DNA shuffling技术提高干扰素比活性的研究  

The research of using DNA shuffling technology to improve the specific activity of interferon

作　　者：尼钢钢[1] 范清林 胡伟伟[1] 宋礼华 

机构地区：[1]安徽大学生命科学学院,合肥230039 [2]安徽安科生物股份有限公司,合肥230088

出　　处：《生物学杂志》2013年第5期14-18,共5页Journal of Biology

基　　金：安徽省科技攻关项目(06013063A)

摘　　要：DNA shuffling技术可以定向进化干扰素,获得比rhIFN-α2b标准品更高比活的干扰素。rhIFN-α2b基因与Infergen基因用DNaseI酶切成30～50 bp小片段,回收之后进行无引物PCR重聚和有引物PCR扩增基因。将重组基因连接到载体pUC19中,转化至DH5α并挑选阳性克隆测序。将测序正确的突变基因连接到载体pET30a中并转化至BL21(DE3)中,然后诱导蛋白表达、SDS-PAGE凝胶电泳和Western blot分析。经过表达细胞诱导、细胞裂解、包涵体变性、复性和单克隆抗体亲和层析之后,获得高纯度的重组干扰素。经测定纯化后的重组干扰素比活达到5.8×108IU/mg,比rhIFN-α2b标准品高出5倍左右。实验结果证明此实验方法对提高干扰素的比活是有效的,可以获得比标准品更高比活的干扰素。The DNA shuffling method can direct evolution to the interferon and get the interferon with higher specific activity than the standard of rhIFN-et2b. The genes of rhIFN-a2b and Infergeu were digested with DNaseI into about 30 - 50 bp. The fragments were re- covered and reunited by PCR without primers and amplified with primers. The recombinant genes were ligated to the pUCI9 vector and transformed them into DHSa and selected the positive clones to sequence. The mutant genes were ligated to the pET30a vector and transformed them into BL21 （DE3）. The proteins were induced to expression and analyzed by SDS-PAGE and Western-blot. The re- combinant interferon with higher purity was got after the expression cells induced, cells lysis, inclusion body denatured, natured and monoclonal antibody affinity chromatography. The specific activity of recombinant interferon reached 5.8 108IU/mg and it was 5 times higher than the standard of rhIFN-a2b. The result showed that the experiment was effective to improve the specific activity of interferon and it could obtain the interferon with high specific activity than the standard.

关 键 词：DNA SHUFFLING 干扰素 酶切 比活 

分 类 号：Q78[生物学—分子生物学] Q511