Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines.
A novel plasmid pGS/2SS-M4GFP was constructed in the present study by recombination of GS/2SS gene and enhanced green fluorescent protein (M4GFP) sequence. The GS/2SS fusion gene encoding two copies of somatostatin genes was firstly introduced into pVAX-asd vector in which the kanamycin resistance cassette was replaced by the asd cassette. The M4GFP gene was then fused into 3' end of GS/2SS gene in the proper reading frame. After purified, plasmid pGS/2SS-M4GFP was transfected into different cell lines derived from pig kidney and human cancer cells. The transcription process of GS/2SS gene was confirmed by RT-PCR, and the localization as well as expression of GS/2SS-M4GFP fusion protein was observed by confocal microscopy and ELISA. Transfection results revealed that sole M4GFP was localized within the cytosol and the nucleus, while fusion protein GS/2SS-M4GFP was localized only in the cytoplasm. Furthermore, it should be noted that subcellular localization of GS/2SS-M4GFP was not specific to one cell line, but appeared to be common across a variety of cell lines. These results provide for the first time valuable evidence that M4GFP is a versatile tool to trace GS/2SS protein and pave the way for further study on its tissue distribution and immunological mechanism in vivo.
Liang
A X
AX
Key Lab of Education Ministry of China in Agricultural Animal Genetics, Breeding and Reproduction, Huazhong Agricultural University, Wuhan 430070, PR China.
Han
L
L
Hua
G H
GH
Geng
L Y
LY
Sang
L
L
Liu
X B
XB
Guo
A Z
AZ
Yang
L G
LG
D000818:Animals; D002460:Cell Line / Q000378:metabolism*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006801:Humans; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013004:Somatostatin / Q000235:genetics; D013552:Swine; D014018:Tissue Distribution; D014162:Transfection; D019076:Transgenes
D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013004:Somatostatin