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

摘要:

[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℃）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 kDa。Western blot表明，39℃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℃）；ELISA表明，39℃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℃）。[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。

关键词:结核分枝杆菌;Rv3194c蛋白;透明质酸;硫酸软骨素;Ι型胶原蛋白

Abstract:

[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 Rv3194c gene from Mycobacterium tuberculosis, and its ability to adhere M. tuberculosis was studied.[Methods] Rv3194c protein was expressed in prokaryotic system. Rv3194c protein was separately incubated with hyaluronic acid, chondroitin sulfate and collagen Ι overnight at different temperature (37, 38, 39, 40℃). Then component changes of culture supernatant were tested by Western blot and ELISA.[Result] Western blot showed that Rv3194c protein expressed in prokaryotic system, with a molecular weight of about 35 kDa, was mainly in soluble form. Western blot showed that His-Rv3194c protein in supernatant of 39℃ experimental group was significantly less than that of other experimental groups (37, 38, 40℃)(***P<0.001). ELISA showed that hyaluronic acid, chondroitin sulfate and collagen Ι in supernatant of 39℃ experimental group was significantly less than that of other experimental groups (37, 38, 40℃)(***P<0.001).[Conclusion] For the first time it was affirmed that Rv3194c protein with detected activity of adhesions in this study will be targeted to the development of the new anti-M. tuberculosis drug.

Key words:Mycobacterium tuberculosis;Rv3194c protein;hyaluronic acid;chondroitin sulfate;collagen