[Expression and localization of strong epitope hAFP(542-550); on eukaryotic cytoplasmic membrane].
OBJECTIVE
To construct the recombinant plasmid of pGPC3-EGFP containing human AFP(542-550) gene, EGFP gene and GPC3 gene to express fusion protein GPC3-hAFP(542-550)-EGFP and to discover its localization on cytoplasmic membrane.


METHODS
GPC3 gene was obtained from total RNA of human placental tissues by RT-PCR; After the enhanced green fluorescent protein (EGFP) gene was amplified from pEGFP-N1 plasmid and the gene segment of-KOZAK-GPCN + afp(542-550)-was chemically synthesized, the recombinant plasmid pcDNA3.1(+)/GPCN+afp(542-550)-EGFP-GPCC (pGPC3-EGFP) containing three chimeric genes of strong epitope hAFP(542-550), GPI-anchored protein GPC3 and EGFP was constructed. The fusion protein (GPC3-hAFP(542-550)-EGFP) was detected on RNA and protein levels at 24 h and 48 h after pGPC3-EGFP was transfected into HepG2 (GPC3(+)AFP(+)) via lipofectamine 2000. EGFP expression was observed by fluorescent microscopy after pGPC3-EGFP was transfected into HepG2 using pEGFP-N1 plasmid transfection as a positive control. The fusion protein in both membrane proteins and soluble proteins extracted from the transfected 293 cells (GPC3(-)AFP(-)) was detected by Western blot using GPC3 monoclonal antibody as primary antibody.


RESULTS
The recombinant plasmid pGPC3-EGFP was successfully constructed through restriction endonuclease digestion and sequencing; pGPC3-EGFP expression in HepG2 cells was detected not only by RT-PCR using specific primers (GPCN-F and EGFP-r) but also by Western blot using GFP polyclonal antibody and GPC3 monoclonal antibody. Green fluoresce was mainly found around pGPC3-EGFP transfected HepG2 cell periphery beside sporadic distribution in cytoplasm, but that of pEGFP-N1 transfected HepG2 cell was evenly distributed in the whole cell. Moreover, the fusion protein was not detected in soluble proteins but membrane proteins extracted from transfected 293 cells.


CONCLUSIONS
The recombinant plasmid of pGPC3-EGFP based on protein engineering theory can express fusion protein (GPC3-hAFP(542-550)-EGFP) in eukaryotic cells. Furthermore, the fusion protein is still located on cytoplasmic membrane, which is a characteristic of GPI-anchored membrane protein, and is a new GPI-reanchored protein.
Yang
Dong-ye
DY
Department of Digestive Diseases at Xiangya Second Hospital, Central South University, Changsha 410011, China. dongyeyang@yahoo.com.cn
Li
Cai-hong
CH
Lan
Fang
F
Zhang
Yang-qing
YQ
Lu
Fang-gen
FG
Yang
Zheng-rong
ZR
Huang
Lai-qiang
LQ
D002462:Cell Membrane / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics*; D053673:Glypicans / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection; D000509:alpha-Fetoproteins / Q000235:genetics* / Q000276:immunology
D000939:Epitopes; D053673:Glypicans; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins