纤维粘连蛋白片段真核表达载体pcDNA3.1(+)-EDA的构建

The construction of eukaryotic expression plasmid pcDNA3.1(+)-EDA containing the EDA segment of fibronectin gene

目的构建纤维粘连蛋白(fibronectin,FN)基因的EDA外显子的真核表达载体,以研究EDA对涎腺腺样囊性癌(slivary adenoid cystic carcinoma,SACC)生物学行为的影响。方法以RT-PCR技术从口腔鳞状细胞癌(oral squamous cell carcinoma)细胞株KB中扩增出外显子EDA,克隆入pcDNA3.1(+)真核表达载体,用PCR和限制性内切酶酶切、DNA测序鉴定真核表达载体pcDNA3.1(+)-EDA。结果PCR、酶切、DNA测序鉴定证明成功构建了pcDNA3.1(+)-EDA真核表达载体,DNA测序显示重组质粒含有与EDA片段基因序列完全相等的基因。结论成功构建了纤维粘连蛋白(fibronectin,Fn)基因的EDA外显子的真核表达载体pcDNA3.1(+)-EDA。

Objective To construct the eukaryotic expression plasmid pcDNA3.1 （ ＋ ）-EDA containing the exon EDA of fibronectin gene for the study of effect of EDA on biological feature of slivary adenoid cystic carcinoma（SACC）. Methods EDA was amplified from the cDNA of oral squamous cell carcinoma cell strain KB by RT-PCR, and the exon EDA was inserted into the appropriate site of pcDNA3. 1 （ ＋ ） vector. The positive clones were identified with restrictive enzymes and PCR. Results The target segment EDA （ about 270bp ） was searched for alignment with NCBI Blast program,which was obtained successfully. Homology of the nucleotide of this segment EDA between the sequence from Genbank was 100%. Conclusion The recombinant plasmid of pcDNA3.1 （ ＋ ）-EDA was successfully constructed.