摘要
通过PCR从三黄肉鸡的肝脏基因组中扩增了鸡α干扰素(ChIFN-α)全长基因。序列分析表明ChIFN-α基因全长582bp,亚克隆其成熟蛋白编码基因(489bp),利用基因重组技术构建了E.coli/pET-28a(+)-IFNα,使IFN-α置于pET-28a的T7启动子下游并同6×His(多聚组氨酸标签)-Tag融合。经酶切鉴定,DNA测序证实重组质粒构建正确;将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE,Western-blot分析证实表达出22kD左右的融合蛋白,表达的蛋白以不溶性的包涵体形式存在并且具有良好的免疫学活性。提纯的包涵体纯度可达70%以上,用镍亲和层析方法纯化蛋白则可达到95%。经透析复性后的蛋白在鸡胚成纤维细胞上能够抑制H9N2禽流感病毒的复制。鸡胚试验中重组干扰素抗病毒效果好在H9N2禽流感病毒攻毒组中,能保护鸡胚并使其孵出率达到100%的重组干扰素最小蛋白含量为2μg;重组干扰素对新城疫病毒复制也有一定的抑制能力,延迟该病毒复制时间为12h至48h;雏鸡试验表明重组干扰素也能较好地抵抗H9N2禽流感病毒对雏鸡的感染。两组试验均表明,亲和层析纯化蛋白是包涵体蛋白活性的20倍左右。
The full length of chicken interferon alpha （ChIFN-α）gene was amplified by the polymerase chain reaction（PCR） from total liver genome of Sanhuang meat-chicken and sequenced. The amplified gene was about 582bp. The coding region for mature protein （489bp） was subcloned into pET-28a（ ＋ ）. The recombinant plasmid pET-28a（ ＋ ）-IFNα was identified by enzyme digestion and DNA sequencing. Data of SDS-PAGE and Western-blot indicated that a 22kD fusion protein was expressed in the form of inclusion bodies with good immunity. The purity of inclusion bodies was above 70% and that of protein purified by nickel affinity chromatography was 95 %. The recombinant protein could inhibit H9N2 avian influenza virus （H9N2 AIV） replication on chick embryo fibroblast. 2μg of recombinant IFN-α could completely protect Chick embryo from HgN2 AIV infection. The recombinant IFN-α can also delay Newcastle disease virus （NDV） replication on chick embryo for 12 - 48h. Chicken administered recombinant IFN-α can resist the H9N2 AIV infection. The bioactivities of recombinant IFN-α purified by affinity chromatograph were 20 times higher than that of inclusion bodies.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第5期737-743,共7页
Chinese Journal of Biotechnology
