摘要
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。
To investigate the distribution and sequence conservation of colA gene in different serogroups of L. interrogans, as well as to determine the collagenase activity, expression and secretion of cola gene product of the spirochete during infection of host cells, PCR and its product sequencing were performed in seven L. interrogans serogroups that prevailing in China and the colA gene product of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was expressed by a prokaryotic expression system and extracted by Ni-NTA affinity chromatography. By using speetrophotometry, the ability of the recombi- nant protein（rColA） to hydrolyze I-IV type natural collagen proteins and two synthetic substrates（Azocoll and Pz-peptide） were determined, and the Km and Kcat values of rColA were also measured. Real-time fluorescent quantitative RT-PCR and Western Blot assay were applied to detect the colA-mRNA level change and ColA secretion of L. interrogans strain Lai during infection of HUVEC, BEAS-2B, L-02 or HEK293 cells. The results showed that the entire colA gene segments were amplified from the different L. interrogans serogroups with high nucleotide and amino acid sequence identities（99.4%-100%）. The rColA could hydrolyze the six substrates in varying extents, in which the hydrolytic ability on type-III collagen showed to be the strongest （P〈0.05）, with 2.16 mg/mL Km and 35.6 h i Kcat values. The colA-mRNA level of L. interrogans strain Lai was significantly increased （P〈0. 01） and the secreted ColA could be detectable in the supernatant of L. interrogans-cell co-cultures during infection of different host cells. These results indicated that the colA gene of L. interrogans is a sequence-conserved and extensively distributed collagenase-encoding gene. The up-regulated expression and external secretion of the gene product during infection of host ceils imply its practical role in the process of L. interrogans infection.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2016年第1期7-12,共6页
Chinese Journal of Zoonoses
基金
国家自然科学基金(81261160321和81171534)
浙江省自然科学基金(LQ14H190001)~~
