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目的构建人源尖吻蝮蛇毒蛋白抗体单链可变区片段(scFv)噬菌体文库。方法采用Trizol试剂提取尖吻蝮蛇咬伤后恢复期患者的外周血淋巴细胞的总RNA,用Oligotex誖mRNA Mini Kits纯化获得总mRNA,逆转录合成总cDNA,针对人抗体轻链和重链可变区基因设计一系列特异性引物,以合成的总cDNA为模板,扩增得到抗体轻链和重链可变区基因库,再通过重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)将轻链和重链拼接得到scFv文库;将scFv基因双酶切后,插入T7噬菌体骨架进行包装,以尖吻蝮蛇蛇毒蛋白抗原进行4轮淘选,建立蛇毒蛋白抗体scFv噬菌体文库,采用噬斑法检测scFv噬菌体文库滴度并进行PCR及测序鉴定。结果成功地将轻链和重链可变区基因库混合拼接得到scFv文库;经4轮淘选,蛇毒蛋白抗体scFv噬菌体文库滴度为6.0×1014PFU/ml,抗体基因在噬菌体中表达的阳性率达50%以上,表达的scFv片段均为轻链和重链的杂合体,可较好的重现可变区的多样性。结论已成功构建了人源尖吻蝮蛇毒蛋白抗体scFv噬菌体文库,为下一步噬菌体文库的克隆构建和筛选奠定了基础。
Objective To construct a single chain variable region fragment (scFv) phage library of human Agkistrodon acutus venom protein. Methods Trizol reagent was used to extract the total RNA of peripheral blood lymphocytes from convalescents after Agkistrodon halysginae convalescent. The total mRNA was purified with Oligotex mRNA Mini Kits, and the total cDNA was reverse transcribed for the light and heavy chains of human antibodies A series of specific primers were designed for the variable region genes. The total cDNA of the antibody was used as a template to amplify the gene library of the variable region of the light and heavy chains of the antibody, and then amplified by overlap extension PCR (SOE-PCR) Light chain and heavy chain splicing to obtain scFv library; scFv gene double digestion, insert T7 phage scaffold packaging, scavenger protein snake venom protein 4 panning, the establishment of snake venom protein scFv phage library, using plaques Method to detect scFv phage library titer and PCR and sequencing identification. Results The scFv library was successfully cloned from the gene library of variable region of light chain and heavy chain. After 4 rounds of panning, the titer of scFv phage library was 6.0 × 1014 PFU / ml. The positive rate of antibody gene expression in phage Up to 50%. The expressed scFv fragments are hybrids of light chain and heavy chain, which can better reproduce the diversity of variable regions. Conclusion The scFv phage library of human Agkistrodon versicolor protein antibody has been successfully constructed, which lays the foundation for the cloning and screening of the phage library.