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【目的】使人源抗菌肽LL-37在真核表达载体中获得高效表达.【方法】采用RT-PCR技术,从人的血样中扩增出LL-37基因片段,并将其连接至pcDNA3.1-His-V5(+)载体.通过脂质体介导法将重组质粒转入奶牛乳腺上皮细胞,G418筛选获得稳定阳性细胞株,并且利用RT-PCR和Western-blot检测目的基因在转录及翻译水平的表达情况.【结果】RT-PCR结果显示,经转染的乳腺上皮细胞能够扩增出523bp的LL-37基因.qRT-PCR结果发现,LL-37mRNA在转染后的不同时期均有表达,经稳定转染的表达量最高.通过Western-blot检测,获得了约17kU的目的蛋白.【结论】结果表明,LL-37基因成功整合至奶牛乳腺上皮细胞并能够正确表达,这为构建分泌LL-37蛋白的奶牛乳腺生物反应器提供了依据.
【Objective】 The aim of this study is to obtain high expression of human antimicrobial peptide LL-37 in eukaryotic expression vector. 【Method】 LL-37 gene fragment was amplified from human blood samples by RT-PCR and ligated into pcDNA3 The recombinant plasmids were transformed into mammary gland epithelial cells by liposome-mediated method, and stable positive cell lines were screened by G418 screening, and the target gene was detected by RT-PCR and Western-blot RT-PCR results showed that the LL-37 gene of 523bp was amplified by transfected mammary epithelial cells.Results of LLRT-PCR showed that LL-37 mRNA was expressed at different stages after transfection And the highest expression level was obtained by stable transfection.The protein of about 17 kU was obtained by Western-blot assay. 【Conclusion】 The results showed that the LL-37 gene was successfully integrated into mammary gland epithelial cells and correctly expressed It provided a basis for constructing cow mammary gland bioreactor that secreted LL-37 protein.