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目的以乳头瘤病毒(human papillomavirus,HPV)18型E6基因为靶标,构建小发夹状RNA(small hairpinRNA,shRNA)表达载体。方法人工方法合成针对靶序列的两条寡核苷酸链,两端加入酶切位点。寡核苷酸链退火后与酶切的线性化表达载体连接。连接产物转化大肠埃希菌,卡那霉素筛选阳性克隆。结果重组质粒酶切片段约为400 bp,与插入片段大小一致。DNA序列分析证实插入片段序列与预期一致。结论成功构建HPV shRNA表达载体pHPV1、pHPV2。
Objective To construct small hairpin RNA (shRNA) expression vector targeting E6 gene of human papillomavirus (HPV) type 18. Methods Two oligonucleotide chains targeting the target sequence were synthetically synthesized, and the restriction sites were added at both ends. Oligonucleotide strands were annealed and ligated to the digested linearized expression vector. The ligation products were transformed into Escherichia coli, kanamycin screening positive clones. Results The recombinant plasmid was about 400 bp, which was consistent with the size of the inserted fragment. DNA sequence analysis confirmed that the insert sequence was as expected. Conclusion The HPV shRNA expression vectors pHPV1 and pHPV2 were successfully constructed.