Purification of Human CD40-Ig Fusion Protein and Its Effect on Human Peripheral Blood CFU-T Formation In Virto
- Author:
Chun-Mei HOU
1
;
He-Zhong LIU
;
Xiu-Sen LI
;
Fei-Zi JIANG
;
Ning MAO
Author Information
1. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
- Publication Type:Journal Article
- From:
Journal of Experimental Hematology
2001;9(3):212-214
- CountryChina
- Language:Chinese
-
Abstract:
CD40/CD40L interactions play a pivotal role in T cell activation, and take part in many physiologic and pathologic procedures and different levels. In this article, stable CHO transformants secreting human CD40-Ig fusion protein were established through transfection and selection with Lipofectamaine and G418, respectively. In order to obtain great valume of recombinant protein, big batch serum-free cultures of engineered CHO cells were performed in roller-bottle using CHO-II-SFM medium. After cultures, the cell-culture supernatants were harvested, concentrated through ultra-filtration, and finally purified by affinity choromatography with Protein G Sepharose Fast Flow. Human peripheral bloods were collected freshly and seperated with Ficoll, CFU-T was cultured in semi-solid culture system with peripheral blood mononuclear cells (PBMNC). Effect of human CD40-Ig fusion protein on the formation of CFU-T was observed in vitro. The results showed that the yield of human CD40-Ig fusion protein was 30 mg in total 3 liter CHO-II-SFM culture supernatant, and it supposed that the expression level of CD40-Ig in CHO cells was more than 10 micro g/ml. The purity of purified fusion protein is above 95%. Furthermore, compared with human IgG, human CD40-Ig fusion protein significantly inhibited the formation of CFU-T at dose 0.25, 1.0, 4.0, and 10 micro g/ml, it lays a good foundation to evaluate its potential functions in vivo.
