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目的检测微型核酶对篮氏贾第鞭毛虫(简称贾第虫)组织蛋白酶B基因体外转录体的切割效率。方法用RNA draw软件对贾第虫组织蛋白酶B基因序列二级结构进行模拟分析,并按照微型核酶设计的原则,选取合适的核酶切割位点,设计微型核酶序列,构建含贾第虫组织蛋白酶B基因短反义序列和长反义序列的两个微型核酶嵌合型犬贾第虫病毒重组质粒(CRzS和CRzL),以三磷酸甘油醛脱氢酶基因的微型核酶(GRzL)和无核酶的组织蛋白酶B基因(PCB)重组质粒作对照。将重组质粒线性化后体外转录为mRNA,然后用微型核酶体外切割组织蛋白酶B基因转录体,采用实时荧光定量PCR检测切割效率。结果微型核酶CRzS和CRzL对组织蛋白酶B基因转录体切割效率分别为75.42%和83.14%,微型核酶GRzL的切割效率为0.02%,无核酶PCB切割效率为23.0%。结论微型核酶可有效切割贾第虫组织蛋白酶B基因转录体,为开展体内抑制组织蛋白酶B基因表达试验奠定了基础。
Objective To detect the cleavage efficiency of microzyme in vitro transcript of cathepsin B gene of Giardia lamblia (Giardia lamblia). Methods RNA draw software was used to simulate the secondary structure of Giardia cathepsin B gene sequence. According to the design principle of mini ribozyme, we selected the appropriate ribozyme cleavage site, designed the mini ribozyme sequence, Two mini-ribozyme Giardia canis recombinant plasmids (CRzS and CRzL) with short antisense and long antisense sequences of cathepsin B and GRzL ) And nuclease-free cathepsin B gene (PCB) recombinant plasmid as a control. The recombinant plasmids were linearized and then transcribed into mRNA in vitro. Then the cathepsin B gene transcripts were cleaved in vitro with the mini-ribozyme, and the cutting efficiency was detected by real-time fluorescence quantitative PCR. Results The efficiency of cleavage of cathepsin B gene transcripts by mini ribozymes CRzS and CRzL was 75.42% and 83.14%, respectively. The cleavage efficiency of the minizygote GRzL was 0.02% and the ribonuclease PCB cleaving efficiency was 23.0%. Conclusion The mini-ribozyme can effectively cleave Giardia cathepsin B gene transcript, which lays the foundation for the in vivo inhibition of cathepsin B gene expression.