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为了构建新疆多浪羊真核表达载体pc DNA3.1-IL-1β,试验采用重组质粒p MD-18TIL-1βPCR和质粒pc DNA3.1双酶切的方法,获得目的基因白细胞介素-1β(IL-1β)和载体片段pc DNA3.1(+),通过连接、转化宿主菌大肠杆菌DH5α感受态细胞,挑取阳性克隆,再通过菌液PCR和双酶切来验证阳性克隆,并测序。结果表明:试验已成功构建真核表达载体pc DNA3.1-IL-1β,完成了pc DNA3.1-IL-1β的菌液PCR和酶切鉴定。通过对IL-1β功能的了解,以及熟悉质粒提取和质粒连接的方法,完成重组质粒pc DNA3.1-IL-1β的构建。
In order to construct eukaryotic expression vector pcDNA3.1-IL-1β in Xinjiang Duolang sheep, the recombinant plasmid p MD-18TIL-1βPCR and plasmid pcDNA3.1 double digestion were used to obtain the target gene interleukin-1β IL-1β) and pcDNA3.1 (+). The positive clones were transformed by competent E. coli DH5α, and the positive clones were confirmed by bacterial liquid PCR and double digestion. The positive clones were verified by sequencing. The results showed that the eukaryotic expression vector pcDNA3.1-IL-1β was successfully constructed and the PCR and restriction enzyme digestion of pc DNA3.1-IL-1β were completed. The construction of recombinant plasmid pcDNA3.1-IL-1βwas accomplished through the understanding of IL-1β function and familiar with plasmid extraction and plasmid ligation.