| 摘要: |
| 参照GenBank上已发表的鸡白细胞介素2序列设计引物,运用RT-PCR技术,从经ConA诱导的20～35日龄固始鸡脾细胞总RNA中扩增出目的片段IL-2基因,将其插入到pGEM-T载体上,构建了克隆质粒pGEM-T-chIL-2.测序结果表明,本试验克隆的固始鸡白细胞介素2基因与GenBank上已发表的序列相比,存在2个核苷酸变异.重新设计表达引物,以克隆质粒pGEM-T-chIL-2为模板PCR扩增表达片段,对表达片段进行HindⅢ和BamHⅠ双酶切,连接到作同样双酶切的pET28a表达载体上,鉴定后转化大肠杆菌BL21并进行IPTG诱导表达,然后对表达产物进行SDS-PAGE电泳鉴定,结果表明,表达蛋白主要以包涵体的形式存在,表达蛋白分子质量约为18 ku;目的蛋白表达量占总量的18%.体外活性检测表明,重组蛋白具有促进淋巴细胞增殖的活性. |
| 关键词: 固始鸡 白细胞介素2 原核表达 生物活性 |
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| 基金项目:国家863计划项目(2002AA242021)；河南省科技厅重大攻关项目(0322010600) |
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| Cloning and expression of gushi-chicken interleukin 2 gene and detection of its biological activity |
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| Abstract: |
| Two pairs of primers of RT-PCR and expressing were designed and synthesied according to the published gene sequence of chicken interleukin-2.About 680 bp target DNA sequence were cloned by RT-PCR depending on the template of total RNA isolated from ConA-stimulated spleen cell and inserted into the plasmid pGEM-T.The sequence coded ChIL-2 was subcloned to the pET28a vector.The expressing plasmid, recombined pET28a-ChIL-2,identified by enzyme digecting and DNA sequencing,was transformed into BL21(DE3) and induced with IPTG.SDS-PAGE illuminated that the expressed protein was 18 ku.recombinant protein was extracted roughly from dissolved BL21,and purified by Ni+ affinity column.MTT colorimetric assay indicated that the recombinant protein could induce chicken spleen T lymphocytes in vitro.Prepression was made for the advanced reserch of the usage of recombinant IL-2 protein as adjuvant to accine and antigene to monoclonal antibody. |
| Key words: Gushi chicken interleukin-2 expression biological activity |
