[Preparation and bioactivity evaluation of SA-mIL15 fusing protein].
OBJECTIVE
To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins.


METHODS
pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate SA/mIL15 fusion proteins. SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography, and mIL15-SA fusion protein through anion exchange chromatography, followed by refolding. The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer. MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA.


RESULTS
Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta (DE3) at about 20% of total bacterial proteins. They exhibited the bi-functionality: proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1x10(6); IU/mg for SA-mIL15 or 2 x 10(5); IU/mg for mIL15-SA, and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency.


CONCLUSIONS
SA/mIL15 bi-functional fusion proteins were generated, which made feasible the development of mIL15-surface-modified cancer cell vaccine.
Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou, China. chenyanli198402@163.com
Yan-li
YL
Chen
Xiao-ling
XL
Xu
Jia
J
Tang
Xiao-xia
XX
Nie
Zhi-chun
ZC
Song
Zhi-min
ZM
Hu
Ji-min
JM
Gao
D000818:Animals; D015153:Blotting, Western; D005434:Flow Cytometry; D019409:Interleukin-15 / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D019809:Streptavidin / Q000235:genetics*
D019409:Interleukin-15; D011993:Recombinant Fusion Proteins; D019809:Streptavidin