[The expression and application of human Fas ligand in E.coli].
OBJECTIVE
To express recombinant human FasL molecule in E.coli.


METHODS
RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2.1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E.coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice.


RESULTS
The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M(r)40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits.


CONCLUSIONS
The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.
Li
Ning-li
NL
Department of Immunology, Fourth Military Medical University, Xi'an 710032, China. ningli@shsmu.edu.cn
Nie
Hong
H
Yu
Qi-wen
QW
Bo
Jun
J
Ma
An-lun
AL
Zhang
Ji-ying
JY
Shen
Bai-hua
BH
Wang
Li
L
Zhang
Dong-qing
DQ
D000818:Animals; D017209:Apoptosis; D003001:Cloning, Molecular; D003922:Diabetes Mellitus, Type 1 / Q000097:blood*; D004926:Escherichia coli / Q000378:metabolism*; D053222:Fas Ligand Protein; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D019169:Jurkat Cells / Q000166:cytology; D007938:Leukemia / Q000097:blood; D008180:Lupus Erythematosus, Systemic / Q000097:blood; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013274:Stomach Neoplasms / Q000097:blood; D048069:Tumor Necrosis Factors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007106:Immune Sera; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D048069:Tumor Necrosis Factors