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为进一步研究NPC1L1在代谢性疾病尤其是脂代谢紊乱中具体的分子机制,本研究以pEGFP-C1质粒为模板,通过PCR扩增出Neo~r/Kan~r基因片段,并对pLiv-11质粒和PCR扩增产物进行酶切,用T4DNA连接酶连接Neo~r/Kan~r、pLiv-11和h NPC1L1基因片段,最终构建了含有表达质粒的重组菌株JM109(pLiv-11-Neo~r/Kan~r-hNPC1L1)。酶切鉴定和序列分析结果表明,构建的p Liv-11-Neo~r/Kan~r-hNPC1L1重组质粒含有hNPC1L1(human Niemann-Pick C1 like 1)基因且基因序列和开放阅读框架均正确。本研究成功构建了肝特异性表达hNPC1L1的真核表达载体,为培育h NPC1L1表达转基因巴马小型猪奠定了一定基础,有利于阐释NPC1L1的调控机制以及揭示肝脂肪代谢相关疾病的发病机制。
In order to further study the specific molecular mechanism of NPC1L1 in metabolic diseases, especially lipid metabolism disorder, we used the pEGFP-C1 plasmid as a template to amplify the Neo ~ r / Kan ~ r gene fragment by PCR and cloned the pLiv-11 plasmid And PCR amplification products were digested with T4 DNA ligase Neo ~ r / Kan ~ r, pLiv-11 and h NPC1L1 gene fragment, the recombinant plasmid containing the recombinant plasmid JM109 (pLiv-11-Neo ~ r / Kan ~ r-hNPC1L1). Restriction endonuclease analysis and sequence analysis showed that the constructed recombinant plasmid pLiv-11-Neo ~ r / Kan ~ r-hNPC1L1 contained the hNPC1L1 gene (human Niemann-Pick C1 like 1) gene and the open reading frame was correct. This study successfully constructed a liver-specific expression of hNPC1L1 eukaryotic expression vector for the development of h NPC1L1 expression of transgenic Bama mini-pigs laid the foundation for the interpretation of NPC1L1 regulatory mechanisms and reveal the pathogenesis of hepatic fat metabolism-related diseases.