表达α-烯醇化酶重组腺病毒的构建及其在籽鹅卵泡颗粒细胞中的过表达

Construction of recombinant adenovirus expressing α-enolase and overexpression in granular cell of Zi goose

为构建表达籽鹅α-烯醇化酶(ENO1)基因的重组腺病毒,本研究利用RT-PCR技术,从籽鹅卵巢组织中扩增ENO1基因,克隆至pShuttle-CMV穿梭载体中,并利用I-CeuI与I-SceI将ENO1基因的表达框切下,连接到腺病毒骨架的pAdxsi载体中。采用PacⅠ酶切,将其线性化,并转染至293(R)细胞进行病毒的包装,制备重组腺病毒。实验结果显示,制备的表达ENO1重组腺病毒的滴度达到1.1×1011pfu/mL。将重组腺病毒感染籽鹅卵泡颗粒细胞后,荧光定量RT-PCR与western blot检测显示细胞中ENO1基因和蛋白表达水平显著高于正常细胞(p<0.05),这些结果为研究ENO1生物学活性及功能奠定了基础。

α-Enolase （ENO1） is a metalloenzyme and also involves in regulation of cell differentiation. To construct recombinant adenovirus for expressing ENO1 of Zi goose and overexpress in follicular granular cell, The ENO1 gene was amplified from total RNA of Zi goose ovary issues by RT-PCR and cloned into shuttle plasmid pShuttle-CMV to constructed pShuttle-CMV-ENO1. In addition, the CMV-ENO1 cassette, from pShuttle-CMV-ENO1 digested with I-CeuI and I-SceI, was inserted into adenovirus vector of pAdxsi which was linearized by digestion with PacI and transfected into 293 （R） cells for packaging to generate the recombinant adenovirus of Ad-CMV-ENO1. Furthermore, the expression level of ENO1 was higher than normal in Zi goose granular cell identified by real time qRT-PCR and western blot when infected with the recombinant Ad-CMV-ENO1. The study laid a foundation of further study on biological activity and function of ENO1.