【作者】 张海峰；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 根据GenBank 上发表的犬(canine familiars)干扰素-γ(Interferon-γ,IFN-γ)和白细胞介素-2 (Interteukin-2,IL-2 )基因序列,设计了两对引物。采用RT-PCR 技术以ConA刺激的犬脾淋巴细胞为材料,从总RNA 中扩增出犬IFN-γ和IL-2 基因。分离纯化扩增片段,克隆入pMD18-T 载体,经酶切鉴定、PCR 鉴定及DNA 测序分析,表明扩增片段为犬IFN-γ和IL-2 基因。测序结果显示克隆的IFN-γ基因全长426 个核苷酸,编码141 个氨基酸,与GenBank 中CaIFN-γ比较,核苷酸序列同源性为99.8%,推导的氨基酸序列同源性为99.3%;克隆的IL-2 基因全长468 个核苷酸,编码155 个氨基酸,将该序列与GenBank中D30710 CaIL-2 比较,核苷酸序列同源性为99.36%,推导的氨基酸序列同源性为98.7%。应用DNA 重组技术,将犬IFN-γ和IL-2 基因分别插入到原核表达载体PJLA605 中,构建原核表达质粒PJLA605-CaIFN-γ和PJLA605-CaIL-2,转化大肠杆菌DH5α,并通过改变诱导时机和诱导表达时间来优化表达条件。结果表明,工程菌PJLA605-CaIFN-γ和PJLA605-CaIL-2 在30℃培养至对数生长后期(OD600 为1.5)转温诱导4h,菌体湿重收得量分别达18.0g/L 和17.8g/L,目标蛋白表达量分别占菌体总蛋白的27.4%和29.4%,这两种基因工程菌得到了理想表达。SDS-PAGE 电泳显示,NaCl 和TritonX-100 可洗去部分杂蛋白,表达产物以包涵体形式存在。尿素溶解包涵体并通过Sephacry1S-200 分子筛初步纯化,包涵体的纯度均达到90%以上。复性后的IL-2 蛋白用MTT 法检测生物活性,结果显示稀释复性的IL-2 蛋白淋巴细胞增殖系数可达2.01,表明此种蛋白具有IL-2 的生物活性。更多还原

【Abstract】 Two pairs of oligonucletide primers were designed to amplify the genes coding for the IL-2 and IFN-γproteins of canine,respectively. The primers were chosen by the analysis of canine relative sequences available in GenBank. The IL-2 and IFN-γgenes of canine were amplified from RNA extracted directly from canine spleen cells which were stimulated with Concavadin A by RT-PCR and cloned into pMD 18-T vector,respectively. The IFN-γand IL-2 genes of canine were sequenced and sequence analysis indicated that the IFN-γcDNA encompassesd an open reading frame of 426 nucletides,encoding a 141-amino acid protein while the IL-2 gene ORF was 468 bases in length,encoding 155 amino acids.Sequence comparision with the selected sequences from GenBank revealed that the IFN-γgene had a high sequence homology of 99.8% and the encoded protein shared a 99.3% amino acid while the nucletide sequence of IL-2 gene is highly similar to the selected sequence,having a nucletide homology of 99.36% and the encoded protein shared a 98.7% amino acid identity with D30710. The PCR products of pMD 18-T-IFN-γand pMD 18-T-IL-2 were inserted into PJLA605 vector,respectively.The recombinant expression plasmids thus constructed,designated PJLA605-CaIFN-γand PJLA605-CaIL-2,were used to express the two proteins. The induction opportunity and inducing time were optimized for higher expression level. The results showed that when induced for 4 hours in logarithmic anaphase,18.0g and 17.8 of wet bacteria per liter could be obtained and the derivative of CaIFN-γand CaIL-2 were about 27.4% and 29.4% of total protein in the host,respectively. The two engineering bacterial strains were highly expressed. The analysis of protein expression revealed that the expression protein only exist in the inclusion bodies. The amounts of bacteria proteins in the inclusion body can be decreased by washing with NaCl and TritonX-100. The dissolved inclusion body was purified with Sephacry1S-200 and the purify was above 90%.The bioactivities of refolding proteins of IL-2 was detected with MTT method.The results showed that biological activity in stimulating the proliferation lymphblats of IL-2 was 2.01,which indicated that the bioactivities of the protein was obvious.更多还原