Expression And Purification Of Avian Influenza Virus H5N1HA1and Mouse IgG Fc Fragment Fusion Protein

Expression of eukaryotic protein often require modification after translation process to get a complete biological activity of protein. Although the prokaryotic expression system have many advantages, but it can’t modify the protein after the translation process, so its application is restricted in many eukaryotic expression. Baculovirus expression vector system is an eukaryotic expression system which was gradually developed, and widely used. It has the function of modification, thus forming the protein highly similar to the natural and soluble. At present, baculovirus expression vector system has been successfully applied to many research fields, and it still continue to improve and perfect.The former research in our lab expressed HA1of the Avian Influenza Virus H5N1and mouse IgG2a Fc fusion protein using baculovirus expression system:HA1-wFc and HA1-mFc. Among them, HA1-wFc make complement Clq key binding sites of for IgG Fc fragement for mutation, HA1-mFc make C1q key binding sites and FcRn binding sites at the same time for mutation, the purpose is to research on FcRn mediated IgG pathways of transport antigen of mucosal immunization. But the protein has always been to unsoluble, it existed in the form of pellet gathered in cell fragments. Thus, it can’t be purified and go on animal immune experiments. This experiment is based on the former, after analysis, the protein expressed unsoluble is related to the lack of signal peptide. So, we add the signal peptide to HA1-Fc, and express it in incect cells once again.Then we express the two soluble fution protein, and purified.Express of the two fution protein is for immune experiment in the future, we will use the two fusion protein as the antigen immune mice. It establishs the basis to study that IgG transcytosis by FcRn mediated transport antigen across the respiratory mucosal barrier. This research has achieved main results as follows:1. The establishment of the fusion protein expression soluble in insect cellsPrimers for HA1-wFc and add two signal peptide:the own signal peptide of HA and honey bee melittin signal peptide, to find out the method of expressing the fusion protein in soluble using Bac-to-Bac. Result, adding two signal peptide can both make the target protein express in soluble in insect cells, and there seems no significant differences in the yield.2. Expression of sHA1-wFc and sHA1-mFc fusion proteinUsing sHA1-wFc and sHA1-mFc fusion protein expression carrier with HA’s own signal peptide, and form the recombined baculovirus after transformation. The fusion protein sHAl-wFc and sHAl-mFc was expressed in HighFive cells. Detecting it by SDS-PAGE and Western Blot, the protein size is about80KD, larger than the predicted size (66.4KD), it showed that the both protein were glycosylated.3. Purification of sHAl-wFc and sHAl-mFcThe two fution protein sHA1-wFc and sHA1-mFc were expressed using Bac-to-Bac in incect cells in large qutities, and then purified by protein A and his affinity chromatography.