固始鸡白细胞介素2基因的克隆、表达及其表达蛋白活性的检测

Cloning and expression of gushi-chicken interleukin 2 gene and detection of its biological activity

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一2为模板PCR扩增表达片段,对表达片段进行 Hind Ⅲ和BamH Ⅰ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。

Two pairs of primers of RT-PCR and expressing were designed and synthesied according to 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.