vector pFastBac to form the recombinant donor plasmid pFastbac-sPDGFPβ/Fc, which was transformed into Escherichia coli DH10Bac. By transposition, sPDGFRβ/Fc gene was integrated into Bacmid, and a recombinant shuttle vector rBacmid-sPDGFRβ/Fc was constructed. Then the rBacmid-sPDGFPβ/Fc recombinant genome DNA was used to transfect Sf9 mediated by lipidbody, and the recombinant baculovirus rBacmid-sPDGRβ/Fc was obtained, which was further used to infect the serum-free cell St9 to express sPDGFRβ/Fc. Then the recombinant protein was purified with Protein A chromatography and analyzed by Western blotting and MTT assay. [ Results ] Western blotting analysis showed a specific band about 97 kDa, consistent with the target protein, 75% pure sPDGFRβ/Fc was obtained by using Protein A chromatography with a yield of 1 μg/mL culture medium, which was confirmed to be able to inhibit PDGF-induced proliferation of Balb/c 3T3 cell. [ Conclusion ] The recombinant protein sPDGFRβ/Fc with activity of inhibiting PDGF-induced cell proliferation can be successfully expressed in insect cell.