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目的:用生物克隆的方法表达肺炎支原体(Mycoplasmsa pneamoniae,Mp)渗透压诱导蛋白C(Osmotically inducible protein C,Osm C)并测定其生物活性活性。方法:根据Mp的Osm C基因序列设计特定引物,PCR扩增Osm C基因。通过构建重组质粒p ET28a-Mp Osm C,并转化入大肠埃希菌BL21(DE3),来诱导目的蛋白表达。我们采用镍柱亲和层析法对Osm C蛋白进行纯化,再用氧化铁二甲酚橙试剂(FOX)测定Osm C蛋白的活性。结果:我们用生物克隆的方法得到Mp的Osm C基因片段,并成功构建重组质粒p ET28a-Mp Osm C。重组质粒转化入大肠埃希菌BL21(DE3)后经过表达我们得到重组Mp Osm C蛋白,再经亲和层析得到高纯度的目的蛋白,且具有分解H2O2的活性。结论:重组Mp Osm C蛋白具有过氧化物酶的活性,为研究肺炎支原体的抗氧化机制提供了理论依据。
OBJECTIVE: To express Mycoplasma pneamoniae (Mp) osmotic inducible protein C (Osm C) by biological cloning and determine its bioactivity. Methods: According to the Osm C gene sequence of Mp, specific primers were designed and PCR amplified Osm C gene. The recombinant protein p ET28a-Mp Osm C was constructed and transformed into Escherichia coli BL21 (DE3) to induce the expression of the target protein. Osm protein C was purified by Nickel column affinity chromatography and Osm protein C activity was determined using the Oxidized xylenol Orange reagent (FOX). Results: We obtained the Osm C gene fragment of Mp by biological cloning and successfully constructed the recombinant plasmid p ET28a-Mp Osm C. Recombinant plasmids were transformed into E. coli BL21 (DE3) and expressed to obtain recombinant Mp Osm C protein. The recombinant protein was purified by affinity chromatography to obtain the target protein of high purity, and had the activity of decomposing H2O2. CONCLUSION: The recombinant Mp Osm C protein has the activity of peroxidase, which provides a theoretical basis for studying the anti-oxidation mechanism of Mycoplasma pneumoniae.