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目的 微小隐孢子虫地方株cDNA文库构建及P2 3、CP15 60基因的克隆。 方法 提取微小隐孢子虫总RNA、mRNA ,逆转录合成cDNA。将cDNA与 pUC18DNA连接 ,导入DH5α宿主细胞中生成cDNA文库。根据文献分别设计并合成两对PCR引物 ,从上述文库中筛选保护性基因 ,对PCR产物克隆、测序。 结果 文库容量为 1.9× 10 6个重组子 ,文库中cDNA插入片段大小介于 0 .4× 10 3～ 6.5× 10 3bp。从该文库中克隆出编码 2 3kDa、15 60kDa子孢子表面蛋白的核苷酸序列。 结论 成功地用 pUC18质粒载体构建了C .parvumcDNA文库。
Objective To construct the cDNA library of Cryptosporidium parvum and clone the P2 3 and CP15 60 genes. Methods Total RNA and mRNA of Cryptosporidium parvum were extracted and cDNA was reverse transcribed. The cDNA was ligated into pUC18 DNA and introduced into DH5α host cells to generate a cDNA library. According to the literature, two pairs of PCR primers were designed and synthesized respectively. The protective genes were screened from the above libraries, and the PCR products were cloned and sequenced. Results The library had a capacity of 1.9 × 10 6 recombinants. The size of cDNA inserts in the library ranged from 0.4 × 10 3 to 6.5 × 10 3 bp. From this library, a nucleotide sequence encoding a 23 kDa, 15 60 kDa sporozoite surface protein was cloned. Conclusion The C.parvum cDNA library was successfully constructed with pUC18 plasmid vector.