猪α1-干扰素的基因改造与高效原核表达  

Gene Modification and High Prokaryotic Expression of Porcine Interferon Alpha-1

作　　者：曹瑞兵[1] 徐学清[1] 周斌[1] 陈德胜[1] 陈溥言[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室,南京210095

出　　处：《生物工程学报》2004年第2期291-294,共4页Chinese Journal of Biotechnology

摘　　要：poIFN α1基因中含有大量的大肠杆菌稀有密码子 ,为了获得高表达 ,使用了大肠杆菌的偏爱密码子 ,人工合成了poIFN α1成熟蛋白编码基因。在保留编码蛋白序列的同时 ,使其 5′端A +T的含量增加到最大限度 ,并将其终止密码子改为TAA。将合成的poIFN α1成熟蛋白编码基因插入原核单纯表达载体pRLC中 ,转化大肠杆菌DH5α。实现了poIFN α1在大肠杆菌中的高效表达 ,表达产物以包涵体形式存在。纯化的包涵体经含DTT的 6mol L盐酸胍的变性液溶解及含GSH GSSG的复性液复性处理 ,复性后的表达产物浓缩后经凝胶层析纯化 ,细胞病变抑制法测定表明 ,重组poIFN α1具有较高的抗病毒活性 ,约为 6 4x10 6 u mg。There are many E.coli rare codons in the gene of porcine interferon al pha-1. In order to obtain high expression of poIFN-α1 in E.coli, the cDNA encod ed poIFN-α1 mature protein was synthesized using biased codons of E.coli witho u t changing the original amino acid sequence and the terminator was changed as TA A. At the same time, Adenine and Thymine were used to the largest extent near th e 5′ terminus of poIFN-α1 mature protein gene. The synthesized gene was inser te d into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-α1. The poIFN-α1 is highly expressed in E.coli DH5α when t he induction was carried out at 42℃. The expressed poIFN-α1 account for 24 5 % of the total cellular proteins and existed as inclusion body. The poIFN-α1 i nclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subse quently the denatured poIFN-α1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured p oIFN-α1 was most efficiently re-natured in refolding buffer containing 1mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natur ed poIFN-α1 was purified by Sephacryl S-200 chromatography. As a result, the p urified poIFN-α1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesi cular stomatitis virus in MDBK cells, which is about 6 4×10 6u/mg. This study pav ed the way for large-scale production of recombinant poIFN-α1 and its usage i n virus disease control of pigs.

关 键 词：猪 α1-干扰素 基因 原核表达 poIFN-α1 大肠杆菌 包涵体 抗病毒活性 

分 类 号：Q786[生物学—分子生物学]