人骨形态发生蛋白4多拷贝表达盒的构建及其表达

Construction and Expression of Multiple-copy Expression Cassette of Human Bone Morphogenetic Protein 4

为了了解在毕赤酵母中人骨形态发生蛋白4基因剂量对其表达量的影响,利用毕赤酵母表达载体pAO815质粒上的2个同尾酶BglⅡ和BamHⅠ在体外构建了人骨形态发生蛋白4成熟肽序列1,3,6,9个拷贝表达盒,并对它们进行表达量研究.结果表明,在毕赤酵母中表达人骨形态发生蛋白4蛋白时,1个拷贝基因能达到最大表达量,增加人骨形态发生蛋白4基因的拷贝数并不会增加人骨形态发生蛋白4的表达量,基因拷贝数的增加反而导致人骨形态发生蛋白4在毕赤酵母中的表达水平下降.

To understand the effect of gene dosage of human bone morphogenetic protein 4（hBMP4） on expression levels in Pichia pastoris,recombinant plasmids carrying 1,3,6,9 expression cassette copies of hBMP4 mature peptide DNA sequence were constructed with unique Bgl Ⅱ and BamH Ⅰ sites shared 4 bases in common between their recognition sites in pAO815 vector in vitro.Then these recombinant plasmids were transformed into Pichia pastoris to analyze expression levels of hBMP4 in Pichia pastoris.The results showed that the highest expression levels of recombinant hBMP4 were achieved by a 1-copy strain,expression levels of hBMP4 were found to decrease for high copy strains in Pichia pastotis.