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目的利用噬菌体展示技术构建大鼠矽肺噬菌体单链抗体(Sc Fv)库,为后续筛选矽肺特异性Sc Fv奠定基础。方法无特定病原体级雄性SD大鼠24只,予质量浓度为100 g/L的二氧化硅混悬液1.0 m L经支气管一次性灌注染尘,构建矽肺模型。于造模后第3、6、9和12周分别取6只大鼠周围血淋巴细胞混匀,以Trizol法提取总RNA,逆转录合成cDNA;利用简并引物进行聚合酶链式反应(PCR),扩增抗体重链可变区(VH)和轻链可变区(VL)的基因,用T4 DNA连接酶连接获得ScFv基因片段,将其与噬菌粒PCANTAB-5e重组,采用氯化钙转化法转化至感受态大肠埃希氏菌(E.coli)TG1中,经M13K07辅助噬菌体超感染,构建矽肺模型大鼠噬菌体ScFv库;随机挑取10个菌落进行质粒双酶切鉴定。结果矽肺模型大鼠周围血总RNA琼脂糖凝胶电泳可见2条明显28S和18S条带,总RNA完整性较好;VH基因片段大小约为400 bp,VL基因片段大小约为350 bp,重组后Sc Fv基因片段长度约为750 bp;M13K07辅助噬菌体扩增后铺双层琼脂平板,见小米粒大小、透亮的噬菌斑,噬菌体滴度为1.35×1016pfu/L;以重组噬菌粒转化感受态E.coli TG1后铺氨苄青霉素抗性固体平板,计算菌落数为8.0×10~9cfu/L;阳性克隆质粒PCR和双酶切产物经琼脂糖凝胶电泳显示阳性插入率为90.0%,所建Sc Fv库库容为7.2×10~9cfu/L。结论成功构建矽肺模型大鼠噬菌体Sc Fv库,其库容量及多样性可为后续筛选提供保障。
OBJECTIVE: To construct phage display scFv library by phage display technique and lay a foundation for subsequent screening of silicosis-specific Sc Fv. Methods Twenty-four male Sprague-Dawley (SD) rats without pathogen-specific pathogen were dosed with 1.0 mL L-1 silica suspension at a concentration of 100 g / L through a single perfusion of bronchial tubes to establish a silicotic model. At the 3rd, 6th, 9th and 12th week after operation, 6 peripheral blood lymphocytes were harvested, and the total RNA was extracted by Trizol method and reverse transcribed into cDNA. The degenerate primers were used for polymerase chain reaction (PCR) ), Amplified the heavy chain variable region (VH) and light chain variable region (VL) genes of the antibody, and obtained the ScFv gene fragment by T4 DNA ligase, recombined with the phagemid PCANTAB-5e, The plasmids were transformed into competent E. coli TG1 by calcium conversion method. The phage ScFv library of silicotic model rats was constructed by superinfection with M13K07 helper phage. Ten colonies were randomly selected for plasmid double enzyme digestion. Results Two bands of 28S and 18S were observed in the total RNA of peripheral blood of silicosis model rats. The total RNA was of good integrity. The size of VH gene fragment was about 400 bp and the size of VL gene fragment was about 350 bp. After Sc Fv gene fragment length of about 750 bp; M13K07 helper phage amplified bilayer agar plate, see the size of millet, translucent plaque, phage titer of 1.35 × 1016pfu / L; recombinant phagemid transformation The competent E. coli TG1 was coated with ampicillin-resistant solid plate, and the number of colonies was calculated as 8.0 × 10-9cfu / L. The positive cloning plasmid PCR and double digestion products showed the positive insertion rate of 90.0% by agarose gel electrophoresis, The constructed Sc Fv library volume was 7.2 × 10 ~ 9cfu / L. Conclusion The construction of phage Sc Fv library of silicosis model rat has been successfully established. The capacity and diversity of the phage library can be used for the subsequent screening.