以蛋白激酶A为靶点的抗结核药物高通量筛选模型的建立和应用

目的 建立以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型，应用该模型筛选具有特异性酶活抑制活性的微生物发酵液粗提物样品。 方法 以结核分枝杆菌 H37Rv 基因组 DNA 为模板，扩增目的基因片段 pknA，构建表达载体 pET43.1a-pknA，在大肠杆菌中克隆表达了重组 MTB PknA 蛋白；采用三步级联的反应方法，利用还原型烟酰胺腺嘌呤二核苷酸到氧化型烟酰胺腺嘌呤二核苷酸这一反应最大吸光值波长的变化，建立和优化蛋白激酶 A 抑制剂高通量药物筛选模型。 结果 成功构建了表达载体 pET43.1a-pknA；建立了稳定灵敏，可用于靶向结核分枝杆菌蛋白激酶 A 的抗结核药物高通量筛选模型；利用该模型对4000个微生物发酵液粗提物样品进行筛选，最终得到21个抑制蛋白激酶 A 活性的阳性样品，阳性率0.53%；以耻垢分枝杆菌和海分枝杆菌为检定菌，平板纸片法检测阳性样品的抗分枝杆菌活性，然后对阳性样品的细胞毒性和酶活抑制特异性进行评价后，最终得到8个阳性样品，其中 I10AA-02916、I09AA-02717、I09AB-02729、I08AB-00801这4个阳性样品酶活抑制特异性、抗菌活性均较好，且细胞毒性较低。 结论 建立了高稳定性的以蛋白激酶 A 为靶点的抗结核药物高通量筛选模型，应用该模型所得到的发酵液阳性样品值得进一步研究。

Establishment and application of a high throughput screening assay for inhibitors of Mycobacterium tuberculosis PknA

Objective To establish and validate a high throughput assay for screening the inhibitors of Mycobacterium tuberculosis PknA. Methods A pknA gene from the genome DNA of Mycobacterium tuberculosis H37Rv was amplified and recombinant plasmid pET43.1a-pknA was constructed to express the PknA protein in E.coli. Using purified PknA, a high throughput screening model was established and optimized to screen for PknA inhibitors. The microbial fermentation extracts were screened using the screening assay, and the anti-bacterial activity, cytotoxicity and specific inhibitory effect of positive samples were further assessed. Results 21 out of 4000 microbial fermentation extracts were identified with inhibitory effect on activity of PknA, among which 4 samples showed antibacterial activity, lower cytotoxicity and better specificity of inhibitory effect. Conclusion The establishment of high throughput screening method and these 8 positive microbial fermentation extracts will facilitate the development of novel antitubercular agents.