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目的了解不同问号钩端螺旋体(简称钩体)血清群colA基因分布和序列保守性、表达产物胶原酶活性以及感染细胞时colA基因表达水平变化和产物分泌情况。方法采用PCR及其产物测序法检测我国主要流行的7个问号钩体血清群代表株中colA基因并了解其序列保守性。构建问号钩体黄疸出血群赖型赖株colA基因原核表达系统,Ni-NTA亲和层析法提取表达的目的重组蛋白rColA。采用分光光度法检测rColA水解I~IV型天然胶原蛋白及Azocoll和Pz-肽合成底物能力并测定其Km和Kcat值。采用实时荧光定量RT-PCR和Western Blot分别检测问号钩体赖株感染HUVEC、BEAS-2B、L-02、HEK293细胞时colA-mRNA水平变化及ColA分泌情况。结果不同血清群问号钩体均能扩增出全长colA基因片段,其核苷酸和氨基酸序列相似性高达99.4%~100%。所构建的colA基因表达系统能有效表达rColA。rColA能不同程度地水解上述6种底物,但水解III型胶原蛋白能力最强(P<0.05),其Km和Kcat值分别为2.16mg/mL和35.6h-1。问号钩体赖株感染各靶细胞时colA-mRNA水平显著升高(P<0.01),问号钩体-细胞共培养物上清中可检出ColA。结论问号钩体colA基因为序列保守、分布广泛的胶原酶编码基因,感染细胞时该基因产物表达上调并外分泌,从而在问号钩体感染宿主过程中发挥实际作用。
Objective To understand the colA gene distribution and sequence conservation of serogroups of Leptospira interrogans (Leptospira interrogans), the expression of collagenase activity and the expression of colA gene in infected cells and the product secretion. Methods The colA gene of seven serogroups of serogroups of Leptospira interrogans (Leptospira interrogans) was detected by PCR and its product sequencing method and its sequence conservation was studied. To construct the prokaryotic expression system of colA gene of Leptospira interrogans strain Leuconostoc meristem, and to extract the expressed recombinant protein rColA by Ni-NTA affinity chromatography. Spectrophotometry was used to determine the ability of rColA to hydrolyze natural collagen type I ~ IV and the substrates of Azocoll and Pz-peptide to synthesize Km and Kcat values. Real-time fluorescent quantitative RT-PCR and Western Blot were used to detect colA-mRNA levels and ColA secretion in HUVEC, BEAS-2B, L-02 and HEK293 cells infected with Leptospira interrogans. Results The full-length colA gene fragment was amplified from different serogroups, and its nucleotide and amino acid sequence similarity was as high as 99.4% -100%. The constructed colA gene expression system can effectively express rColA. rColA hydrolyzed the above six substrates to different extents, but hydrolyzed Type III collagen was the strongest (P <0.05), with Km and Kcat values of 2.16 mg / mL and 35.6 h-1, respectively. ColA-mRNA levels were significantly increased (P <0.01) when infected with Leptospira interrogans, and ColA was detected in the co-culture supernatant of Leptospira interrogans. CONCLUSIONS The colA gene in C. interrogans is a conserved and widely distributed collagenase-encoding gene. The expression of this gene is up-regulated and exocrine in infected cells, and thus plays an important role in host infection.