title: [Cloning and expression of human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells].
abstract: OBJECTIVE
  To clone and express human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells.
  
  
  METHODS
  Primers were designed based on the coding sequence for the human Oct4 cDNA. The fragment containing Oct4 gene, amplified by reverse transcription PCR (RT-PCR) from human HeLa cell cDNA as the template, was subcloned into pET-22b(+) and pcDNA3.1(+) vectors to construct the gene recombinant prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)-his-Oct4, respectively. Thereafter, the double enzymes digestion and the target gene sequencing were performed for identification. The prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)- his-Oct4 from the positive clones were respectively transformed into E.coli BL21 (DE3) and the embryo HEK293T cells for protein expression.
  
  
  RESULTS
  DNA fragments (1100 bp) containing the coding region of Oct4 gene were obtained from RT-PCR. The recombinant expression plasmids were confirmed by enzymes digestion identification and sequencing analysis, proving both of the expression vectors were successfully constructed. SDS-PAGE analysis showed that the molecular mass of OCT4 expressed in the host E.coli BL21 (DE3) was 38 000, which was consistent with the theoretical prediction. Western blotting confirmed the expression of pcDNA3.1(+)-his-Oct4 transfected into HEK293T.
  
  
  CONCLUSIONS
  Human Oct4 cDNA was cloned and its protein was successfully expressed in E.coli and HEK293T cells.
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China.
authors.forename: Bibo
authors.initials: B
authors.lastname: Ruan
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China.
authors.forename: Wuyu
authors.initials: W
authors.lastname: Fu
authors.affiliation: Central Laboratory, Guangdong Pharmaceutical University, Guangzhou 510006, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Rui
authors.affiliation: Biomedical Research and Development Center, Jinan University, Guangdong Key Laboratory of Bioengineering Pharmaceutics, Guangzhou 510632, China.
authors.forename: Haixuan
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China.
authors.forename: Pengjun
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China.
authors.forename: Shuangye
authors.initials: S
authors.lastname: Liao
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Wu
authors.affiliation: Biomedical Research and Development Center, Jinan University, Guangdong Key Laboratory of Bioengineering Pharmaceutics, Guangzhou 510632, China.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathogen Biology and Immunology, School of Basic Course, Guangzhou 510006, China. *Corresponding author, E-mail: hychen1208@126.com.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Chen
mesh_terms: D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D050814:Octamer Transcription Factor-3; D011994:Recombinant Proteins