野生型和突变型人白介素13哺乳细胞表达质粒的构建

Construction of the wild-type and mutant interleukin13 expression plasmids in mammalian cells

目的:构建野生型及突变型人IL-13(hIL-13)哺乳细胞表达质粒。方法:采用融合PCR扩增人生长激素(hGH)-hIL13融合蛋白编码序列。PCR产物和pcDNA3.1表达载体经HindⅢ和EcoRⅠ双酶切处理后,将PCR产物定向插入pcDNA3.1真核表达载体中,构建质粒pcDNA3.1(+)/hGH-hIL13-a和pcDNA3.1(+)/hGH-hIL13-g。将其转化到DH5α感受态细胞内进行扩增,阳性克隆鉴定,质粒抽提,进行测序验证。结果:重组质粒pcDNA3.1(+)/hGH-hIL13-a和pcDNA3.1(+)/hGH-hIL13-g经测序验证,hGH-hIL13融合蛋白编码序列与实验设计一致,且已与pcDNA3.1(+)真核表达载体正确重组。结论:成功构建了哺乳细胞表达质粒pcDNA3.1(+)/hGH-hIL13-a和pcDNA3.1(+)/hGH-hIL13-g,为后续野生型及突变型hIL-13哺乳细胞的重组表达奠定了基础。

Objective：To construct the expression plasmids of wild-type and mutant interleukin-13 expression plasmids in mammalian cells.Methods：The hGH-hIL13 fusion protein gene was amplified by fusion PCR.Both the PCR product and the vector pcDNA3.1（＋） were digested by the Hind Ⅲ and EcoRⅠ.Then the PCR product was cloned in the eukaryotic expression vector of pcDNA3.1（＋）.The plasmids were constructed and transformed into E.coli competent cells DH5α.The positive clones were selected,and tested by sequencing.Results：The hGH-hIL13 fusion protein gene was in accordance with the design and correctly inserted into the vector proved by sequencing.Conclusion：The successful construction of the pcDNA3.1（＋）/hGH-hIL13-a and pcDNA3.1（＋）/hGH-hIL13-g plasmids lays the foundation for the wild-type and mutant interleukin-13 expression in mammalian cells.