【摘要】：研究在大腸桿菌中表達重組人胱抑素C(Cystatin C,Cys C)重鏈單域抗體(Variable domain of the heavy chain of heavy-chain antibody,VHH),經(jīng)純化和復(fù)性后,建立測定人血清Cys C的膠乳增強免疫比濁法(Particle enhanced turbidimetric immunoassay,PETIA)。根據(jù)大腸桿菌編碼蛋白的特性設(shè)計Cys C重鏈單域抗體編碼基因序列,人工合成目的基因克隆至PET32a(+)載體中,構(gòu)建重組人Cys C重鏈單域抗體原核表達質(zhì)粒PET32a(+)-Cys C重鏈單域抗體,測序鑒定正確后轉(zhuǎn)入大腸桿菌BL21中,異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)表達,所獲得的目的抗體經(jīng)Ni-NTA親和層析法進行純化,通過稀釋復(fù)性,SDS-PAGE鑒定其純度。Cys C重鏈單域抗體化學(xué)偶聯(lián)膠乳顆粒,建立PETIA法測定人血清Cys C。SDS-PAGE電泳分析顯示,表達的Cys C重鏈單域抗體相對分子質(zhì)量約為53 k,經(jīng)Ni-NTA親和層析法純化后抗體純度達90%以上,復(fù)性回收率為60%左右。用Cys C重鏈單域抗體建立的測定人血清Cys C的PETIA,能檢測到血清中的Cys C含量。成功構(gòu)建Cys C重鏈單域抗體原核表達系統(tǒng)并獲得了有活性的Cys C重鏈單域抗體,該抗體可用于建立測定Cys C的PETIA法,為下一步開發(fā)Cys C的免疫檢測試劑盒奠定了基礎(chǔ)。
[Abstract]:To study the expression of recombinant human cystatin C (Cystatin C (Cys C) heavy chain single domain antibody (Variable domain of the heavy chain of heavy-chain antibody,VHH) in E. coli, after purification and renaturation, To establish a latex enhanced immunoturbidimetric assay (Particle enhanced turbidimetric immunoassay,PETIA) for the determination of Cys C in human serum. The Cys C heavy chain single domain antibody encoding gene sequence was designed according to the characteristic of E. coli protein, and the target gene was synthesized and cloned into PET32a () vector. The recombinant human Cys C heavy chain single domain antibody prokaryotic expression plasmid PET32a ()-Cys C heavy chain single domain antibody was constructed, identified by sequencing and transferred into E. coli BL21. Isopropyl-尾-D-galactogalactoside (IPTG) was induced and expressed. The purified antibody was purified by Ni-NTA affinity chromatography, and its purity was identified by dilution and renaturation by SDS-PAGE. Cys C heavy chain single domain antibody was chemically coupled with latex particles. PETIA method was established for the determination of human serum Cys C.SDS-PAGE electrophoresis. The relative molecular weight of Cys-C heavy chain single domain antibody was about 53 kg. the purity of the antibody purified by Ni-NTA affinity chromatography was more than 90%, and the recovery rate of renaturation was about 60%. The content of Cys C in human serum Cys C can be detected by PETIA, established by using heavy chain single domain antibody against Cys C. The prokaryotic expression system of Cys-C heavy chain single domain antibody was successfully constructed and the active Cys-C heavy chain single domain antibody was obtained. The antibody can be used to establish PETIA assay for the determination of Cys-C and lay a foundation for the further development of Cys-C immune detection kit.
【作者單位】： 南京理工大學(xué)環(huán)境與生物工程學(xué)院;中國人民解放軍第八一醫(yī)院檢驗科;南京大學(xué)生命科學(xué)學(xué)院;
【基金】：973項目資助(No.2014CB744501)
【分類號】：R943

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