抗环胍氨酸肽单链噬菌体抗体库的建立

Construction of Single-chain Fragment V Antibody pHEN2 Phagemid Library of Anti-cyclic Cit- rullinated Peptide

【目的】拟构建并鉴定抗环胍氨酸肽（cyclic citrutlinated peptide，CCP）单链片段V（Single chain Fragment V，ScFv）噬菌体抗体库，为进一步可溶性表达和检测类风湿性关节炎（RA）关节滑膜的CCP表达情况，揭示CCP及其抗体在RA发病中作用奠定基础。【方法】①分离抗CCP抗体阳性患者外周血单个核细胞，提取mRNA并反转录cDNA。扩增免疫球蛋白重链可变区（VH）和轻链可变区（VL）。通过重叠聚合酶链反应（PCR）用连接片段将VH和Vκ／Vλ体外连接成单链抗体基因；SfiⅠ和NotⅠ酶切并与噬菌粒PHEN2连接；将pHEN2ScFv电转至感受态大肠杆菌TG1，建立抗CCp-ScFv噬茵体抗体库；②检测抗CCP-ScFvPHEN2噬菌体的库容、插入效率；【结果】抗CCP-ScFv噬菌体抗体库库容8．76×10^8CFUcfu／mL，插入频率83．3％；插入率83．33％。所插入DNA片断约1180bp。MSPⅠ酶切指纹图谱鉴定也显示多样性。【结论】所构建抗环胍氨酸肽单链噬茵体抗体库库容可满足进一步可溶性表达的要求。

[Objective] To construct and identify of single-chain fragment V antibody（ScFv） pHEN2 pha- gemid library of anti-cyclic citrullinated peptides（CCP）, and to further explore the soluble expression of CCP in synovium of rheumatoid arthritis（RA） so as to provide the basis for revealing the role of CCP and antibody in the pathogenesis of RA. [Methods]Peripheral blood mononuclear cells in patients with anti-CCP antibody were isolated for the extraction of mRNA and reverse transcription of cDNA. Immunoglobulin heavy chain variable region（VH） and light chain variable region（VL） were amplified. VH and Vκ/Vλ were linked into ScFv gene with junction segment by using overlapping PCR in vitro. Sfi Ⅰ and Not Ⅰ endonuclease were linked to phagemid pHEN2 which was electrically transformed to competence Escherichia coli. TG1. Anti-CCP-ScFv phagemid antibody library was constructed. The content and insertion rate of anti-CCP-ScFv pHEN2 phagemid were detected. [Results]The content of anti-CCP-ScFv pHEN2 phagemid antibody library was 8.76×10^8 CFU cfu/ml, and the insertion rate was 83. 330//oo. The inserted DNA fragment was ll80bp. MSP Ⅰ endonuclease fingerprinting identification showed the diversity. [Conclusion]The content of ScFv pHEN2 phagemid library of anti-CCP can meet the demand of further soluble expression.