小鼠黏蛋白1(MUC1)基因的体外转录及其在NIH/3T3细胞中的表达

Preparation of in vitro transcribed mouse MUC1 mRNA and its expression in NIH/3T3 cells

目的体外转录获得小鼠黏蛋白1(MUC1) mRNA并在NIH/3T3细胞中表达。方法通过反转录PCR从小鼠4T1细胞中扩增MUC1基因,然后插入真核表达载体pc DNA3. 1(+)中构建重组表达载体pc DNA3. 1(+)-MUC1。双酶切鉴定正确后,以该质粒为模板,下游引物中引入血凝素标签(HA TAG)序列,PCR扩增MUC1-HA TAG融合基因片段,然后克隆至表达载体pc DNA3. 1(+),重组载体经双酶切鉴定和DNA序列测定后作为模板,PCR扩增获得含T7启动子和MUC1-HA TAG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、纯化最终获得修饰的MUC1 mRNA。通过多种转染试剂将体外转录MUC1-HA TAG mRNA转入NIH/3T3细胞,Western blot法检测融合蛋白MUC1-HA TAG的表达。结果反转录PCR扩增的MUC1基因长度约为1900 bp,酶切鉴定和序列分析证实MUC1-HA TAG融合基因已成功插入pc DNA3. 1(+)质粒。插入序列与Gen Bank中小鼠的MUC1基因序列完全一致,HA TAG正确插入MUC1下游,读框正确。Western blot法分析证实体外转录修饰的MUC1-HA TAG mRNA能在NIH/3T3细胞中表达。结论体外转录修饰的小鼠MUC1-HA TAG mRNA能在哺乳动物NIH/3T3细胞中翻译表达。

Objective To obtain in vitro transcription of mouse mucin 1( MUC1) mRNA and express it in NIH/3 T3 cells.Methods The MUC1 gene was amplified from mouse 4 T1 cells by reverse transcription PCR( RT-PCR),and then insertedinto the eukaryotic expression vector pc DNA3. 1( +) to construct the recombinant vector pc DNA3. 1( +)-MUC1. After identificationby restriction enzyme cutting,the recombinant plasmid was used as a PCR template for the amplification of the MUC1-HATAG fusion gene fragment with the reverse primer containing hemagglutinin tag( HA TAG) sequence. Subsequently,theMUC1-HA TAG fusion gene was cloned into the expression vector pc DNA3. 1( +). After double restriction enzyme digestionand DNA sequence identification,the recombinant plasmid encoding MUC1-HA TAG was used as a PCR template for theamplification of a PCR product containing T7 promoter and the MUC1-HA TAG fusion gene fragment as an in vitro transcriptiontemplate to obtain the modified MUC1-HA TAG mRNA by in vitro transcription,tailing and purification. The resultingMUC1-HA TAG mRNA was transfected into NIH/3 T3 cells by various transfection reagents and the expression of the fusionprotein MUC1-HA TAG was detected by Western blot analysis. Results The MUC1 gene amplified by RT-PCR was about1 900 bp in length. The restriction enzyme digestion and sequence analysis confirmed that the MUC1-HA TAG fusion genehad been successfully inserted into pc DNA3. 1( +),and the insertion sequence was identical to the MUC1 gene sequence ofthe mouse in GenBank database. HA TAG was correctly inserted in the downstream of MUC1 gene and the reading framewas correct. Western blot analysis indicated that the in vitro transcriptionally modified MUC1-HA TAG mRNA could beexpressed in NIH/3 T3 cells. Conclusion In vitro transcriptionally modified mouse MUC1-HA TAG mRNA can be translatedand expressed in mammalian NIH/3 T3 cells.