摘要
目的：建立一个能表达人βＩＶＳ－Ⅱ－ｎｔ６５４Ｃ→Ｔ突变基因（β６５４基因）的ＨｅＬａ细胞模型。方法：应用长片段多聚酶链反应（ＰＣＲ）技术从一例β６５４突变基因纯合子的基因组ＤＮＡ中扩增出β６５４基因全长片段，重组进ｐｃＤＮＡ３．１真核表达载体质粒中。经ＤＮＡ测序证明序列中确实含有β６５４突变后，将重组表达质粒用脂质体方法转化ＨｅＬａ细胞，用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）方法检测β６５４突变基因在转染细胞中表达的ｍＲＮＡ。结果：该重组表达质粒能在ＨｅＬａ细胞中转录β６５４突变基因，并剪接成正常和异常两种ｍＲＮＡ（对应于１８３ｂｐ和２５６ｂｐ两种ＲＴ－ＰＣＲ产物），而对照组无扩增条带出现。结论：重组表达质粒转化的ＨｅＬａ细胞能表达β６５４基因。
Objective: To establish a cell model expressing human βIVS Ⅱ nt 654C→T allele (β 654 mutant). Methods: DNA fragment of entire β globin gene encompassing the codon region and poly(A) signal of the allele was amplified by long PCR from the genomic DNA of a homozygote with β 654 mutation. The amplified fragments were cloned into the Hind Ⅲ and Xba Ⅰ sites of the pcDNA3.1 vector. After reidentification of the clone with β 654 mutant by DNA sequencing, the recombinant plasmid was transfected into HeLa cell using liposome method. The expression of the β 654 mutant gene in the transfected cells was detected by RT PCR. Results: Both the normally processed β globin mRNA (183bp) and aberrant processed β globin mRNA (256bp) were identified in the transfected HeLa cells,while no RT PCR product was detected in the controls.Conclusion: The transfected HeLa cells can express human β 654 allele.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
1997年第10期532-534,共3页
Chinese Journal of Hematology
基金
国家自然科学基金
上海市卫生局青年科研基金
