白细胞介素-10基因多拷贝表达盒的构建及在毕赤酵母中的表达

目的构建白细胞介素-10(IL-10)基因多拷贝表达盒,提高IL-10在毕赤酵母中的表达水平。方法体外构建重组表达载体αIL-10/pAO815,BamHⅠ和BglⅡ双酶切获得目的基因表达盒(AOX-αIL-10),再连接到BglⅡ酶切位点处,依次构建多拷贝重组载体n(AOX-αIL-10)/pAO815。从质粒pPIC9K上用NdeⅠ和SalⅠ双酶切获得Kan抗性基因,重组到多拷贝表达载体n(AOX-αIL-10)/pAO815上。重组载体n(AOX-αIL-10)/pAO815电转化毕赤酵母,PCR筛选含有IL-10基因的酵母转化子,甲醇诱导表达,对表达量高的转化子再次经重组载体n(AOX-αIL-10)/pAO815-Kan电转化,高浓度G418抗性筛选二次酵母转化子,使用甲醇诱导表达。ELISA测定IL-10含量,MC/9细胞测定IL-10活性。结果所构建的8拷贝表达盒的重组载体8(AOX-αIL-10)/pAO815和4拷贝表达盒4(AOX-αIL-10)/pAO815-Kan,转化子分泌表达IL-10水平最高,为(8.25±1.65)mg/L,比活性为1.465×105U/mg。结论已成功构建了高拷贝表达盒,并提高了IL-10在毕赤酵母中的表达水平。

Construction of Multiple Copy Expression Cassette for Expression of Human Interleukin-10 in Pichia pastoris

Objective To construct the multiple copy expression cassette for human interleukin-10(IL-10) gene and improve the expression level of the gene in Pichia pastoris.Methods Construct recombinant plasmid αIL-10/pAO815 and digest with BamHⅠ and BglⅡ to obtain expression cassette AOX-αIL-10 for target gene,then link to BglⅡ site of αIL-10/pAO815 to construct multiple copy expression vector n(AOX-αIL10)/pAO815.Digest plasmid pPIC9K with NdeⅠ and SalⅠ to obtain Kan-resistant gene and insert into n(AOX-αIL-10)/pAO815.Transform expression vector n(AOXαIL-10)/pAO815 to Pichia pastoris by electrotransformation,and screen the transformants carrying IL-10 gene by PCR for expression under induction of methanol.The transformants with high expression level were further transformed to Pichia pastoris by electrotransformation using expression vector n(AOX-αIL10)/pAO815-Kan,and new transformants were screened with G418 at a higher concentration for expression under induction of methanol.Determine the content of expressed IL-10 by ELISA,and the activity with MC/9 cells.Results Eight copy expression cassette 8(AOX-αIL-10)/pAO815 and four copy expression cassette 4(AOX-αIL-10)/pAO815-Kan were constructed.The highest expression level of IL-10 was(8.25±1.65)mg/L,and the specific activity was 1.465×10~5 U/mg.Conclusion Multiple copy expression cassettes for IL-10 were successfully constructed,and the expression level of IL-10 in Pichia pastoris was significantly improved.