人小肠三叶因子在毕赤酵母中克隆表达及对肠粘膜修复作用的研究

Expression of Human Intestinal Trefoil Factor in Pichiav pastoris and Its Biological Activity on Intestinal Epithelium in vitro

摘要:

为了研究人肠三叶因子（hITF）对肠粘膜的保护作用，利用RT-PCR从肠粘膜中扩增出hITF基因片段，与诱导分泌型毕赤酵母载体pPIC9连接构建了重组质粒pPIC9hITF，重组质粒转化至宿主菌GS115，经过PCR鉴定和转化子发酵筛选，得到一个重组毕赤酵母高产菌株GS115/pPIC9hITF。在5L发酵罐中用基本盐培养基培养重组菌株，添加甲醇诱导表达hITF，离心收集的上清液通过离心交换层析纯化得到hITF。质谱鉴定结果表明纯化的hITF与天然提取产品在N端序列上完全相同。细胞实验和动物实验结果表明重组hITF能够促进细胞迁移，并可以保护肠粘膜免受有害因子的侵袭，保持了较好的生物学活性。

Abstract:

In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection，positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product，electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore，the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5_L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study，we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction，the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.