摘要
为研究分枝杆菌PPE13基因在宿主细胞的生物功能,本试验利用成簇的规律间隔的短回文重复序列干扰(clustered regularly interspaced short palindromic repeat interference,CRISPRi)构建海分枝杆菌PPE13诱导型敲低菌株。CRISPRi主要包括表达失活的Cas9蛋白(dead cas9,dCas9)和特异性单向导RNA(single guide RNA,sgRNA),通过形成复合体特异性识别相应DNA序列并抑制目的基因的转录。用无水四环素(anhydrotetracycline,ATC)诱导海分枝杆菌PPE13诱导型敲低菌株,提取细菌RNA,利用定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)技术检测dCas9、PPE13基因的表达情况,观察细菌在不同时间D600 nm的变化,分析诱导剂质量浓度和目的基因转录水平之间的关系。使用PPE13敲低菌株感染巨噬细胞并检测胞内菌的存活情况。结果表明,在ATC诱导后,dCas9表达水平显著增高。针对PPE13基因设计的4个sgRNA中,sgRNA1对PPE13基因转录的抑制作用最为明显,为70%~90%。生长曲线结果显示,敲低PPE13菌株在48 h内D600 nm无明显变化。诱导剂的质量浓度越高,对PPE13基因的转录水平的抑制作用越强。敲低PPE13可降低细菌在细胞内的增殖能力。本研究构建了海分枝杆菌PPE13敲低菌株,为深入研究分枝杆菌PPE13感染宿主细胞的致病机制奠定了基础。
In order to study the biological function of Mycobacterium PPE13 gene in host cells,CRISPRi was used to construct Mycobacterium marinum PPE13 inducible knockdown strain.CRISPRi mainly includes the expression of dead cas9(dCas9)and specific single guide RNA(sgRNA),which specifically recognize the corresponding DNA sequence and inhibit the transcription of the target gene by forming a complex.Anhydrotetracycline(ATC)was used to induce the PPE13 inducible knockdown strain of Mycobacterium marinum,and the bacterial RNA was extracted.The expression of dCas9 and PPE13 genes was detected by quantitative polymerase chain reaction(qPCR).The changes of D_(600 nm)of bacteria at different time were observed,and the relationship between the concentration of inducer and the transcription level of target gene was analyzed.Macrophages were infected with PPE13 knockdown strain,and the survival of intracellular bacteria was detected.The results showed that the expression level of dCas9 was significantly increased after ATC induction.Among the four sgRNAs designed for PPE13 gene,sgRNA1 has the most obvious inhibitory effect on PPE13 gene transcription,about 70%-90%.The results of growth curve showed that there was no significant change in D_(600 nm)within 48 h.The higher the concentration of inducer,the stronger the inhibitory effect on the transcription level of PPE13 gene.Knockdown of PPE13reduces the proliferative capacity of bacteria inside the cell.The construction of Mycobacterium marinum PPE13knockdown strain provides a basis for further study of the pathogenic mechanism of Mycobacterium PPE13infection in host cells.
作者
王垚垚
毕斯琪
陈标
宋厚辉
杨杨
WANG Yaoyao;BI Siqi;CHEN Biao;SONG Houhui;YANG Yang(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology,College of Animal Science and Technology/College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2023年第8期1670-1676,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(32272951)
浙江省自然科学基金资助项目(LY21C180001)
浙江省属高校基本科研业务费资助项目(2020YQ008)。
