摘要
目的为筛选微小隐孢子虫保护性抗原基因,构建了微小隐孢子虫T7噬菌体展示文库。方法用Trizol试剂提取微小隐孢子虫总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上定向EcoRⅠ/HindⅢ接头,用EcoRⅠ和HindⅢ消化接头,使形成两端分别带有EcoRⅠ和HindⅢ粘性末端的双链cDNA。经Mini Column纯化,收集400bp以上的双链cDNA片段,将其连接于带有EcoRⅠ和HindⅢ末端的T7Select 10-3b载体,经体外包装后,以BLT5403为受体菌构建T7噬菌体展示文库。结果经测定,库容量为1.2×107pfu/mL,重组率为96.7%,扩增后文库滴度为2.4×1010pfu/mL。对随机挑取的100个噬菌斑进行PCR鉴定,92%的插入片段大于400bp。结论成功构建了微小隐孢子虫T7噬菌体展示文库。
To screen protective antigen gene of Cryptosporidium parvum, the T7 phage display library from C. parvum was constructed, and the total RNA was extracted from C. parvum by Trizol reagent. The mRNA was isolated from total RNA by PolyATract mRNA Isolation Kit and the ds eDNA was synthesized by reverse transcription. In addition the directional EcoR Ⅰ/Hind m linkers were ligated into the ends of ds eDNA and the ds eDNA was digested with EcoR Ⅰ and Hind Ⅲ, which resulted in ds eDNA with EcoR Ⅰ and Hind Ⅲ ends. And the ds eDNA fragments longer than 400bp in length were fractionated by Mini Column, and then ligated into the T7 Select 10-3b vertor with EcoR Ⅰ and Hind m ends.After packaging in vitro, the T7 Select 10-3b vertor was transformed into BLT5403 to construct the T7 phage display library. The experimental results showed that the library contained 1.2 × 10^7 clones, and approximately 96.7% of the library were recombinant. The titer of the amplied library was 2.4 × 10^10 pfu/mL. The eDNA fragments longer than 400bp in length were 92 % of 100 plaques by PCR identification. These results suggests that the T7 phage display library from C. parvum is constructed successfully.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第1期26-28,42,共4页
Chinese Journal of Zoonoses
基金
国家自然科学基金资助项目(No.30400324)
