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

中文关键词: 秦川牛  PDHB基因  腺病毒  肌内前体脂肪细胞

英文关键词: Qinchuan cattle  PDHB gene  Adenovirus  Intramuscular preadipocytes

中文摘要:

      [目的]旨在通过构建秦川牛丙酮酸脱氢酶β亚基(Pyruvate dehydrogenase β subunit,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,本实验克隆获得的牛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1.66×10⁹ PFU.mL^-1。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]成功克隆了秦川牛PDHB基因并构建了重组腺病毒质粒pAd-PDHB,并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。

英文摘要:

      【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 293A 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_^-1.【Conclusion】In this study, we constructed the recombinant recombinant adenovirus plasmid pAd-PDHB and acquired the high titer recombinant adenovirus Ad-PDHB.