共表达鸡IBDV VP2和IL-2基因的重组毕赤酵母的构建及免疫原性  

Expression of a Fusion Protein of Chicken IL-2 and IBDV VP2 in Pichia Pastoris

作　　者：丁轲[1] 余祖华[1] 张春杰[1] 程相朝[1] 刘一尘[1] 王臣[1] 

机构地区：[1]河南科技大学动物科技学院,河南洛阳471003

出　　处：《河南科技大学学报（自然科学版）》2010年第3期74-78,共5页Journal of Henan University of Science And Technology:Natural Science

基　　金：国家"十一五"科技支撑计划项目(2007Z06-017);河南省自然科学基金项目(04240500101)

摘　　要：通过重叠延伸PCR扩增IL2-VP2串联基因,克隆到pMD18-T载体上,亚克隆正向插入到毕赤酵母表达质粒pPICZαA中,经PCR和双酶切鉴定表明成功构建了酵母重组表达载体pPICZαA-IL2-VP2。将该重组质粒线性化后电击转化入感受态毕赤酵母X-33菌株,构建成分泌型表达IL2-VP2的酵母工程菌株。经甲醇诱导后,通过SDS-PAGE、Western-blot活性检测分析表明IL2-VP2基因长度为1.85kb,包含缺失了TAA终止密码子的IL-2基因和缺失了ATG起始密码子的VP2基因,两个基因之间以高度柔韧性的(Gly-Gly-Gly-Ser)编码核苷酸相连接,并在该体系中得到分泌表达,分子量约为50kD,且表达产物对传染性法囊病毒具有较好的反应原性。The IL2-VP2 fusion gene was amplified by overlap extension PCR(SOE-PCR) and was cloned into pMD18-T.The IL2-VP2 gene was digested and inserted into Pichia Pastoris expression vector pPICZaA.The expression plasmid pPICZαA-IL2-VP2 was obtained.The recombinant plasmid was lineared and transformed into competant Pichia Pastoris X-33 strain by electroporation,and the recombinant transformants were selected by Zeocin and identified by PCR.The results showed that a 1.85 kb long DNA sequence of IL2-VP2 gene was cloned.The IL2-VP2 gene included the gene of chicken IL-2 deleted stop codon TAA and IBDV VP2 gene deleted start codon ATG.The IL2 gene and VP2 gene were connected with high flexible short peptide Gly-Gly-Gly-Ser.The expressed fusion products were identified by SDS-PAGE and Western blot,and the relative molecular mass of the fusion protein was about 50 kD.The products could be secreted into the medium,and had specific IBDV binding activity.

关 键 词：重叠延伸PCR 鸡IL-2 IBDV VP2 融合表达 毕赤酵母 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]