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目的构建婴幼儿小肠组织全长cDNA文库,为进一步通过酵母双杂交方法搜寻与轮状病毒VP4蛋白相互作用的宿主细胞受体蛋白提供必要条件。方法2006年11月至2007年9月从婴幼儿小肠组织中提取总RNA,并纯化mRNA,反转录合成第1链,连接EcoRI人工接头,分级分离后,除去小于500bp cDNA片段,与质粒pUra-MΔpolyA连接后,氯化钙法转化感受态细胞DH5α,测定文库大小并利用EcoRI-XhoI双酶切鉴定插入cDNA片段的大小。结果构建成含有6×107重组子的婴幼儿小肠组织cDNA文库,插入片段的大小范围在0.5~2.0kb之间,平均长度1.5kb。结论构建的文库合格,适合用于筛选目的cDNA克隆。
Objective To construct a full-length cDNA library of small intestine of infants and young children, and to provide the necessary conditions for further searching the receptor protein of host cell interacting with rotavirus VP4 protein by yeast two-hybrid method. Methods From November 2006 to September 2007, total RNA was extracted from small intestine tissues of infants and young children. The mRNA was purified and the first strand was reverse transcribed to EcoRI artificial linker. After fractionated, the cDNA fragments less than 500 bp were removed and ligated with plasmid pUra -MΔpolyA, the competent cells DH5α were transformed by calcium chloride method, and the size of the library was determined. The size of the inserted cDNA fragment was identified by double enzyme digestion with EcoRI-XhoI. Results The cDNA library of small intestine tissue containing 6 × 107 recombinants was constructed. The insert size ranged from 0.5 to 2.0 kb with an average length of 1.5 kb. Conclusion The constructed library is qualified and suitable for screening the cDNA clones of interest.