采用TOPOTA克隆技术构建人CC10质粒并在BEAS-2B细胞中的表达

CC10 Plasmid was Constructed by Using the TOPO TA Technology and Expressed in BEAS-2B Cells

目的:采用TOPO TA克隆技术构建及鉴定人CC10基因表达载体,并在支气管上皮细胞系BEAS-2B细胞中获得稳定表达,初步探讨CC10在呼吸道上皮细胞炎症反应中的作用。方法:提取人下鼻甲组织的总RNA,逆转录反应生成c DNA,再用PCR方法扩增出含人CC10编码区全长的DNA片段,将PCR产物直接连接到pc DNA3.1/V5-His TOPO TA载体中,转化至大肠杆菌后经筛选鉴定出CC10表达载体,用脂质体法将CC10质粒转染到BEAS-2B,细胞免疫荧光法检测CC10蛋白的表达。随后用促炎细胞因子IL-1β刺激转染空质粒和CC10质粒的BEAS-2B细胞,用实时定量RT-PCR和ELISA检测炎性趋化因子RANTES m RNA和蛋白的表达。结果:目的基因与TOPO TA载体在室温下5 min的连接反应效率91.7%,用酶切法鉴定质粒并测序,人CC10基因成功克隆到真核细胞表达载体pc DNA3.1中,CC10蛋白在BEAS-2B细胞中无表达,但在体外转染CC10质粒后CC10蛋白表达明显增高。转染CC10质粒的BEAS-2B细胞可抑制IL-1β诱导的RANTES m RNA和蛋白的表达。结论:利用TOPO TA克隆技术可高效、快速的构建人CC10基因表达载体,并能够在BEAS-2B细胞中获得稳定表达,CC10蛋白在呼吸道上皮细胞中可发挥抗炎作用。

Objective： To construct and identify human Clara cell 10-kd protein（CC10） gene expression vector expression vector by using the TOPO TA cloning technology, and CC10 plasmid could stability expressed in bronchial epithelial cell line BEAS-2B cells.Preliminary discuss the role of CC10 in airway epithelial cells inflammation. Methods： The human total RNA were extracted from human inferior turbinate tissue and RNA generated to c DNA by reverse transcription reaction. Then the DNA fragment which contains the full coding region of CC10 gene was produced by the means of PCR reaction. Next the PCR products which include CC10 gene was directly ligated to pc DNA3.1/V5-His TOPO TA vector and transformated to e. coli, after screening and identified, the human CC10 expression vector was acquired. Finally, the CC10 plasmid was transfected to bronchi epithelial cell line BEAS-2B by using the liposome method.The expression level of CC10 protein was detected by the techniques of immunofluorescence. Then we used the proinflammatory cytokines IL-1β to stimulate the empty plasmid and CC10 plasmid transfected BEAS-2B cells, and detected inflammatory chemokines RANTES m RNA and protein expression by the methods of real-time quantitative RT-PCR and ELISA. Results： The ligation reaction between target gene and TOPO TA vector can completed in 5 minutes at room temperature and the efficiency was 91.7 %. It was simple operation and time saving to construct plasmid by using TOPO TA cloning technology compared with traditional method. The recombinant CC10 plasmid was identified by the method of restriction enzymes and PCR analysis, and then it was sequenced and confirmed which contained the correct nucleotide sequence of the CC10 gene. CC10 protein was not expressed in BEAS-2B cells naturally, but expressed obviously in the cells after being transfected with reconstructive CC10 plasmid. CC10 plasmid transfection could inhibit IL-1β induced RANTES m RNA and protein expression in BEAS-2B cells. Conclusion： The human CC10 expression plasmid was rapidly and successfully constructed with high efficiency by using the TOPO TA cloning technology, and CC10 protein was able to gain stably expressed in BEAS-2B cells. CC10 protein can play an anti-inflammatory role in airway epithelial cells.