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 Ni

2+

affin

ity 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 35×10

3

mainly existing in the inclusion body of E.coli.

that could be purified through Ni

2+

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.