[Expression and functional analysis of CRT-E2-EGFP fusion protein in B16 cells].
OBJECTIVE
To detect the expression of the Calreticulin and HPV E2 Fusion Protein in B16, and study the effects on proliferation and apoptosis of B16 cell lines in vivo.


METHODS
To construct eukaryotic fluoresce expression vector pEGFP-CRT-E2, pEGFP-CRT and pEGFP-E2. Then the recombinant plasmids were transfected into B16 cells by Lipofectamine 2000. The expression of proteins was detected by Western blot. The location of different GFP fusion proteins in B16 was tested by inverted fluoresce microscope. Flow cytometry was applied to detect the effects of fusion proteins on the growing of B16 and then the apoptosis effects of B16 induced by different proteins were observed.


RESULTS
The correctly constructed recombinant plasmid pEGFP-CRT-E2 and the expression of CRT-E2 gene could be detected in B16 cells. Apoptosis of B16 cells could be detected after the transient transfection. Meanwhile, the apoptosis rate of B16 cells transfected by pEGFP-CRT-E2 was much higher than that of control cells. And cell cycle G1/G0 arrest was also found (P<0.01).


CONCLUSIONS
The eukaryotic expression plasmid pEGFP-CRT-E2 is successfully constructed and it could correctly express the fusion protein in B16 cells. And the B16 cells transfected by plasmid pEGFP-CRT-E2 could induce apoptosis.
Yang
Jian-Lin
JL
Institute of Molecular and Biology, Three Gorges University, Yichang 443002, China. mmgbb2002@126.com
Liu
Chang-Bai
CB
Li
Dan-Li
DL
Han
Li
L
D000818:Animals; D017209:Apoptosis; D037282:Calreticulin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D037282:Calreticulin; D004268:DNA-Binding Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C076036:oncogene protein E2, Human papillomavirus type 1; D049452:Green Fluorescent Proteins