结核分枝杆菌低氧反应蛋白HRP1（Rv2626c）的分泌致病机制初探

  目的  验证结核分枝杆菌（Mycobacterium tuberculosis, Mtb）基因Rv2626c编码的低氧反应蛋白1（hypoxic response protein 1, HRP1）为分泌蛋白。  方法  从Mtb H37Rv标准毒力株基因组上扩增目的基因并添加His标签；运用扩增产物构建重组质粒，电转入耻垢分枝杆菌（Mycobacterium smegmatis, Ms）（MC2 155）构建重组菌；热诱导重组菌表达蛋白，检测培养滤液及细菌裂解液中蛋白的表达情况，将10 kDa培养滤液抗原（CFP-10）（Ms）和CFP-10（Mtb）设为阳性对照，将胞质蛋白热休克蛋白65（GroEL2）（Mtb）作为阴性对照。  结果  PCR成功扩增 HRP1 、GroEL2（Mtb）、CFP-10（Mtb）、CFP-10（Ms），并且重组质粒测序峰单一且信号稳定，鉴定重组质粒构建成功。成功构建重组Ms并实现蛋白GroEL2（Mtb）、CFP-10（Mtb）、CFP-10（Ms）、HRP1在Ms中的表达。在重组菌裂解液中和培养基滤液中均检测到目标蛋白HRP1；结果与阳性对照CFP-10一致；阴性对照GroEL2（Mtb）仅在细菌裂解液中检测到，在培养滤液在未检测到。  结论  Mtb基因Rv2626c编码的蛋白HRP1可以通过Ms的分泌系统分泌到细菌外，作为Mtb分泌蛋白，在Mtb致病机制中发挥作用。

Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)

  Objective  To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c.  Methods  The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC2 155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls.  Results  The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate.  Conclusion  The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.