摘要:
目的：构建过表达西藏小型猪GHR基因显负性突变体的慢病毒载体，并对其进行功能验证。方法：首先以pRLG3A为模板，PCR扩增CAG启动子，In-Fusion克隆至Cla I/BamH I双酶切的pCD550A-1载体中，替换其EF1α启动子，获得pCAGGP；然后从西藏小型猪肝脏组织cDNA中PCR扩增GHR基因的显负性突变体片段(dnGHR)；最后将dnGHR片段插入pCAGGP多克隆位点，最终得到过表达猪GHR显负性突变体的慢病毒载体pCdnGGP。对所构建的质粒进行测序和酶切鉴定。将所构建的pCdnGGP载体转染293T细胞，并将包装好的慢病毒感染猪的胚胎成纤维细胞(PEFs)，倒置荧光显微镜检测GFP表达，收集细胞并提取总RNA检测dnGHR基因在293T细胞和PEFs中的表达，以对pCdnGGP进行体外功能验证。结果：测序和酶切证实成功构建了pCdnGGP。pCdnGGP转染293T细胞以及病毒感染PEFs之后，倒置荧光显微镜下可见绿色荧光，RT-PCR检测证实dnGHR在293T细胞和PEFs中均能正常表达。结论：成功构建过表达猪GHR基因显负性突变体的慢病毒载体，为相关后续研究打下了良好基础。
Abstract:
Objective: To generate lentiviral vector harboring the dominant-negative mutant of porcine growth hormone receptor, and in vitro confirm the functionality of the resulting plasmid. Methods: Firstly, CAG promoter was amplified from pRLG3A, and subsequently subcloned into pCD550A-1 at the Cla I/BamH I sites by In-Fusion cloning, designated pCAGGP, as verified by restriction enzyme digestion and sequencing. Secondly, the fragment of dominant-negative mutant of growth hormone receptor (dnGHR) was amplified from liver cDNA of Tibet minipig, and then inserted into pCAGGP at the multiple cloning site (MCS) by In-Fusion cloning to generate pCdnGGP, as verified by restriction enzyme digestion and sequencing. To in vitro confirm the functionality of the resulting plasmid (pCdnGGP), pCdnGGP was transiently transfected into 293T cells to produce lentiviurs LV- CdnGGP, and LV-CdnGGP was used to infect pig embryonic fibrolast (PEFs), followed by EGFP assay under inverted fluorescence microscope and detecting dnGHR transgene expression by RT-PCR. Results: Enzyme digestion and DNA sequencing demonstrated that pCdnGGP was successfully constructed. 293T cells transiently transfected with pCdnGGP and PEFs infected by LV-CdnGGP displayed the green fluorescence under inverted fluorescence microscope and the increased expression of dnGHR. Conclusion: The lentiviral vector harboring pig dnGHR gene was successfully constructed, which will lay a solid foundation for further research.