结核分枝杆菌Rv3194c蛋白的表达、纯化及活性鉴定

Expression, purification and characterization of Rv3194c protein from Mycobacterium tuberculosis

【目的】Rv3194c基因编码的是结核分枝杆菌的PDZ信号蛋白,本研究对其是否具有黏附素特性进行了探索。【方法】对Rv3194c进行原核表达,然后Rv3194c蛋白分别与透明质酸、硫酸软骨素、Ι型胶原蛋白在不同温度(37、38、39、40°C)的缓冲液中孵育过夜,然后用Western blot和ELISA分析其上清液中组分含量的变化。【结果】Western blot鉴定结果表明,His-Rv3194c蛋白以可溶形式表达,蛋白质的分子质量大小为35 k Da。Western blot表明,39°C实验组的上清中His-Rv3194c蛋白含量(***P<0.001)显著小于其它实验组(37、38、40°C);ELISA表明,39°C实验组的上清中透明质酸(HA)、硫酸软骨素(CS)、Ι型胶原蛋白(CollagenΙ)的含量(***P<0.001)极显著小于其它实验组(37、38、40°C)。【结论】首次证实了Rv3194c蛋白具有黏附素特性,可成为研发新型抗结核药物的靶点蛋白。

[Objective] PDZ [Post-synaptic density-95（PSD-95）, Drosophilia tumor suppressor protein diskslarge-1（DLG）, the tight junction protein zonula occludentes 1（ZO-1）] signal protein was encoded by Rv3194 c gene from Mycobacterium tuberculosis, and its ability to adhere M. tuberculosis was studied. [Methods] Rv3194 c protein was expressed in prokaryotic system. Rv3194 c protein was separately incubated with hyaluronic acid, chondroitin sulfate and collagen Ι overnight at different temperature（37, 38, 39, 40 °C）. Then component changes of culture supernatant were tested by Western blot and ELISA. [Result] Western blot showed that Rv3194 c protein expressed in prokaryotic system, with a molecular weight of about 35 k Da, was mainly in soluble form. Western blot showed that His-Rv3194 c protein in supernatant of 39 °C experimental group was significantly less than that of other experimental groups（37,38, 40 °C）（＊＊＊P〈0.001）. ELISA showed that hyaluronic acid, chondroitin sulfate and collagen Ι in supernatant of 39°C experimental group was significantly less than that of other experimental groups（37, 38, 40 °C）（＊＊＊P〈0.001）.[Conclusion] For the first time it was affirmed that Rv3194 c protein with detected activity of adhesions in this study will be targeted to the development of the new anti-M. tuberculosis drug.