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目的构建小鼠Setd8第1-214aa的截短片段p CMV-HA-Setd8NO.214真核表达载体,并在293T细胞中验证其表达。方法利用PCR方法扩增出Setd8的截短片段Setd8NO.214基因片段,通过酶切将其定向插入到p CMV-HA载体中,构建真核表达质粒p CMV-HA-Setd8NO.214,通过限制性内切酶酶切和DNA测序鉴定正确后,应用脂质体转染法转染入293T细胞中,应用Western blot鉴定蛋白的表达。结果酶切电泳及测序结果证明,构建的真核表达质粒p CMAHA-Setd8NO.214序列正确无误,Western blot法证明了Setd8NO.214蛋白能够高效表达。结论成功构建了p CMV-HASetd8NO.214真核表达载体,并证明了其在真核细胞293T中的表达,为进一步研究Setd8在真核细胞中的功能提供了前提基础。
Objective To construct the eukaryotic expression vector pCMV-HA-Setd8NO.214 of truncated fragment 1-214aa of mouse Setd8 and verify its expression in 293T cells. Methods The Setd8 NO.214 gene fragment of Setd8 was amplified by PCR and inserted into pCMV-HA vector by restriction enzyme digestion. The eukaryotic expression plasmid pCMV-HA-Setd8NO.214 was constructed, After restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into 293T cells by lipofection method. Western blot was used to identify the protein expression. Results The results of enzyme digestion electrophoresis and sequencing proved that the constructed eukaryotic expression plasmid pCMAHA-Setd8NO.214 was correct, and Western blot confirmed that Setd8NO.214 protein was highly expressed. Conclusion The eukaryotic expression vector pCMV-HASetd8NO.214 has been successfully constructed and its expression in eukaryotic 293T cells has been demonstrated. This study provides a precondition for the further study of the function of Setd8 in eukaryotic cells.