Expression Of Fusion Protein HBsAg/GM-CSF In Pichia Pastoris And It's Immunogenicity

Recombinant hepatitis B(HB) vaccine is the only widely used genetically engineered vaccine.It has many characters,such as easily obtained material;high yield;safe and efficient,so plays a important role in preventing the infection of hepatitis B virus(HBV).However,10% of healthy people do not develop adequate protection after the standard three-dose HB vaccine.Generally,if vaccine cann't provide effective protection,we'll try to improve the effection of the vaccine from the body and the vaccine.In this study,we based on the theory of ameliorating immunogenicity of vaccine,expressed fusion protein HBsAg/GM-CSF in the Yeast Pichia pastoris by genetic engineering and tested its immunogenicity,in order to search for a new method to enhance immunogenicity of HBsAg.The genes of HBsAg and GM-CSF were obtained by PCR.Two fusion genes composed of HBsAg and GM-CSF,GM-s and s-GM, were linked through a linker of 15 amino acids,then the fusion genes were cloned into yeast shuttle vector pPIC9K by genetic engineering methods.Restriction enzyme analysis and sequence analysis showed the recombinant plasmids contained fusion genes were same as design.Recombinant plasmids were electrotransformed into Pichia pastoris GS115 and screened multiple inserts by G418,then induced by methanol and expressed. SDS-PAGE analysis showed two fusion proteins GM-CSF-HBsAg(GS) and HBsAg-GM-CSF(SG) can expressed in the Yeast Pichia pastoris,the highest expression level was 55% of the total protein in supernatant,and molecular weight was 32 KD.Westen blot analysis showed two fusion proteins exhibited specific reaction with both anti-HBsAg and anti-GM-CSF antibody.The supernatant was purified by ion exchang columns afer ultrafiltration, and obtained the fusion proteins in a purity more than 90% one step.Inoculated mice with the purified fusion proteins.Mice were divided into five groups,?control group injected Saline,(2)S group injected HBsAg which was expressed in the Yeast Pichia pastoris and saved in our laboratory,(3)S+GM group added GM-CSF on base of S group,?GS group injected fusion protein GS,(5)SG group injected fusion protein SG.Inoculate at 0,2,4 week,blood from caudal artery every two weeks after the first inoculation,collect serum,and the level of anti-HBsAg in mice serum was tested by ELISA.Anti-HBsAg appeared at fourth week after the first inoculation.At the sixth week the positive rate of S group,S+GM group,GS group,SG group were 50%,100%,66%,100%,and the level of anti-HBsAg antibody induced by GS and SG fusion protein was higher than other groups.This is the first description of generating HBsAg/GM-CSF fusion protein in the Yeast Pichia pastoris and testing its immunogenicity.Our experimental result is that fusing HBsAg and GM-CSF can improve the immunogenicity of HBsAg.It's a new idea and method for enhancing the immunity effect of HBV vaccine.