小鼠Klf11基因真核表达载体的构建、结构功能预测与初步验证

Eukaryotic expression vector construction,structure and function prediction and preliminary verification of mouse Klf11 gene

【目的】旨在克隆小鼠Kruppel样转录因子家族成员11(KLF11)基因并构建其真核表达载体,在小鼠AML12肝实质细胞系中过表达Klf11后探究其对脂代谢相关基因的调控作用。【方法】以小鼠肝脏组织cDNA为模板,通过PCR扩增小鼠Klf11基因的蛋白编码区(CDS),随后将其连接至pcDNA3.1-Puro-N-3HA载体,构建重组质粒。利用酶切、PCR及测序对重组质粒进行鉴定,将鉴定正确的质粒命名为pcDNA3.1-mKLF11。利用在线软件对Klf11序列及其编码蛋白进行生物信息学分析。使用免疫组化技术检测KLF11在小鼠肝脏组织中的表达。将pcDNA3.1-Puro-N-3HA与pcDNA3.1-mKLF11质粒分别转染至HEK293T和AML12细胞,利用实时荧光定量聚合酶链反应(qPCR)和蛋白质印迹技术(Western blotting)对小鼠Klf11基因的表达效果进行检测。最后通过qPCR和油红O染色,检测游离脂肪酸(FFA)处理后Klf11的过表达对AML12细胞脂质代谢的影响。【结果】(1)PCR、酶切及测序结果显示,成功克隆了小鼠Klf11基因的CDS区片段并构建了pcDNA3.1-mKLF11重组质粒;(2)生物信息学分析结果显示,小鼠Klf11基因的编码区序列与大鼠和人类的相似性较高,小鼠KLF11蛋白表现出亲水性特征,二级结构包括无规卷曲、α-螺旋、延伸链和β-转角,不存在信号肽,且不存在跨膜区;小鼠KLF11蛋白的三级结构与人类和大鼠的KLF11蛋白相比均存在显著差异;(3)免疫组化结果表明,KLF11主要表达于小鼠肝细胞细胞核;(4)qPCR和Westren blotting结果显示,在HEK293T和AML12细胞中,pcDNA3.1-mKLF11转染组Klf11基因的mRNA和蛋白表达水平均显著高于对照组;此外,在FFA处理的条件下,Klf11过表达后小鼠AML12细胞中脂代谢相关基因Fasn、Acaca和Plin2的表达量显著升高,并且过表达组中细胞内脂滴数量增加。【结论】成功构建了小鼠Klf11基因的真核表达载体,对小鼠Klf11基因及其编码蛋白进行了生物信息学分析。免疫组化结果表明KLF11主要表达于小鼠肝细胞细胞核。此外,在AML12细胞中成功过表达KLF11蛋白,并证明Klf11的过表达显著促进了AML12细胞的脂质沉积,为后续探究Klf11基因调控哺乳动物肝脏脂质代谢的作用机制提供依据,为开发家畜脂质代谢障碍疾病防治的新药物提供新思路。

[Objective]This study aims to clone the Kruppel-like transcription factor family member 11(KLF11)gene from mice,construct its eukaryotic expression vector,and investigate its regulatory effects on lipid metabolism-related genes after overexpression of Klf11 in the mouse AML12 hepatocyte cell line.[Method]Using cDNA from mouse liver tissue as a template,the coding sequence(CDS)of the mouse Klf11 gene was amplified by PCR.The amplified fragment was then ligated into the pcDNA3.1-Puro-N-3HA vector to construct a recombinant plasmid.The recombinant plasmid was verified through restriction enzyme digestion,PCR,and sequencing,and the correct plasmid was named pcDNA3.1-mKLF11.Bioinformatics analysis of the Klf11 sequence and its encoded protein was performed using online software.Immunohistochemistry was used to detect KLF11 expression in mouse liver tissue.The pcDNA3.1-Puro-N-3HA and pcDNA3.1-mKLF11 plasmids were transfected into HEK293T and AML12 cells.The expression of the mouse Klf11 gene was detected using quantitative real-time PCR(qPCR)and Western blotting.Finally,the effects of Klf11 overexpression on lipid metabolism in AML12 cells treated with free fatty acids(FFA)were assessed using qPCR and Oil Red O staining.[Result]The results of PCR,restriction enzyme digestion,and sequencing confirmed the successful cloning of the CDS region of the mouse Klf11 gene and the construction of the pcDNA3.1-mKLF11 recombinant plasmid.Bioinformatics analysis revealed high sequence similarity in the coding region of the Klf11 gene between mice,rats,and humans.The mouse KLF11 protein exhibited hydrophilic characteristics,with its secondary structure comprising random coils,α-helices,extended strands,andβ-turns.It lacked signal peptides and transmembrane regions.The tertiary structure of the mouse KLF11 protein showed significant differences compared with KLF11 proteins of humans and rats.Immunohistochemistry results indicated that KLF11 was primarily expressed in the nuclei of mouse hepatocytes.qPCR and Western blotting results showed that in both HEK293T and AML12 cells,the mRNA and protein levels of Klf11 were significantly higher in the pcDNA3.1-mKLF11 transfection group than those of the control group.Furthermore,under FFA treatment,Klf11 overexpression significantly increased the expression of lipid metabolism-related genes Fasn,Acaca,and Plin2,and increased the number of intracellular lipid droplets in AML12 cells.[Conclusion]This study successfully constructed an eukaryotic expression vector for the mouse Klf11 gene and conducted bioinformatics analysis of the mouse Klf11 gene and its encoded protein.Immunohistochemistry results show that KLF11 is primarily expressed in the nuclei of mouse hepatocytes.Additionally,KLF11 protein is successfully overexpressed in AML12 cells,and Klf11 overexpression significantly promotes lipid accumulation in AML12 cells.This research lays the groundwork for further investigation into the role of the Klf11 gene in regulating lipid metabolism in mammalian livers and provides new insights for developing therapies for lipid metabolism disorders in livestock.