[Construction, bioactivity identification and structural characteristics analysis of a novel recombinant immunosuppression protein B7-2-L-PE40KDEL].
OBJECTIVE
To construct a novel recombinant B7-2-L- PE40KDEL fusion protein used to selectively kill T cells expressing high levels of CD28 so as to induce immune tolerance and prevent graft versus host disease (GVHD) and host versus graft disease (HVGD).


METHODS
The cDNA encoding human B7-2 was ligated with cDNA encoding PE40KDEL by using sequence overlapping extension (SOE) techniques. The gene of interest was subcloned into a high output expression vector pRSETA and transformed into E. coli cells. Its molecular structural characteristics, such as flexibility, antigenicity, hydrophilicity, and epitope were analyzed. The purification protocol of expressed protein was established and its cytotoxicity to selectively kill T cells expressing high levels of CD28 was measured by MTT method.


RESULTS
B7-2-L-PE40KDEL fusion protein was expressed at high levels in E. coli cells and the purified product attained over 95% of purity. The structural characteristics of B7-2-L-PE40KDEL were not significantly changed in comparison with B7-2 and PE40KDEL. In cytotoxicity assay, B7-2-L-PE40KDEL fusion protein specifically killed Jurkat cells which express high level CD28 receptor and was non-cytotoxic to CD28 receptor-negative cell line Raji.


CONCLUSIONS
B7-2-L-PE40KDEL novel fusion protein can selectively kill the T cells which express CD28 receptor and may become a kind of new effective drug for inducing T cell immune tolerance and preventing GVHD and HVGD.
Yuan
Zhihong
Z
Affiliated Hospital of Academy of Military Medical Science, National Center of Biomedical Analysis Laboratory of Immunoassay, Beijing 100039, China.
Xi
Yongzhi
Y
Zhang
Huili
H
Kong
Fanhua
F
Guan
Hairong
H
Guo
Siqi
S
Liu
Nan
N
Liang
Fei
F
Sun
Yuying
Y
Cui
Jianwu
J
D015703:Antigens, CD / Q000235:genetics*; D051940:B7-2 Antigen; D001426:Bacterial Proteins / Q000235:genetics*; D018106:CD28 Antigens / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018106:CD28 Antigens; C497964:CD86 protein, human; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa