表达载体sHLA-A2-IgG1-Fc的构建与鉴定

Construction and identification of eukaryotic expression vectors of soluble HLA-A2 and human IgG1-Fc fragment

目的构建表达载体pcDNA3.0-sHLA-A2-IgGl-Fc,为进一步真核表达可溶性HLA-A2-IgG1-Fc蛋白奠定基础。方法从T2细胞中提取总RNA,借助RT-PCR技术扩增包括信号肽的可溶性HLA-A2的cDNA序列并把其插入真核表达载体pcDNA3.0,然后经酶切和测序法鉴定;用PCR的方法从含IgG1-Fc基因的PIG质粒中扩增目的基因IgG1-Fc段,然后经酶切后插入前面构建好的表达载体pcDNA3.0-sHLA-A2中,最后经酶切和测序法鉴定。结果经酶切鉴定及测序分析,证实已将目的基因HLA-A2和IgG1-Fc片段插入载体pcDNA3.0。结论本研究成功构建表达载体pcDNA3.0-sHLA-A2-IgG1-Fc。

Objective To construct a recombinant plasmid pcDNA3.0-sHLA-A2-IgG1-Fc,and to provide a firm basis on construction of soluble HLA-A2-IgG1-Fc protein by eukaryotic expression system. Methods Total cell RNA was extracted from the cell line T2 and the eDNA sequences with encoding signal peptide were amplified by RT-PCR. The cDNA fragment was inserted into the eukaryotic expression vector pcDNA3.0 and the recombinant plasmid was identified by restriction endonuclease digestion and sequencing. The gene of objective IgG1-Fc fragment was amplified from plasmid PIG by PCR. After restriction endonuclease digestion, the eDNA frogment was inserted into the constructed vector pcDNA3.0-sHLA-A2 and the recombinant plasmid was identified by resriction endonuclease digestion and sequencing. Results After restriction endonuclease treatment and sequencing, it was confirmed that HLA-A2 and IgG1-Fc has been inserted into vector pcDNA3.0. Conclusion The recombinant plasmid pcDNA3.0-sHLA-A2- IgG1-Fc has been constructed successfully.