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目的:构建人类白细胞抗原G5(HLA-G5)的表达载体,并进行慢病毒包装,为进一步研究HLA-G5功能提供基础工具。方法:人工合成HLA-G5全长序列经测序正确后,定向接入真核表达载体pCDH-CMV-MCS-EF1-copGFP,转化入大肠杆菌,酶切鉴定并测序正确后进行慢病毒包装和滴度测定。结果:经琼脂糖凝胶电泳鉴定、PCR鉴定及测序,HLA-G5表达质粒质量合格,序列比对与设计序列符合率100%,HLA-G5慢病毒滴度测定为7.06×108。结论:成功地构建了HLA-G5表达载体并完成了慢病毒包装,为今后的研究工作提供了基础工具。
OBJECTIVE: To construct human leukocyte antigen G5 (HLA-G5) expression vector and packaging with lentivirus to provide basic tools for further study of HLA-G5 function. Methods: The full-length sequence of HLA-G5 was sequenced correctly and then inserted into eukaryotic expression vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid was transformed into E.coli and identified by restriction analysis and lentivirus packaging Degree measurement. Results: The quality of HLA-G5 expression plasmid was confirmed by agarose gel electrophoresis, PCR identification and sequencing. The coincidence rate of sequence alignment and designed sequence was 100%, and the titer of HLA-G5 lentivirus was 7.06 × 108. Conclusion: The successful construction of HLA-G5 expression vector and the completion of lentivirus packaging provide the basic tools for future research.