牛白细胞介素2基因原核表达及多克隆抗体制备  

Prokaryotic Expression of Bovine Interleukin-2 Gene and its Polyclonal Antibody Preparation

作　　者：盖仁华[1,2] 王衡[3] 郎跃深 李广兴[1] 张瑞莉[1] 马德星[1] 张国庆[1] 杨贵君[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]浙江大学药物安全评价研究中心,浙江杭州310058 [3]华南农业大学兽医学院,广东广州510642 [4]河北秦皇岛青龙满族自治县职教中心,河北秦皇岛066500

出　　处：《中国畜牧兽医》2011年第12期71-75,共5页China Animal Husbandry & Veterinary Medicine

基　　金：黑龙江省教育厅科学研究项目(10531005);黑龙江省自然科学基金重点项目(ZJN0702-01)

摘　　要：根据GenBank发表的牛白细胞介素2(BoIL-2)基因序列,设计1对特异性引物,应用RT-PCR技术扩增出重组牛白细胞介素2(rBoIL-2)基因。将克隆片段与pMD18-T载体连接,并经过酶切及PCR鉴定,测序结果显示,克隆的BoIL-2基因与GenBank发表的BoIL-2基因序列同源性为98.7%。将测序正确的克隆片段与pET30a(+)载体连接,构建重组表达质粒pET30a(+-)rBoIL-2,通过双酶切及PCR鉴定阳性的重组质粒进行序列测定后在大肠杆菌的表达,分子质量约为23.49ku;表达产物主要分布在包涵体中。Western blotting证实所得到的重组蛋白为BoIL-2重组蛋白。用镍离子亲和树脂对所得的BoIL-2重组蛋白进行纯化,并免疫新西兰兔成功制备兔抗rBoIL-2多克隆抗体,为下阶段的研究提供了重要的试验材料。Specific primers for bovine IL-2（BoIL-2） cDNA were designed and synthesized according to the previously published sequence in GenBank.The total cell RNA,isolated from ConA-stimulated peripheral blood lymphocyte of bovine,was used as template to generate complementary DNA by reverse transcription.The DNA fragments were amplified by polymerase chain reaction（PCR） and digestion,and cloned into the pMD18-T vector.The fragment was confirmed bovine interleukin-2 by DNA sequencing.The sequence analysis showed that it was similar to the sequence of bovine IL-2 publised in GenBank,the homology was 98.7% in nucleotide acid.Then the prokaryotic expression plasmid pET30a/rBoIL-2 was constructed and transformed into BL21 cell.The recombinant rBoIL-2 was expressed efficiently in forms of inclusion body.SDS-PAGE and Western blotting analysis showed that the recombinant fusion protein had a molecular weight 23.49 ku.The inclusion body was purified by QIAGEN Ni-NTA resin.The protein was purified and used to immunize rabbit,and polyconal antibody was prepared.The preparation of its polyclonal antibody will provide solid basis for the biological activity study and application of BoIL-2.

关 键 词：白细胞介素-2 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]