CAJ | 학술논문

目的构建系统性红斑狼疮(SLE)、类风湿性关节炎(RA)、强直性脊柱炎、干燥综合症(SS)、原发性胆汁性肝硬化(PBC)等自身免疫性疾病的噬菌体抗体库.方法从多例确诊的高滴度自身免疫病患者外周血淋巴细胞中抽提总RNA,用RT-PCR方法分别扩增免疫球蛋白分子轻链κ、λ基因及重链Fd基因.经SacI和XbaI限制性双酶切后,在T4连接酶的作用下,将轻链基因片段克隆入同样双酶切的pComb3Hss载体中以构建轻链文库,随后将重链Fd段PCR产物经XhoI和Spe I双酶切后载入同样处理的κ/λ-pComb3Hss载体,然后将重组质粒κ/λ-Fd-pComb3Hss转化大肠杆菌XL1-Blue.用辅助噬菌体M13 KO7感染XL1-Blue,则可在噬菌体表面表达出随机的重组抗体库.结果提取的淋巴细胞总RNA及合成的cDNA质量好,PCR扩增了长度为660 bp的轻链κ、λ基因及重链Fd基因,成功构建了库容为4×104的轻链抗体库和库容为6×104的Fab片段噬菌体抗体库,酶切及PCR鉴定均正确无误.结论成功构建了自身免疫性疾病的噬菌体抗体库,为进一步筛选出高亲和力抗体打下了基础.
목적 구건계통성홍반랑창(SLE)、류풍습성관절염(RA)、강직성척주염、간조종합증(SS)、원발성담즙성간경화(PBC)등자신면역성질병적서균체항체고.방법 종다례학진적고적도자신면역병환자외주혈림파세포중추제총RNA,용RT-PCR방법 분별확증면역구단백분자경련κ、λ기인급중련Fd기인.경SacI화XbaI한제성쌍매절후,재T4련접매적작용하,장경련기인편단극륭입동양쌍매절적pComb3Hss재체중이구건경련문고,수후장중련Fd단PCR산물경XhoI화Spe I쌍매절후재입동양처리적κ/λ-pComb3Hss재체,연후장중조질립κ/λ-Fd-pComb3Hss전화대장간균XL1-Blue.용보조서균체M13 KO7감염XL1-Blue,칙가재서균체표면표체출수궤적중조항체고.결과 제취적림파세포총RNA급합성적cDNA질량호,PCR확증료장도위660 bp적경련κ、λ기인급중련Fd기인,성공구건료고용위4×104적경련항체고화고용위6×104적Fab편단서균체항체고,매절급PCR감정균정학무오.결론 성공구건료자신면역성질병적서균체항체고,위진일보사선출고친화력항체타하료기출.
Objective To construct the phage antibody library against autoimmune diseases,such as systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, Sjogren syndrome and primary billary cirrhosis. Methods The total RNA was extracted from peripheral blood lymphocytes of some patients who suffered from autoimmune diseases. The heavy chain Fd and light chain κ,λ gene were amplified by RT-PCR. The amplification products were digested by ScaI and XbaI, then ligated into the phagemid vector pComb3Hss to construct light chain library via T4 ligase. The amplification products of Fd and the light chain library were digeseted by XhoI and Spe I. The ligated sample of Fd and light chain library was then transformed into competent E. coli XL1-Blue. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody. Results The quantity of total RNA and cDNA were qualified. Human immunoglobulin light chain and heavy chain Fd genes (660 bp) were amplified successfully. The light chain library with the capacity size of 4×104 and the human recombinant Fab antibody library with the capacity size of 6 × 104 were also successfully obtained, while both the digestion by enzymes and PCR identification showed the right results. Conclusion A phage antibody library against autoimmune diseases is successfully constructed,which lays a foundation for the following panning of high affinity antibody.