新城疫病毒HN基因真核细胞定位表达的重组质粒的构建

目的 构建定位表达于细胞不同部位的新城疫病毒HN(hemagglutinin-neuraminidase)基因.方法 应用反转录聚合酶链反应(RT-PCR)技术,以新城疫病毒D90株RNA为模板,分别扩增出含有HN基因全长和不含终止子的HN基因片段,将克隆的HN基因片段经双酶切后定向插入真核表达质粒pcDNA3.1(+)中,经过定向插入组织凝血酶原激活物(tPA)的前导序列和/或A型流感病毒血凝素(HA)基因跨膜区(TM)片段进行修饰分别构建胞浆型、跨模型和分泌型DNA质粒.结果 所有重组质粒经酶切和测序鉴定插入正确.经过体外转染真核细胞,间接免疫荧光和SDS-PAGE蛋白质电泳检测,证明构建的新城疫病毒HN基因定位表达于真核细胞的胞浆、胞膜和细胞外.结论 成功构建了定位DOI:10.3760/cma.j.issn.1673-4394.2009.04.003作者单位:150040,哈尔滨医科大学附属肿瘤医院内科(隋红,李乐静,白玉贤);150001 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪病研究室(李曦,张永欣,肖晶,符芳)通信作者:李曦,E-mail:lx2005@126.com表达于细胞不同部位的HN蛋白的DNA质粒即胞浆型、跨模型和分泌型.

Construction of the expression recombinant plasmid containing Newcastle disease virus HN gene targeting to different cellular compartments

Objective To construct DNA plasmids expressing three different hemagglutinin-neuraminidase (HN) of Newcastle disease viruse(NDV), namely cytoplasmic, secreted and membrane-anchored. Method The genomic DNA of NDV strain D90 was used as template in a high-fidelity PCR to amplify the HN full length and without a stop codon HN without a stop codon gene via RT-PCR technology. The digested PCR products were inserted into the corresponding sites of PcDNA3.1 (+) modified with the tissue plasminogen activator (tPA) leader sequence and/or DNA fragment containing the transmembrane anchor domains (TM) of influenza virus hemagglutinin (HA) amplified by PCR. All constructs were verified by appropriate restriction enzyme di-gestion and DNA sequencing. Meanwhile, an indirect immunofluorescence assay and SDS-PAGE were per-formed to determine the modified HN gene expressed and targeted into the expected cell compartments of the transfected cells. Results All constructs were proved with correctly inserted fragments and accuratly expressed in the different cellular compartments. Conclusion Three DNA constructs expressing different HN proteins, namely cytoplasmic, secreted and membrane-anchored were constructed successfully.