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目的研究重组人磷脂酶D2(rhPLD2)在体内外的免疫学活性,尤其是研究其抗炎活性。方法将编码rh-PLD2的cDNA片段经RT-PCR克隆至pET-30a载体中,并纯化来自包涵体中由大肠杆菌表达的rhPLD2重组蛋白。通过测定豚鼠慢性哮喘模型的支气管肺泡灌洗液(BALF)和血液中嗜酸性粒细胞的数量及肺组织中IL-5、MMP-9的表达来评定rhPLD2重组蛋白的抗炎活性。结果从人Daudi细胞中获得了不含膜结合部位及信号肽的,可编码631个氨基酸的rh-PLD2 cDNA序列(GenBank登录号:AY178289)。rhPLD2重组蛋白的纯度约76%,其生物活性达到50.9745U/L(0.9212g/L)。用rhPLD2治疗慢性哮喘动物模型证实了其的抗炎功效。包括:能下调炎性细胞因子IL-5的表达。结论由大肠杆菌表达的rhPLD2重组蛋白具有抗炎活性。
Objective To study the immunological activity of recombinant human phospholipase D2 (rhPLD2) in vitro and in vivo, especially its anti-inflammatory activity. Methods cDNA fragments encoding rh-PLD2 were cloned into pET-30a vector by RT-PCR and purified from rhPLD2 recombinant protein expressed in E. coli in inclusion bodies. The anti-inflammatory activity of rhPLD2 recombinant protein was assessed by measuring the number of bronchoalveolar lavage fluid (BALF) and blood eosinophils in guinea pig chronic asthma model and the expression of IL-5 and MMP-9 in lung tissue. Results The rh-PLD2 cDNA sequence encoding 631 amino acids (GenBank accession number: AY178289) was obtained from human Daudi cells without membrane binding site and signal peptide. The purity of rhPLD2 recombinant protein was about 76%, and its bioactivity reached 50.9745U / L (0.9212g / L). Animal models of chronic asthma treated with rhPLD2 demonstrated its anti-inflammatory efficacy. Including: can down-regulate the expression of inflammatory cytokine IL-5. Conclusion The rhPLD2 recombinant protein expressed by E. coli has anti-inflammatory activity.