抗A型流感病毒聚合酶碱性蛋白1多克隆抗体的制备和鉴定

Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody

摘要:

流感病毒属于正黏病毒科，为有包膜包裹的单股负链RNA病毒。它的8个基因片段编码至少16种病毒蛋白，其中3个蛋白组成流感病毒的聚合酶复合体。流感病毒聚合酶碱性蛋白1 (PB1) 是该复合体的组分之一，在病毒的转录、复制及重配中发挥重要的作用。为研究其功能，构建His-PB1 (aa 550–755) 融合蛋白原核表达质粒，IPTG诱导融合蛋白表达，通过镍柱亲和将其纯化，然后作为抗原免疫家兔。对抗血清进行间接ELISA检测，表明抗体效价可达1∶100 000。兔源PB1蛋白抗血清经亲和纯化后，用于免疫印迹检测流感病毒WSN毒株PB1蛋白以及外源转染的FLAG-PB1蛋白，结果表明该PB1抗体具有良好的特异性，同时也能特异性识别其他亚型的A型流感病毒的PB1蛋白，为进一步研究流感病毒PB1蛋白的功能奠定了基础。

Abstract:

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648–2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL21. The expression of His tagged PB1 protein was induced by IPTG, and His-PB1 proteins were purified by Ni-NTA resin. For preparation of PB1 protein antiserum, rabbits were immunized with His-PB1 fusion protein 3 times. Then the titer of PB1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB1 protein from WSN virus infected or pCMV FLAG-PB1 transfected cells. Meanwhile, PB1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.