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目的构建天然人源甲状腺未分化癌噬菌体单链抗体库并初步筛选出抗甲状腺未分化癌抗体。方法从甲状腺未分化癌患者癌周淋巴结组织中提取总RNA,采用RT-PCR技术扩增抗体可变区基因VH和VL基因片段,并分别与相应Linker连接,再通过重叠延伸PCR技术将其拼接组装成sc Fv基因片段,并引入酶切位点SfiⅠ和NotⅠ。将双酶切后的sc Fv重组基因片段与噬菌粒栽体p CANTAB-5E连接,产物经化学转化转入大肠杆菌TG1,超感染噬菌体,构建噬菌体单链抗体库。以甲状腺未分化癌细胞(ARO细胞株)为抗原对该抗体库进行4轮“吸附-洗脱-扩增”的筛选,并行ELISA鉴定抗体特异性。结果成功获得约为750 bp的sc Fv基因。用p UC19标准质粒测定转化效率达到108 cfu/μg,双酶切鉴定sc Fv基因的阳性插入率为86.4%(19/22)。筛选过程中,甲状腺未分化癌单链抗体得到富集,收获率不断提高,第4轮为第1轮的77倍。ELISA结果显示筛选出的抗体能与甲状腺ARO细胞株特异性结合。结论成功构建了人源甲状腺未分化癌噬菌体单链抗体库,并从中筛选出具有甲状腺未分化癌细胞特异性的人源噬菌体单链抗体,为进一步放免研究打下基础。
Objective To construct natural single-chain antibody library of human undifferentiated thyroid carcinoma and to screen out the anti-thyroid undifferentiated carcinoma antibody. Methods Total RNA was extracted from the peritumoral lymph nodes of patients with undifferentiated thyroid carcinoma. The VH and VL gene fragments of the variable region of the antibody were amplified by RT-PCR and ligated with the corresponding Linker, respectively, and then spliced by overlap extension PCR Assembled into sc Fv gene fragment, and introduced restriction sites SfiI and NotI. The double-digested sc Fv recombinant gene fragment was ligated with the phagemid vector pCTATAB-5E, and the product was transformed into E. coli TG1 by chemical transformation. The bacteriophage was over-infected to construct phage scFv library. The antibody library was screened by 4 rounds of “adsorption - elution - amplification” with an undifferentiated thyroid carcinoma cell line (ARO cell line) as an antigen and the specificity of the antibody was identified by ELISA. Results The sc Fv gene of about 750 bp was successfully obtained. The transformation efficiency was 108 cfu / μg when using pUC19 standard plasmid and 86.4% (19/22) by double enzyme digestion. During the screening process, single-chain antibody of undifferentiated thyroid carcinoma was enriched and the yield was continuously increased. The fourth round was 77 times of the first round. The ELISA results showed that the selected antibodies could specifically bind to the thyroid ARO cell line. Conclusion Human phage display single-chain antibody library of human undifferentiated human thyroid carcinoma has been successfully constructed and single-chain human phage scFv against human thyroid carcinoma cells has been screened out for further study on radioimmunoassay.