Construction and identification of the two lentiviral eukaryotic expression vectors of pEGFP-N1-HIF-lα and pEGFP-N1-muHIF-1α
10.3760/cma.j.issn.1002-0098.2012.s1.055
- VernacularTitle:两个慢病毒真核表达载体pEGFP-N1-HIF-1α和pEGFP-N1-muHIF-1α的构建及检测
- Author:
Shu-Hong WANG
1
;
Xiong ZHANG
;
Jin-E ZHANG
;
Hua-Yan GUO
;
Na GUO
;
Yuan-Liang HUANG
Author Information
1. 杭州市第一人民医院口腔科
- Keywords:
Point mutation;
Lentivirus;
Hypoxia-inducible factor;
Carrier;
Reorganization
- From:
Chinese Journal of Stomatology
2012;47(z1):244-247
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct two lentiviral eukaryotic expression vectors of pEGFP-N1-HIF-1α and pEGFP-N1-muHIF-1α and identificate the gene sequences.Methods Primers were designed according to wild-type human HIF-1 α gene sequence information and determined restriction sites of human HIF-1α point mutant sequence,taking human HIF-1α and constructed HIF-1α gene mutant clone as a template for polymerase chain reaction(PCR) amplification.Then,the target gene PCR product and purpose vector were digested.Adapter-ligated fragments of the target fragment with purpose vector were transformed into DH-5α competent cells using the recombinant PCR methods.Finally,the positive clones were selected and sequenced to verify whether the sequence of insert fragments in recombinant clones was consistent with oligo sequences designed or not.Results Through recombinant clone colony PCR,enzyme digestion of the recombinant clones and plasmid cloning and sequencing,the fragment sequences inserted in recombinant clones were consistent with the designed oligo sequences.Conclusions The two lentiviral eukaryotic expression vectors of pEGFP-N1-HIF-1α and pEGFP-NI-muHIF-1α were successfully constructed and applicable for follow-up experiments on transfection of bone marrow mesenchymal stem cells.
