植物偏爱密码子优化HPV18L1全长基因的重叠PCR合成  

Full-length of human papillomavirus type 18L1 gene optimized using the plant prefered condons and synthesized by overlapping PCR

作　　者：赵莹[1] 张丽仪[1,2] 刘昌政[1] 周晓红[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广东广州510515 [2]中山大学肿瘤防治中心腹科,广东广州510060

出　　处：《南方医科大学学报》2009年第3期387-392,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(30872204、30400367);广东省科技计划项目(73007)

摘　　要：目的以重叠PCR方法合成植物偏爱密码子优化后的人乳头瘤病毒18L1全长基因。方法自GenBank获取国内外所发布的HPV18L1全长基因序列,经生物信息学分析后选定目标序列,经Synthetic Gene Designer及JCat(JavaCodon Adaptation Tool)分析软件将此序列原始密码子改造为植物偏爱密码子,在C端加入蛋白纯化标签His-tag,获得改造后的HPV18L1全长序列(mHPV18L1)。结合酶切位点分析将mHPV18L1设计为204～477bp的五个大片段LS1-LS5,再将每个大片段分割成57～59bp的5～11个寡聚核苷酸短链,设计合成5对内引物和1对外引物,经重叠PCR扩增获得mHPV18L1全长基因,纯化后将其克隆入pMD18T载体,酶切、测序鉴定插入子。结果以重叠PCR成功扩增获得mHPV18L1全长,pMD18T-mHPV18L1重组质粒经酶切、PCR鉴定均得到预期1749bp大小的片段,并经测序验证。结论获得以植物偏爱密码子进行序列优化的mHPV18L1全长基因及其重组质粒pMD18T-mHPV18L1,为后续mHPV18L1的植物转化研究奠定了一定的工作基础。Objective The Human papillomavirus type 18L1 （HPV18L1） gene was synthesized by overlapping PCR after optimization using plant preferred codons. Methods The gene sequences of HPV18L1 were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. The target sequence was selected and modified using plant preferred codons by the Synthetic Gene Designer and JCat （Java Codon Adaptation Tool）with the addition of a His-tag to the C-terminus to construct the full-length modified HPV18L1 （mHPV18L1）. mHPV18L1 was divided into 5 large segments, namely LS1 to LS5, with sizes ranging from 204 to 477 bp. Forty-three small oligonucleotide fragments with sizes of 57-59 bp and 6 pairs of primers were designed and synthesized, mHPV18L1 was amplified by overlapping PCR and subcloned into pMD18-T vector. The recombinant plasmid was identified by restriction enzymes digestion and sequencing. Results mHPV18L1 was successfully assembled using overlapping PCR. The results of digestion with restriction enzymes and PCR amplification confirmed that the recombinant vector pMD18T- mHPV18L1 contained the inserts with expected size of 1749 bp. mHPV18L1 sequence was confirmed by sequencing. Conclusion mHPV18L1 with plant preferred codons and the recombinant vector pMD 18T- mHPV 18L 1 have been obtained.

关 键 词：HPV18L1 重叠PCR 密码子优化 

分 类 号：Q943.2[生物学—植物学]