提取Vero细胞膜蛋白，利用表达纯化的禽肺病毒（aMPV）F蛋白与人IgG Fc融合蛋白和能与Fc结合的蛋白A进行免疫沉淀，结合质谱分析确定aMPV的受体蛋白。选用两种方法进行受体的功能验证：一种是利用HIV或VSV-G假性反转录系统和aMPV F蛋白表达载体共转染多种细胞系，获得假型病毒，筛选出aMPV非敏感细胞系，然后将推测的受体基因导入非敏感细胞系使其变为敏感细胞系，进行病毒感染试验验证。另一种是利用慢病毒载体携带shRNA沉默受体基因构建缺陷型细胞系，进行病毒感染试验验证。将多种细胞系（包括缺乏受体的）分成三组：病毒高水平复制组，病毒低水平复制组和无病毒复制组，建立实时荧光定量RT-PCR检测这三组细胞系以及实验鸡不同组织部位中受体基因的表达差异，研究受体与aMPV复制的相关性和组织嗜性。通过构建F蛋白突变体，进一步确定F蛋白与受体结合的功能域。

The receptor of aMPV was identified by expressed F protein of AMPV fused in frame with the Fc domain of human immunoglobulin G coupled with Mass-Spec.Two methods were selected to evaluate the function of the identified receptor in support of aMPV entry.The pseudotyped particles should be produced by co-transfection of cell lines with the AMPV F protein expression plasmid and with the pseudotyped HIV or VSV-G plasmid for Screening for non-susceptible cells. Then use lentivirial vector to stably express the identified receptor in this receptor-lacking cell line. After getting this cell line to express your identified protein, do infection with F protein-pseudotyped virus.Another method was to use shRNA to reduce or abolish the expression of this candidate receptor in Vero cells,you see no virus replication or less virus replication.Dozens of cell lines including cell lines lacking the receptor were classified them into different groups: high level virus replication, low-level virus replication, and no virus replication. Real-time PCR was used to check the distribution and level of receptor gene in these cell lines and different tissues of chickens for studying the correlation between the presence of this receptor and virus replication and tissue tropism.Furtherly determine the domain of F protein responsible for binding to the receptor by involving the mutagenesis to generate mutants and Co-Immunoprecipitation experiment.

将扩增的APV F基因亚克隆至真核表达载体pPRE载体，利用重叠PCR技术将HA标签插入到APV F蛋白的C端，构建质粒F-HA，便于蛋白的检测和免疫沉淀。为找到与APV F蛋白作用的细胞蛋白，我们分别将10μg F-HA，PRE-F2-Fc，PRE-IFc，M78-F质粒DNA转染293细胞，PBS洗涤后收集转染细胞，每加入非变性缓冲液裂解细胞，冻融离心，加入抗HA抗体作用后再加入琼脂糖凝胶G，收集胶粒，洗涤离心，进行SDS-PAGE电泳，凝胶进行银染，免疫沉淀结果显示F-HA能拉下一个分子量约72kDa的蛋白。将分子量约72kDa的目的蛋白条带切下来，用胰酶消化，抽提液抽提，进行质谱分析，通过GenBank数据库中搜索确定该蛋白为人的HSPA5蛋白。将APV F基因和人的HSPA5基因分别克隆至载体LC和LN中，构建F基因和HSPA5基因的表达质粒，利用荧光素互补试验进行这两种蛋白互作的验证。证实两种蛋白互相作用后，再进行APV F蛋白和鸡HSPA5蛋白的相互作用。