Prokaryotic expression, purification, identification of human cystatin C and preparation of its antiserum
- VernacularTitle:人胱抑素C的原核表达纯化鉴定及抗血清的制备
- Author:
Tingmei CHEN
;
Jiafu FENG
;
Ju CAO
;
Yangan WEN
;
Zhiguang TU
- Publication Type:Journal Article
- Keywords:
cystatin C;
prokaryotic expression system;
protein purification;
antiserum
- From:Journal of Third Military Medical University
2003;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a prokaryotic expression vector of cystatin C (Cys C), purify Cys C protein produced by the expression system, and prepare its antiserum. Methods Total RNA was isolated from HL-60 cells, and human Cys C gene was amplified with RT-PCR. The cDNA fragment was cloned into pMD18-T vector and which was confirmed by sequencing. The enzyme-digested target fragment was cloned into PET-32(a) expression vector and transfected into E.coli. BL 21(DE3), in which Cys C expression was induced. After the inclusion body protein was purified through Ni2+ affinity chromatography, processed by dialysis, identified by Western blotting, a rabbit was immunized with the fusion protein, and the antiserum was obtained. Results The result of DNA sequence analysis showed that the cloned Cys C gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blotting showed that the expressed Cys C fusion protein was about 35?103, mainly existing in the inclusion body of E.coli., that could be purified through Ni2+ affinity chromatography. The titer of the antiserum to the purified protein was 1∶8 000 by ELISA, and Western blotting confirmed that the antiserum reacted specifically to the Cys C protein. Conclusion A recombinant Cys C protein and the specific polyclonal antibody have been obtained, which provides a basis for establishment of immunoassays of human Cys C.
