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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pkn A,构建表达载体p ET43.1a-pkn A,在大肠杆菌中克隆表达了重组MTB Pkn A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p ET43.1a-pkn A;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。
OBJECTIVE To establish a high-throughput screening model of anti-TB drugs targeting protein kinase A, and to screen crude extracts of microbial fermentation broth with specific inhibitory activity. Methods The genomic DNA of Mycobacterium tuberculosis H37Rv was used as a template to amplify the target gene fragment pkn A. The expression vector p ET43.1a-pkn A was constructed and the recombinant MTB Pkn A protein was cloned and expressed in E. coli. The reaction method utilizes the change of maximum absorbance wavelength of nicotinamide adenine dinucleotide to oxidized nicotinamide adenine dinucleotide to establish and optimize high-throughput screening model of protein kinase A inhibitor. Results The expression vector p ET43.1a-pkn A was constructed successfully. A stable and sensitive high-throughput screening model of anti-TB drugs targeting Mycobacterium tuberculosis A protein kinase A was established. Using this model, 4000 microbial fermentation broths The samples were screened, and finally 21 positive samples that inhibited the activity of protein kinase A were obtained, the positive rate was 0.53%. The anti-branching activity of positive samples was tested by smear mycobacterium smegmatis and mycobacterium maritimeae Eight positive samples were finally obtained, of which four positive samples, I10AA-02916, I09AA-02717, I09AB-02729 and I08AB-00801, were tested for enzyme activity Inhibitory specificity, antibacterial activity are better, and cytotoxicity is lower. Conclusion A high-throughput screening model of anti-TB drug targeting protein kinase A was established. The positive samples of fermentation broth obtained by this model deserved further study.