The Preparation Of Polyclone Antibody Against Human Cystatin C And Establishment Of Immunoassay Kit

Cystatin C(CysC), a non-glycosilated, basic, low moleculor protein(13kD).It is stable produced by all nucleated cells and its production rate isunaltered due to age, sex, muscle mass, infection, dietary factors and liverdisease，etc. Recent studies have validated that the serum concentration ofCysC is an superior marker of glomerular filtration rate (GFR), especiallyfor early renal impairment. However, up to now, the main determination ofserum CysC in clinical laboratories is based on immunoassay kits fromabroad. These kits were so expensive that the popularization of CysC wasrestricted. Therefore, the localization of the immunoassay kits of CysC wasvaluable and meaningful.Objectives:To construct two prokaryotic expression vectors of human cystatin C,pET32a （+）-CysC and pCold TF-CysC, express and purify solubleTrx-CysC and tag-free CysC fusion protein separately. The polyclonalantibody against human CysC was prepared and purified. With the self-madepolyclonal antibody, particle enhanced turbidimetric immunoassay method for CysC was established，and compared with commercial immunoassay kits.The prepared tag-free CysC was utilized as protein standard.Methods:1. A cDNA fragment coding for the full-length CysC was designed andsynthesized based on the synonymous codon bias of E.coli and optimized G+C content. The synthetic CysC was then inserted into pET32a（+）expression vector； the constructed pET32a （+）-CysC plasmid wastransformed to E.coli BL21(DE3) and soluble Trx-CysC fusion protein wasexpressed under induction of IPTG. The recombinant protein was purified byNi-Sepharose6FF affinity chromatography and identified by SDS-PAGEand Western blot.2. Total RNA was extracted from HL60cell line, the cDNA templatewas generated by RT-PCR, and the DNA fragment of CysC was cloned intopCold TF expression vector；the constructed pCold TF-CysC plasmid wastransformed to E.coli BL21(DE3) and soluble TF-CysC fusion protein wasexpressed under induction of IPTG. After purified by Ni-Sepharose affinitychromatography, The TF-CysC was treated with GST-HRV3C protease, andthe TF tags and GST-HRV3C proteases were removed by size-exclusionchromatography（SEC）in one step. The tag-free CysC was identified bySDS-PAGE.3. New Zealand rabbits were immunized with the purified Trx-CysCfusion protein and the polyclonal antibody was purified. 4. The purified polyclonal antibody was incubated with uniformpolystyrene particles，and a particle enhanced turbidimetric immunoassaymethod for human serum CysC was established. The self-made method waspreliminary compared with commercial immunoassay kits.Results:1. Because of the6×his tag, soluble recombinant Trx-CysC protein waspurified by Ni-Sepharose affinity chromatography. New Zealand rabbitswere immunized with the fusion protein and the the polyclonal antibody wasobtained.2. After purified by Ni-Sepharose affinity chromatography, TheTF-CysC was treated with GST-HRV3C protease, and the TF tags andGST-HRV3C proteases were removed by size-exclusion chromatography（SEC）in one step. The tag-free CysC can be used as protein standard.3. A particle enhanced turbidimetric immunoassay method for humanserum CysC was established, and the self-made method was preliminarycompared with commercial immunoassay kits.Conclusions:1. Soluble recombinant Trx-CysC protein was obtained by molecularcloning technique and protein expression and purification. New Zealandrabbits were immunized with the fusion protein and the the polyclonalantibody was obtained.2. Soluble tag-free CysC protein was obtained and it can be used as protein standard.3. With self-made polyclonal antibody, a particle enhancedturbidimetric immunoassay method for human serum CysC was established,which should facilitate the clinical application of serum CysC test.