【摘要】：目的:反義技術(shù)的發(fā)展為研發(fā)新一代抗菌藥物提供了新思路。本研究以大腸桿菌16S rRNA為藥物作用靶標(biāo),運(yùn)用本實(shí)驗(yàn)室建立的MAST技術(shù)平臺(tái)篩選其反義核酸作用靶點(diǎn);通過體外和胞內(nèi)實(shí)驗(yàn)對(duì)篩選靶點(diǎn)的有效性進(jìn)行驗(yàn)證,以期獲得能高效結(jié)合反義寡核苷酸的靶點(diǎn)序列;根據(jù)有效作用靶點(diǎn)合成硫代反義寡核苷酸研究其抑菌效果,為研發(fā)新一代核酸抗菌藥物進(jìn)行有益探索。方法:通過在轉(zhuǎn)錄16S rRNA過程中引入生物素標(biāo)記的UTP,將其固定到親和素磁珠上,保持RNA的自然折疊狀態(tài)。然后與寡核苷酸文庫雜交,篩選能結(jié)合的文庫序列從而闡明靶點(diǎn),該方法即為MAST技術(shù)(mRNA accessible site tagging)。運(yùn)用該技術(shù)篩選獲得16S rRNA6個(gè)反義寡核苷酸結(jié)合靶點(diǎn)。根據(jù)靶點(diǎn),設(shè)計(jì)并合成6條靶點(diǎn)特異的反義寡核苷酸和5條10-23型脫氧核酶(DNAenzyme)。采用反義寡核苷酸依賴RNase H切割技術(shù)對(duì)6條反義寡核苷酸體外對(duì)16S rRNA的結(jié)合能力進(jìn)行檢測(cè),并采用10-23型脫氧核酶催化切割技術(shù)對(duì)其中5條脫氧核酶的體外結(jié)合切割活性進(jìn)行檢測(cè)。經(jīng)體外驗(yàn)證,所篩6個(gè)靶點(diǎn)中5個(gè)靶點(diǎn)為16S rRNA可及位點(diǎn),其中靶點(diǎn)Ⅴ(907-926nt)結(jié)合活性最好。然后,構(gòu)建原核表達(dá)載體用IPTG誘導(dǎo)表達(dá)單倍體錘頭狀核酶(sRZ)和雙倍體錘頭狀核酶(dRz),利用表達(dá)的核酶胞內(nèi)特異降解16S rRNA抑制大腸桿菌生長,對(duì)靶點(diǎn)Ⅴ胞內(nèi)有效性進(jìn)行驗(yàn)證。針對(duì)體外和胞內(nèi)驗(yàn)證有高效結(jié)合活性的靶點(diǎn)Ⅴ,合成硫代反義寡核苷酸與通透性好的大腸桿菌SM101溫育,檢測(cè)其對(duì)大腸桿菌生長的抑制效果,進(jìn)行核酸抗菌素的初步研究。結(jié)果:運(yùn)用MAST技術(shù)篩選獲得位于16S rRNA 179-198、446-465、497-516、887-906、907-926、1236-1255nt6個(gè)靶點(diǎn)(Ⅰ-Ⅵ);反義寡核苷酸依賴RNase H切割活性分析結(jié)果顯示,靶點(diǎn)Ⅱ-Ⅵ為反義寡核苷酸體外高效結(jié)合靶點(diǎn);脫氧核酶催化切割活性分析結(jié)果顯示,靶點(diǎn)Ⅴ體外活性最為顯著;胞內(nèi)驗(yàn)證結(jié)果表明,針對(duì)靶點(diǎn)Ⅴ的單倍體和雙倍體核酶對(duì)大腸桿菌生長均有抑制效果,且雙倍體核酶效果更為顯著;針對(duì)靶點(diǎn)Ⅴ的硫代反
[Abstract]:Objective: the development of antisense technology provides a new idea for the development of a new generation of antibacterial drugs. In this study, Escherichia coli 16s rRNA was used as the target of drug action, and the target of antisense nucleic acid was screened by using the MAST technology platform established in our laboratory. The effectiveness of screening target was verified by in vitro and intracellular experiments in order to obtain the target sequence which could bind antisense oligodeoxynucleotides efficiently. The antibacterial effect of antisense thioate oligodeoxynucleotides (ASODN) was studied according to the effective target, and a useful exploration for the development of new generation of nucleic acid antibacterial drugs was carried out. Methods: biotin-labeled UTP, was introduced into the 16s rRNA transcription process to immobilize it to avidin magnetic beads to maintain the natural folding state of RNA. And then hybridized with the oligonucleotide library to screen the binding library sequence to clarify the target. This method is called MAST technique (mRNA accessible site tagging). 16s rRNA6 antisense oligodeoxynucleotides (ASODN) binding targets were screened by this technique. According to the target, six target specific antisense oligonucleotides and five 10-23 deoxyribozyme (DNAenzyme). Were designed and synthesized. The binding ability of six antisense oligodeoxynucleotides to 16s rRNA in vitro was detected by RNase H cleavage technique. The in vitro binding activity of 5 deoxyribozymes was detected by 10-23 type deoxyribozyme catalytic cleavage technique. Five of the six screened targets were 16s rRNA accessible sites, among which the binding activity of target 鈪，

本文編號(hào)：2470069