秦川牛PDHB基因重组腺病毒载体的构建与鉴定

Construction of the Recombinant Adenovirus of Qinchuan Cattle PDHB Gene

[目的]旨在通过构建秦川牛丙酮酸脱氢酶β亚基(pyruvate dehydrogenaseβsubunit,PDHB)基因的重组腺病毒载体,为研究PDHB基因在牛前体脂肪细胞分化过程中的功能做准备。[方法]根据牛PDHB基因mRNA序列(GenBank Accession No.NM_001035435)设计引物,克隆该基因的编码区(coding sequence,CDS)序列。测序验证后将其重组到穿梭载体pAdTrack-CMV上,经PmeⅠ线性化后,转化到含有pAdEasy-1腺病毒骨架载体的E.coli BJ5183感受态细胞中进行同源重组,以获得重组质粒pAd-PDHB。再将经PacⅠ酶切线性化的pAd-PDHB转染到HEK 293A细胞中,进行病毒包装并扩增高滴度病毒Ad-PDHB,绿色荧光蛋白(GFP)标记法测定病毒滴度。将高浓度的Ad-PDHB病毒感染牛肌内前体脂肪细胞,实时荧光定量PCR(qRT-PCR)检测PDHB的表达量。[结果]经测序验证,实验克隆获得的牛PDHB基因CDS与数据库GenBank收录的序列一致。将PDHB基因CDS与穿梭载体pAdTrack-CMV重组并转染HEK 293A细胞后成功获得了重组腺病毒Ad-PDHB,其滴度为1.66×10^(9) PFU/mL。腺病毒Ad-PDHB侵染牛肌内前体脂肪细胞后,PDHB在mRNA的表达水平比对照组高25.5倍。[结论]成功克隆秦川牛PDHB基因,并构建重组腺病毒质粒pAd-PDHB,获得能够在牛肌内前体脂肪细胞中过表达PDHB基因的高滴度重组腺病毒Ad-PDHB。

[Objective] The aim of this study was to construct the recombinant adenovirus vector with Qinchuan cattle PDHB gene, in order to provide a basis for studying PDHB gene functions in the bovine intramuscular preadipocytes differentiation process. [Method] The PDHB gene was cloned with the primers designed according to the PDHB mRNA sequence(GenBank Accession No.NM_001035435). After sequencing validation, the PDHB gene was cloned into a shuttle vector pAdTrack-CMV. After digested and linearized by PmeⅠ, it was transformed into E. coli BJ5183 competent cells containing backbone vector pAdEasy-1 to obtain recombinant adenovirus plasmid pAd-PDHB by homologous recombination. Further, the recombinant plasmid was digested and linearized by PacⅠ, then transfected into HEK 293 A cells for virus packing, amplifying and titer testing by GFP labelling method. The relative mRNA expression of PDHB gene was detected by qRT-PCR in the bovine intramuscular preadipocytes which were infected with virus Ad-PDHB. [Result] After sequencing validation, the sequence of cattle PDHB CDS cloned in this assay was consistent with the counterpart in GenBank. The recombinant adenovirus Ad-PDHB was successfully constructed. The virus titer of the Ad-PDHB was 1.66×10^(9) PFU/mL. [Conclusion] In this study, we constructed the recombinant recombinant adenovirus plasmid pAd-PDHB and acquired the high titer recombinant adenovirus Ad-PDHB.