大鼠矽肺噬菌体单链抗体库构建

Establishing a genebank for phage single-chain antibody using silicotic rats

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1.35×10^16pfu/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 establish a genebank for phage single-chain antibody for further screening the specificity of single chain fragment variable（ Sc Fv） in lung tissue of silicosis rats by phage display technology. Methods Twenty-four specific pathogen free male SD rats were used to construct silicosis model by one-time bronchial perfusion with 1. 0 m L of silicon dioxide suspension（ mass concentration,100 g / L）. We took periphery blood from 6 rats 3,6,9 and 12 weeks respectively after establishing the model. The peripheral lymphocytes were mixed,and total RNA was extracted using Trizol,and c DNA was synthesized by reverse transcription. The degenerated primers were used to amplify the variable region of heavy chain（ VH） gene and variable region of light chain（ VL） gene by polymerase chain reaction（ PCR）.Then VH and VL genes were assembled to form Sc Fv by T4 DNA linker. The cloning recombinant of Sc Fv and plasmid of PCANTAB-5e were transformed into competence E. coli TG1 by calcium chloride. The Sc Fv genebank of silicosis model was constructed by M13K07 helper phage superinfection. There were 10 bacterial colonies for plasmid restriction dualenzyme digestion randomly selected for confirmation. Results Agarose gel electrophoresis showed that there were two bands of obvious 28 S and 18 S in total RNA of periphery blood lymphocytes of silicosis rats. The total RNA was intact. The size of VH gene fragment was about 400 bp,the size of VL gene fragment was about 350 bp and recombinant Sc Fv gene fragment length was about 750 bp. The helper phage was amplified and placed with double-deck agar plate and observed limpid plaque with the size of a rice grain. The phage titer was 1. 35 × 10^（16） pfu / L. The recombinant plasmids were transformed into E. coli TG1 and total bacterial count was 8. 0 × 10^9 cfu / L in resistant plate. The positive cloned plasmid PCR gel electrophoresis and double enzyme results showed a positive inserting rate of 90. 0%. The capacity of phage single-chain antibody genebank of experimental silicosis was 7. 2 × 10^9 cfu / L. Conclusion The silicosis rat model with phage Sc Fv gnebank could be successfully established,and its capacity and diversity provide support for the follow-up screening.