CAJ | 학술논문

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rhPLD2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 rhPLD2 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50.9745 U/L(0.9212 g/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.
목적 연구중조인린지매D2(rhPLD2) 재체내외적면역학활성,우기시연구기항염활성.방법 장편마rhPLD2적Cdna편단경RT-PCR극륭지Pet-30a재체중,병순화래자포함체중유대장간균표체적rhPLD2중조단백.통과측정돈서만성효천모형적지기관폐포관세액(BALF)화혈액중기산성립세포적수량급폐조직중 IL-5、 MMP-9적표체래평정rhPLD2중조단백적항염활성.결과 종인Daudi 세포중획득료불함막결합부위급신호태적,가편마631개안기산적 rhPLD2 Cdna서렬(GenBank등록호:AY178289). rhPLD2중조단백적순도약76%,기생물활성체도50.9745 U/L(0.9212 g/L).용 rhPLD2치료만성효천동물모형증실료기적항염공효.포괄:능하조염성세포인자IL 5적표체.결론 유대장간균표체적rhPLD2중조단백구유항염활성.
To investigate the immunological activities of the recombinant human phosphatase D2 (rhPLD2) in vitro and in vivo, especially its ability to reduce inflammatory reactions, the cDNA fragment encoding rhPLD2 was cloned into prokaryotic expression vector pET30a by RT-PCR and the recombinant protein rhPLD2 expressed in E.coli was purified from the inclusion bodies, while the anti inflammatory activity of rhPLD2 was determined by the amount of eosinophils in bronchoalveolar fluid(BALF) and blood and the expression of IL-5 and MMP-9 in lung tissues of guinea pig model of chronic asthma. It was found that the rhPLD2 recombinant protein was obtained from human Daudi cells by cloning to E.coli, which contained no membrane-binding site and signal peptide. The cDNA sequence encoded 631 amino acid residues (GenBank Accession Number: AY178289). The purity of the rhPLD2 approached up to 76% with a bioactivity of 50.9745 units/L (0.9212 g/L). In addition, the anti inflammatory effect of rhPLD2 protein could be demonstrated in the guinea pig model of chronic asthma after treatment with rhPLD2 protein, such as down regulation in the expression of the inflammatory cytokine IL-5. It is concluded that the anti-inflammator activity of the recombinant human truncated PLD2 protein produced from the E.coli plasmid can be demonstrated both in vitro and in vivo.