本文關(guān)鍵詞： 肺炎支原體 滲透壓誘導(dǎo)蛋白C 克隆 表達(dá)　出處：《中國病原生物學(xué)雜志》2016年05期 　論文類型：期刊論文

【摘要】：目的表達(dá)肺炎支原體(Mycoplasma pneumoniae)滲透壓誘導(dǎo)蛋白C(OsmC)并測定其活性。方法在Mp中找出編碼OsmC蛋白的基因,根據(jù)Mp OsmC基因序列設(shè)計(jì)特定引物,PCR擴(kuò)增OsmC基因。利用EcoRI和XhoI對其進(jìn)行雙酶切,然后連接到表達(dá)載體pET28a,構(gòu)建重組質(zhì)粒pET28a-MpOsmC,轉(zhuǎn)化大腸埃希菌BL21(DE3),IPTG誘導(dǎo)目的蛋白表達(dá),采用鎳柱親和層析對重組OsmC蛋白進(jìn)行純化,利用氧化鐵二甲酚橙試劑(FOX)測定OsmC蛋白的活性。結(jié)果肺炎支原體Mpn625基因編碼OsmC蛋白。PCR擴(kuò)增OsmC基因片段大小為426bp,連接到原核表達(dá)載體pET28a得到重組質(zhì)粒pET28a-MpOsmC。重組質(zhì)粒轉(zhuǎn)化大腸埃希菌BL21(DE3),IPTG誘導(dǎo)表達(dá)分子質(zhì)量單位為19.4ku的重組Mp OsmC蛋白,與理論值相符。通過親和層析,得到高純度的目的蛋白。FOX試劑法測定Mp OsmC具有分解H_2O_2活性。結(jié)論重組Mp OsmC蛋白具有氧化酶活性,為探討肺炎支原體的抗氧化機(jī)制提供了理論依據(jù)。
[Abstract]:Objective the expression of Mycoplasma pneumoniae (Mycoplasma pneumoniae) osmotic pressure induced protein C (OsmC) and determine its activity. Methods to find the encoding OsmC protein in Mp gene, the specific primers designed according to the Mp sequence of OsmC gene, PCR gene was amplified by OsmC. The double enzyme digestion by EcoRI and XhoI, and then connected to the expression vector pET28a. The recombinant plasmid pET28a-MpOsmC was transformed into E.coli BL21 (DE3), the expression of target protein was induced by IPTG, using nickel affinity chromatography of recombinant OsmC protein was purified using ferric oxide two reagent xylenol orange (FOX) determination of the activity of the OsmC protein. The results of Mycoplasma pneumoniae Mpn625 gene encoding OsmC protein.PCR amplified OsmC gene fragment size is 426bp. Connected to the prokaryotic expression vector pET28a to obtain recombinant plasmid pET28a-MpOsmC. recombinant plasmid was transformed into Escherichia coli BL21 (DE3), IPTG induced expression of a molecular mass of recombinant 19.4ku Mp Osm The C protein is consistent with the theoretical value. By affinity chromatography, a high purity target protein is obtained. The.FOX reagent method for the determination of Mp OsmC has the ability to decompose H_2O_2 activity. Conclusion the recombinant Mp OsmC protein has the activity of oxidase, which provides a theoretical basis for exploring the antioxidant mechanism of Mycoplasma pneumoniae.

【作者單位】： 邵陽醫(yī)學(xué)高等�？茖W(xué)校;南華大學(xué)病原生物學(xué)研究所;
【基金】：湖南省科技計(jì)劃項(xiàng)目(No.2013FJ3067)
【分類號(hào)】：R375.2
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本文編號(hào)：1532628