重组PCR构建不同CAG重复的雄激素受体基因载体

Construction of human androgen receptor gene vectors with different CAG repeats by recombinant PCR

摘要: 目的: 构建不同CAG重复的人雄激素受体(hAR)基因载体用于真核细胞表达。方法: 以CAG重复数目为CAG8,CAG21,CAG34的白细胞基因为模板,扩增含相应CAG重复的中间片段。以克隆有野生型hAR基因的质粒为模板扩增上、下游片段,并与中间片段进行重组PCR,拼接的目的片段经SalⅠ-NruⅠ酶切后置换质粒中相应片段,构建含不同CAG重复的载体。结果: 构建后的载体经酶切和DNA测序,证实CAG重复数目为8,21,34的表达载体大小和序列正确。结论: 重组PCR技术是体外基因重组的一种有效、可行的方法。

Abstract: Objective: To construct human androgen receptor（hAR） gene vectors with different CAG repeats for expression in eukaryocytes.Methods: Middle fragments with 8,21,34 CAG repeats were amplified with corresponding leukocyte gene templates.Upstream and downstream fragments amplified with the plasmid template and cloned with wild type hAR gene were then spliced with the middle fragments to obtain whole target genes.Digested with SalⅠand NruⅠ,Target genes were inserted into the digested plasmid.Results: Restriction enzyme digestion and sequence analysis showed that the size and sequence of reconstructed plasmids with 8,21,34 CAG repeats were correct.Conclusion: The results indicate that recombinant PCR is an effective and feasible way for gene recombination in vitro.