Lin Xuan, Chen Hedan, Xie Ying, Zeng Zhu, Hu Zuquan, Jia Yi, Wang Yun, Tan Chengjian, Liu Lina
Key Laboratory of Biology and Medical Engineering, Immune Cells and Antibody Engineering Research Center of Guizhou Province, Engineering Research Center of Medical Biotechnology, State Key Laboratory of Functions and Applications of Medicinal Plants, School of Biology and Engineering, Guizhou Medical University, Guiyang 550025, China.
Key Laboratory of Biology and Medical Engineering, Immune Cells and Antibody Engineering Research Center of Guizhou Province, Engineering Research Center of Medical Biotechnology, School of Biology and Engineering, Guizhou Medical University, Guiyang 550025, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Oct;34(10):896-901.
Objective To obtain in vitro transcription of mouse mucin 1(MUC1) mRNA and express it in NIH/3T3 cells. Methods The MUC1 gene was amplified from mouse 4T1 cells by reverse transcription PCR (RT-PCR), and then inserted into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant vector pcDNA3.1(+)-MUC1. After identification by restriction enzyme cutting, the recombinant plasmid was used as a PCR template for the amplification of the MUC1-HA TAG fusion gene fragment with the reverse primer containing hemagglutinin tag (HA TAG) sequence. Subsequently, the MUC1-HA TAG fusion gene was cloned into the expression vector pcDNA3.1(+). After double restriction enzyme digestion and DNA sequence identification, the recombinant plasmid encoding MUC1-HA TAG was used as a PCR template for the amplification of a PCR product containing T7 promoter and the MUC1-HA TAG fusion gene fragment as an in vitro transcription template to obtain the modified MUC1-HA TAG mRNA by in vitro transcription, tailing and purification. The resulting MUC1-HA TAG mRNA was transfected into NIH/3T3 cells by various transfection reagents and the expression of the fusion protein MUC1-HA TAG was detected by Western blot analysis. Results The MUC1 gene amplified by RT-PCR was about 1 900 bp in length. The restriction enzyme digestion and sequence analysis confirmed that the MUC1-HA TAG fusion gene had been successfully inserted into pcDNA3.1(+), and the insertion sequence was identical to the MUC1 gene sequence of the mouse in GenBank database. HA TAG was correctly inserted in the downstream of MUC1 gene and the reading frame was correct. Western blot analysis indicated that the in vitro transcriptionally modified MUC1-HA TAG mRNA could be expressed in NIH/3T3 cells. Conclusion In vitro transcriptionally modified mouse MUC1-HA TAG mRNA can be translated and expressed in mammalian NIH/3T3 cells.
目的 获取小鼠黏蛋白1（MUC1）mRNA的体外转录产物，并在NIH/3T3细胞中进行表达。方法 通过逆转录聚合酶链反应（RT-PCR）从小鼠4T1细胞中扩增MUC1基因，然后将其插入真核表达载体pcDNA3.1(+)中构建重组载体pcDNA3.1(+)-MUC1。经酶切鉴定后，将重组质粒作为PCR模板，用含血凝素标签（HA标签）序列的反向引物扩增MUC1-HA标签融合基因片段。随后，将MUC1-HA标签融合基因克隆到表达载体pcDNA3.1(+)中。经双酶切和DNA序列鉴定后，将编码MUC1-HA标签的重组质粒作为PCR模板，扩增出含T7启动子和MUC1-HA标签融合基因片段的PCR产物作为体外转录模板，通过体外转录、加尾和纯化获得修饰后的MUC1-HA标签mRNA。将所得的MUC1-HA标签mRNA用各种转染试剂转染至NIH/3T3细胞中，通过蛋白质免疫印迹分析检测融合蛋白MUC1-HA标签的表达。结果 RT-PCR扩增的MUC1基因长度约为1900 bp。酶切和序列分析证实MUC1-HA标签融合基因已成功插入pcDNA3.1(+)中，插入序列与GenBank数据库中小鼠的MUC1基因序列一致。HA标签正确插入MUC1基因下游且读码框正确。蛋白质免疫印迹分析表明，体外转录修饰的MUC1-HA标签mRNA可在NIH/3T3细胞中表达。结论 体外转录修饰的小鼠MUC1-HA标签mRNA可在哺乳动物NIH/3T3细胞中翻译表达。
