表达H5N1亚型禽流感HA、NA、 M1基因的重组杆状病毒的鉴定

Identification of Recombinant Baculoviruses Expressing HA，NA，M1 Genes of H5N1 Avian Influenza Virus

关键词:  重组杆状病毒  禽流感病毒样颗粒  电镜  Western Blot  ELISA

Key words:  recombinant baculovirus  avian influenza virus-like particles  electron microscope  Western Blot  ELISA

摘要:

为证实插入了禽流感HA、NA和M1基因的重组杆状病毒(VLP01株)能表达HA、NA和M1蛋白并自行组装成具有禽流感形态的病毒样颗粒，对VLP01株的细胞培养上清进行离心纯化或透析处理，获得的表达产物用电镜、Western Blot、ELISA进行鉴定。电镜观察表达产物，可见禽流感病毒样颗粒；以Western Blot方法用HA、NA和M1抗体分别检测表达产物，可见特异性条带；以NA多抗为捕获抗体、HA单抗为检测抗体建立双抗体夹心ELISA方法检测表达产物，结果为阳性，说明表达产物为同时含有HA蛋白和NA蛋白的病毒样颗粒。结果显示，VLP01株能够表达HA、NA和M1蛋白，并自行组装成禽流感病毒样颗粒分泌到细胞培养上清中。

Abstract:

The aim of the work was to confirm the recombinant baculovirus (VLP01 strain) inserted three genes of avian influenza (HA, NA and M1) can express those proteins and self-assemble into virus-like particles with avian influenza morphology. The products of VLP01 were purified by centrifugation or dialysis, the expression product was identified by electron microscopy, Western Blot and ELISA. The avian influenza virus-like particles were observed with electron microscope. The antibodies against the protein HA、NA and M1 respectively，reacted with the products of VLP01 was stained at specific location，the same as the HA、NA and M1 protein.A double antibody sandwich ELISA method was established using NA polyclonal antibody as the capture antibody and HA monoclonal antibody as the detection antibody to detect the products of VLP01, the result was positive, indicating that the expressed products were virus-like particles containing both HA and NA proteins. It is concluded that VLP01 could express protein HA, protein NA and protein M1, and self-assemble into avian influenza virus-like particles to be secreted into the cell culture supernatant.