| Influenza is an infectious disease caused by influenza virus,characterized by sudden,rapid spread,wide spread and a certain seasonality,also known as seasonal flu.Influenza virus is divided into three type A,B,C which type A and B.is further spreade in worldwide,influenza A virus(IAV)classified according to its envelope glycoproteins include hemagglutinin(H1-H18)and neuraminidase(N1-N11).The WHO report shows that influenza infects 5-10% of the world’s population every year,induces 3-5 million cases of severe illness,resulting in 250,000 to 500,000 deaths.H3 subtype influenza A virus is one of the major subtypes which cause seasonal flu.H3 subtype is the key of the prevention and control of influenza for it has potential to be a pandemic strain or provide the possibility of a gene for a pandemic strain.The flu vaccine is the most effective measures to prevent the flu.Influenza virus HA protein can divide into the globular head domain HA1 and the stem domian HA2.HA1 can be combined with the sialic acid receptor on host cell membrane.The membrane fusion happen when the virus is taken into cells by endocytosis and at the low p H of endosomes,a series of conformational change happen to HA2.The high frequency mutation of amino acid sequence and the protective effect of glycosylated modification in HA1 head area are difficult points in vaccine design.Its HA2 stem domain is relatively conservative,but the immune response is weak the short time exposure.The researcher focus on the recombinant protein with trimer HA which can simulatethe influenza virus infect.At the same time,with the research on the HA2 broad-spectrum neutralizing antibody,the use of full-length of HA2 or HA2 conservative epitope as the method of immunogen-induced broad-spectrum neutralizaing antibody was paid attention.Based on these problems and research,our group discovered a conservative HA2 stem domain with conservative sequence analysis: DLWSYNAELLVALENQ.The immunogen which HA2 epitope link to Nov-particle,can induce neutralizing antibody in mice which shows inhibit the conformational change of HA in stem-targeted virus neutralization.In order to slove the Nov-particle pre-existing immunity and further verify the reliability of this epritope,the new carrier is selected.Studies have shown that Ferritin protein can be self-assembled into 24 polymers with a diameter of 12 nm,and its N segments can be inserted into exogenous genes.After the HA protein is inserted,the protein can form a trimer conformation.But Ferritin’s ability to deliver peptides is unclear.Based on the above analysis,we expect to obtain the conservative HA(762aa),HA2(412aa),HA2(90-105)protein sequence to connect Ferritin,forming the immunogen with trimer conformation.Meanwhile,the reliability of HA2(90-105)and the ability of Ferritin to deliver the short peptide were verified.The three sequences were attached to Ferritin proteins,respectively,using SSG linker,facilitating formation of trimer conformation and exposure epitopes In order to study the effect of glycosylation on HA protein,we adopted the expression system of Mammalian cell and E.coil.Length of HA,HA2 in two kinds of expression system,no protein expression,we consider the wrong express system was used.HA2(90-105)expressed few in mammalian cell expression system,which can be a large number of expression in E.coil,after protein purification,particle size analysis,electron microscope for structural characterization,the 12 nm in diameter of immunogen of spherical structure were gained.BALB/c mice were immunized with the recombinant protein in three different doses and two adjuvant Freund’s adjuvant and aluminium hydroxide gel adjuvant.While PBS and Ferritin protein as negative control,H3N2 virus as virus control.After immune 4 times,the HA2(90-105)antigen specific was detected,The Freund’s adjuvant immune group has an antibody titers of about 104,informed that the recombinant protein had certain immunogenicity and was irrelated to the dose.The titers of the aluminum adjuvant group decreased with the increase of dose,and was lower than that of the Freund’s adjuvant group,and no specific antibody was produced in the virus control group.The serum has a certain binding capacity with H3N2 HA protein and the Freund’s adjuvant group is higher than the aluminum adjuvant group.The antibody titer of serum against H3N2 virus was about 105,and the antibody of aluminum adjuvant was lower than Freunds group.After four immunization,H3N2 virus was used to boost the immune system,the titer of HA2(90-105)antigen specific antibody was significantly increased,and the antibody titer was about 103 times higher.The level of antibody in the aluminum adjuvant group was elevated,but was lower than that of the Freund’s group.The results showed that the Freund’s adjuvant could enhance the immunogenicity of recombinant protein.Immune serum should not have the activity of HAI for which the conserve epitope which located in HA2 stem domain The result of HAI support our theory.Ig G1,Ig G2 a,Ig G2 B.in immune sera were significantly improved after 4th immunization by antibody typing test.However,Ig M level did not change,and humoral immunity level was undesirable.Microneutralization test in vitro showed that immune serum with Freund’s adjuvant were significantly improved and the ID50 was 863.7,which was 6.16 times higher than the pre-immune serum.Indicating the serum has neutralization activity.The ID 50 of aluminum adjuvant group with 100 ug immunogen was only 1.21 times higher than the pre-immune serum.The results showed that the serum with neutralization ability was obtained by the immune mice of the Freunds adjuvant,and the aluminum hydroxide adjuvant could not induce the neutralizing antibody in mice.Combined with the results of Elisa experiment,aluminum hydroxide adjuvant cannot effectively enhance the immunogenicity of recombinant protein.Virus challenge was performed after immunization 4 times of recombinant protein Weight of mice was measured every 24 h.The PBS group lost 38 percent,the Ferritin group decreased by 33 percent,and the Freund adjuvantgroup lost less than 5 percent,while the aluminum adjuvant group lost less than 10 percent.It shows that recombinant protein has certain protective ability to mice.In conclusion,the recombinant immunogen of HA2(90-015)with Ferritin as the carrier has certain neutralization ability for H3N2.The HA2(90-105)epitopes are potential broad-spectrum neutralization vaccine candidates.Ferritin’s ability to recurse to HA2(90-105)is weaker than that of Nov-particle,and its ability to transmit epitaph remains to be further studied. |
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