Expression, Purification And Characterization Of Hepatitis C Virus Recombinant E1E2 Glycoprotein Expressed By Pichia Pastoris Yeast

Hepatitis C virus (HCV) is the pathogen of hepatitis C. It infects over 3% of the world population. Development of an effective vaccine may be the key in the control of HCV infection.HCV is hypervariable, so it used to be suspectable to get an efficient vaccine which is able to neutralize different HCV subgenotypes. However, recent studies have shown that HCV envelope proteins contain conserved receptor binding domain, which can induce broadly neutralizing antibodies against HCV. One report showed that E1E2 glycoproteins expressed by mammalian cells can protect chimpanzees from heterogeneous HCV infection as a HCV vaccine. But the yield of HCV envelope glycoprotein expressed by mammalian cells is too low to be applied to mass production. More HCV envelope protein can be produced by E.coli expression system, but the protein hasn't glycan. Hence, this HCV envelope protein is very different from native HCV envelope glycoprotein in structures, functions and antigenicity. Herein, we used Pichia pastoris yeast, an efficient and cheap eukaryotic expression system, to express HCV E1E2 recombinant glycoproteins (rE1E2s).We constructed eukaryotic expression plasmid pPIC9K-E1E2, which encodes HCV E1 (aa187-aa346) and E2 (aa381-aa699). Then It was electroporated into Pichia pastoris strain SMD1168. A single colony of multiple inserted His+ Mut+ SMD1168 recombinants was selected and confirmed. It can express recombinant HCV E1E2 glycoproteins (rElE2s), the yield of rElE2s was 40mg/L. We got purified rElE2s by chromatography with Q-S-FF column, P-S-FF column and G100 column.There are four forms of rE1E2s:72kD,95kD,145kD and over 200kD. SDS-PAGE shows the major rElE2s are the 72kD protein and over 200kD aggregation(Agg). It was shown that the major glycans of rE1E2s are high mannose oligosaccharides and the minor glycans are the complex forms. The complex glycans can affect the recognition of monoclonal antibody A4. To assess whether rElE2s are able to bind CD81 large extracelluar loop(LEL), we employed GST-pulldown assay. We constructed pET-GST-LEL plasmid and transform into E.coli BL21 strain. GST-LEL protein was expressed by BL21 and purified by Glutathione Sepharose 4B beads. GST-pulldown assay shows rE1E2s can bind GST-LEL. It suggest that rE1E2s does not only contain the sequence of HCV E2 CD81 binding domain, but also has the domain with correct conformation. Moreover, rElE2s can assemble into virus-like particles with HCV core protein. We mixed rE1E2s and core protein (aal-137), then renatured and concentrated the proteins. Two kinds of particles were produced, the diameter of smaller particles is about 35nm, and the diameter of bigger particles is about 55nm. So we suggest that the smaller particles are only assembled by core protein and the bigger particles are assembled by core protein and rE1E2s.High humoral response was induced by vaccination with rE1E2s, and could hold two months. Comparing with vaccination with only rElE2s, higher humoral response was induced more rapidly, but more transient by vaccination with Al(OH)3 as adjuvant. Similar results were obtained in immunized mice. Blocking assay showed the rabbit serum induced by rE1E2s can block the interaction between rE1E2s with CD81 as a dose-dependent manner. Furthermore, we employed HCV pseudotype particles(HCVpp) system and HCV cell culture(HCVcc) system to confirm whether the serum can neutralize HCV. The results indicated the rabbit serum which induced by only rE1E2s can neutralize two kinds of HCVpp derived from HCV genotype la or lb,even HCV virions derived from HCV genotype 2a. And the rabbit serum which induced by rElE2s addition with Al(OH)3 as adjuvant can neutralize HCVpp derived from genotype la, can neutralize HCVpp derived from genotype lb with lower efficiency. The ability of neutralizing virus is HCV-specific because of all the serum disable to neutralize VSVpp. The inhibit assay of mouse serum is not complete. As a conclusion, rE1E2s expressed by Pichia pastoris yeast can induce broad neutralizing antibodies against HCV, and is hopeful for mass production as a HCV vaccine.