重组野猪α-干扰素基因的高效表达和下游工艺的研发  

Overexpression and Down-stream Technology of Recombinant Sus scrofa Interferon alpha in Escherichia coli

作　　者：李文辉[1] 刘娣 王君伟[3] 徐冬梅 杨秀琴[1] 

机构地区：[1]东北农业大学动物科技学院,哈尔滨150030 [2]黑龙江省农业科学院,哈尔滨150086 [3]东北农业大学,哈尔滨150030 [4]哈药集团生物工程有限公司,哈尔滨150076

出　　处：《中国生物工程杂志》2008年第6期65-70,共6页China Biotechnology

摘　　要：利用PCR方法,从东北野猪和长白猪的肌肉基因组DNA中分别扩增出一条501bp的基因片段,与GenBank上登载的猪α-干扰素基因序列相比,核苷酸同源性达到98.4%以上。以其中野猪α-干扰素基因片段为参考模板,经过密码子优化、5′和3′区域的mRNA二级结构优化,全基因合成得到编码野猪α-干扰素基因成熟肽的基因。克隆该基因到原核表达载体pWL之中.随后转化至宿主菌DH-5α。经过SDS-PAGE电泳检测,发酵后的重组野猪IFN-α-干扰素基因的蛋白表达量大于25%。复性纯化后的重组蛋白,经过VSV-WISH系统检测,其生物活性为4.45×106IU/mg。A 501bp sequence of Sus scrofa IFN-α gene and Susscrota domestica of changbai IFN-α gene was amplified by normal polymerase chain reaction （PCR） from their genome whose homology are more than 98 4% ,compared with nucleotide sequence published on NCBI . Referring to Sus scrofa IFN-α gene sequence, a new sequence was synthesized after codon bias and mRNA structure optimizaed . Then , this sequence was inserted in pWL vector and the recombinant plasmids were transformed into the E. coli DH-5α After fermentation,the expression level of recombinant protein are 25% by SDS-PAGE analysis. The biologic activity was 4.45 × 10^6 IU/mg by VSV-WISH system analysis after protein refolding and purification.

关 键 词：野猪α-干扰素基因优化发酵工艺 复性 纯化 活性检定 

分 类 号：Q786[生物学—分子生物学]