膜联蛋白A1基因合成及其真核表达载体的构建

摘要

Objective To synthesize annexin Al(ANXA1) gene and construct its eukaryotic expression vector. Methods A 870bp DNA fragment encoding ANXA1 was generated by PCR from a pair of oligoes. The obtained sequences of ANXA1 was cloned into pEGFP-N3 vector by double digestion of restriction enzyme Kpn 1 & BamH I and transformed into E. coli DH5α, and then the recombinant pEGFP-N3-ANXAl was constructed and identified. Results ANXA1 was acquired by PCR amplification, and was proved to be the exact gene by electrophoretogram of recombinant plasmid pEGFP-N3-ANXAl after restriction emzyme digestion. Sequencing analysis was applied to identify the recombinant expression plasrnids. It showed that the sequence homology of ANXA1 cloned with the ANXA1 on GenBank was 99% , and the whole reading frame was correct. Conclusion The eukaryotic expression plasmid pEGFP-N3-ANXAl has been successfully constructed, which lay the foundation of high-level expression of ANXA1 protein and provided solid experiment foundation for further studies on the function of ANXAI.%目的 探讨人膜联蛋白A1( ANXA1)基因的合成并构建其克隆载体.方法 根据ANXA1基因序列设计一对寡聚核苷酸引物,通过PCR反应合成一段长度为870bp的ANXA1基因.真核表达载体pEGFP-N3经Kpn Ⅰ& BamH Ⅰ限制性内切酶双酶切处理后与合成的ANXA1基因进行连接,连接产物转化E.coli DH5α感受态细胞进行扩增,并提取质粒进行鉴定.结果 PCR扩增获得ANXA1基因,对PCR产物及重组质粒酶切后的凝胶电泳鉴定证实为与理论设计目的片段大小( 870 bp)相符的DNA基因片段,并成功地转入克隆载体pEGFP-N3中.测序分析显示合成的目的基因与GenBank中ANXA1基因序列同源性为99％,整个表达载体阅读框正确无误.结论 成功构建了ANXA1基因pEGFP-N3表达载体,为下一步高效表达ANXA1蛋白和进一步研究其作用机理及应用价值打下基础.