[Construction expression and biologic activity of recombinant human sCR1 eucaryotic cell].
OBJECTIVE
To express human soluble complement receptor type 1(sCR1)protein using ferment cell secreting type carrier and study the extraorgan biologic activity of recombinant human sCR1 fusion protein.


METHODS
Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR and them, cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k-sCR1 containing human sCR1.After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMD1168. The ferment cell line of the recombinant sCR1 which was chosen by G418 resistance was identified by PCR, After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blot, purified by Ni(2+)-NTA agarose affinity chromatography, and its biologic activity was identified.


RESULTS
The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line. The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9k-sCR1 induced by methanol. It was a protein band about M(r) 31 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni(2+)-NTA agarose affinity chromatography.


CONCLUSIONS
The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system, which resembles the human natural protein's antigenicity and biologic activity.
Wang
Guang-lan
GL
Center Laboratory Medicine of Jinan Command, PLA 153 Hospital, Zhengzhou 450042, China. herrwangl@yahoo.com.cn
Gong
Cui-cui
CC
Wang
Wen-li
WL
Zhang
Chang-yuan
CY
Liu
Ya-li
YL
He
Pei-xia
PX
Zheng
Shu-na
SN
Chen
Xiang-lin
XL
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005057:Eukaryotic Cells / Q000378:metabolism* / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D010843:Pichia; D017463:Receptors, Complement 3b / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C482970:CR1L protein, human; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D009942:Organometallic Compounds; D017463:Receptors, Complement 3b; D011993:Recombinant Fusion Proteins; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid