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[目的]构建人尿道上皮细胞(SV-HUC-1)T7噬菌体展示c DNA文库,为研究人尿道上皮细胞与生殖道感染病原体的相互作用奠定基础。[方法]用Trizol试剂提取SV-HUC-1细胞总RNA,分离纯化出mRNA,经反转录合成得到其双链c DNA,在双链c DNA末端加上定向的EcoRⅠ/HindⅢ黏性末端,然后收集并纯化200bp以上的双链c DNA片段,连接于T7噬菌体载体,经体外包装后转入BLT5403宿主菌,T7噬菌体展示c DNA文库构建成功。[结果]将文库扩增后,用噬斑试验检测其库容,结果为1.2×106pfu/cm3。用PCR鉴定随机挑取的噬菌斑,计算其重组率达93.75%,且插入片段都大于200bp。[结论]成功构建了SV-HUC-1细胞T7噬菌体展示c DNA文库,为下一步研究泌尿生殖道感染病原体与人尿道上皮细胞的相互作用奠定了前期实验基础。
[Objective] To construct c DNA library of human urethral epithelial cells (SV-HUC-1) T7 phage and lay the foundation for the study of the interaction between human urethral epithelial cells and pathogens of genital tract infection. [Method] The total RNA of SV-HUC-1 cells was extracted with Trizol reagent, and the mRNA was isolated and purified. The double-stranded c DNA was obtained by reverse transcribed, and the directional EcoRⅠ / HindⅢ- Then, the double-stranded DNA fragment of 200 bp or more was collected and purified, ligated to the T7 phage vector, transformed into BLT5403 host bacterium after in vitro packaging, and the T7 phage display c DNA library was successfully constructed. [Result] After the library was amplified, its storage capacity was tested by plaque test, and the result was 1.2 × 106pfu / cm3. Plaque picked at random was identified by PCR. The recombination rate was 93.75%, and the inserted fragments were all larger than 200bp. [Conclusion] The c-DNA library of T7 phage displayed by SV-HUC-1 cells was successfully constructed, which laid the foundation for the preliminary study of the interaction between urogenital tract pathogens and human urethral epithelial cells in the next step.