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.
出处
《河南科技大学学报（自然科学版）》
CAS
北大核心
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)
