九个苯丙氨酸羟化酶基因单点突变重组质粒的构建和鉴定  

Construction and identification of nine single-point mutant recombinant plasmids of phenylalanine hydroxylase gene

作　　者：白晋丽[1] 瞿宇晋[1] 金煜炜[1] 王红[1] 宋昉[1] 

机构地区：[1]首都儿科研究所遗传室,北京100020

出　　处：《解放军医学杂志》2008年第5期527-530,共4页Medical Journal of Chinese People's Liberation Army

基　　金：北京市自然科学基金资助项目(5052008)

摘　　要：目的构建Y154H、R157I、Y206C、G247R、D282G、G346R、S349A、A389G、R400K9个PAH基因突变型重组质粒,为下一步研究这些PAH突变体在哺乳动物细胞中表达蛋白功能奠定基础。方法①选择错义突变位点。结合突变位点所在酶蛋白的结构域(如催化结构域或与辅因子BH4结合位点)及患儿相关临床表型进行了9个突变位点的选择。②构建PAH基因突变型重组质粒。以野生型PAH真核表达载体pcDNA3.0-PAH-wt为模板,应用PrimerPremier5.0软件自行设计了9对突变引物,并在Platin-iumTaqDNA高保真聚合酶的作用下直接利用体外PCR定点突变法构建PAH基因突变型重组质粒。③初筛PAH基因突变型重组质粒。氨苄抗性初步筛选:利用重组基因含氨苄青霉素抗性基因的特点,可筛选出阳性克隆;PCR及酶切技术筛选:根据野生型与突变型靶序列酶切片段的差异进行筛选,其中S349A、D282G、G247R、Y206C、Y154H这5种突变存在MvaⅠ、MvaⅠ、HindⅢ、RsaⅠ、RsaⅠ的天然酶切位点,而R157I、G346R、A389G、R400K与其相应的野生型所在位点周围并不存在天然的酶切位点,需要在目的位点的上/下游设计引物,即人工构建DdeⅠ、HindⅢ、PstⅠ、MvaⅠ酶切位点,然后利用上述限制性内切酶进行突变体的酶切鉴定。④验证PAH基因突变型重组质粒,突变体最终由DNA测序加以验证。结果这9个突变型PAHcDNA序列除特定碱基位点被突变的碱基替换外,其他序列和野生型的完全保持一致。结论成功构建了9个PAH基因突变型重组质粒。体外定点突变技术准确、实用。Objective To perform PCR site-directed mutagenesis of nine novel PAH gene mutations （Y154H, R157I, Y206C, G247R, D282G, G346R, S349A, A389G, R400K） identified in northern Chinese and construct mutant recombinant plasmids of PAH gene Methods 1） Every mutant recombinant plasmid was constructed according to the site of the mutation localized in functional domain of PAH gene and the related clinic phenotype of patients with the gene mutation. 2） Using the wild-type PAH expression vector as a templet, the mutant recombinant plasmids were directly amplified by PCR with Platinium Taq DNA polymerase and nine pairs of primers which were designed according to the human PAH eDNA sequence and the requirement for site-directed mutagenesis technology. 3） The positive strains were selected by Amp resistant test, PCR and restriction endonuclease analysis The Mvα Ⅰ, Mvα Ⅰ, Hind Ⅲ, Rsα Ⅰ, Rsα Ⅰ sites exist in the sequences near the mutant sites of S349A, D282G, G247R, Y206C, Y154H, respectively, but not in the related sequence of wild-type PAH expression vector. Restriction endonuclease digestion could be directly used in identifying the mutant sites. However, the amplification created restriction site （ACRS） analysis was supplied in the followed identification of R157I G346R, A389G, R400K. Finally the sequences of mutant recombinant plasmids of PAH gene were confirmed by DNA sequence analysis Results Every sequence analysis showed that the mutant nucleic acids were introduced at the expected sites of PAH gene, suggesting that the mutant recombinant plasmids of PAH gene were constructed successfully. Conclusion PCR site-directed mutagenesis is accurate and highly efficient. The successfully mutagenized plasmids of PAH gene lay the foundation for the functional analysis of phenylalanine hydroxylase in mammalian cell system.

关 键 词：PAH基因 点突变 质粒 

分 类 号：R34-33[医药卫生—基础医学]