[Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor].
OBJECTIVE
To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF).


METHODS
The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF.


RESULTS
DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE.


CONCLUSIONS
The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.
Ma
Gao-feng
GF
Institute of Digestive Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. gfmj@163.com
Chen
Xue-qing
XQ
Yang
Xiao-qiang
XQ
Wu
Jin-bao
JB
Zhang
Zhen-shu
ZS
D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics*; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor