Construction And Expression Of MBD2/HA1 Fusion Gene Eukaryotic Expression Vector And Its Immunoprotection

ObjectivesInfluenza A viruses (IAV) have caused several large scale epidemics inthe world, and thousands of people died in the influenza history. Until nowthere is none special medicine used for it, so the inoculation of influenzavaccine may be the best way to prevent influenza. At present, DNA vaccinehas come to be the hot spot of influenza vaccine research. Influenza virushemagglutinin (HA) could stimulate protective antibody in vivo. The anti-HAantibody showed great neutralizing effect, which could be attributed to eitherthe prevention of viral entry into susceptible cells by the blocking of virusbinding, or their acting on later stages of viral replication. HA was composedof two residues, HA1 and HA2. The five main antigenic determinants of HAlocated in HA1 domain, therefore HA1 become the optimal target of influenzasubunit vaccine research. Mouseβ-defensin 2 (mBD2) is one kind ofendogenous antibiotic peptides, which can be applied as a natural immuneadjuvant, mainly due to its role in attracting immature DC and activatingAPC.In this study, we want to construct the eukaryotic expression vector formBD2 and influenza virus HA1 fusion gene, named as pcDNA3.1(+)/mBD2-HA1, and observe the expression of mBD2-HA1 fused protein in HEK293 cells. In order to study on the immunoprotection in BALB/c mice,the mice were vaccinated with the pcDNA3.1(+)/mBD2-HA1 DNA,challenged with IAV, and evaluated the feasibility of mBD2 taken as animmune adjuvant.MethodsmBD2 and HA1 gene were amplified from plasmids pcDNA3.1 (+)/mBD2and pcDNA3.1(+)/HA1 respectively, and a polypeptide linker Gly₄Ser wasused to splice mBD2 and HA1 gene by overlap-PCR for constructing themBD2-HA1 fusion gene. mBD2-HA1 fragment was inserted into eukaryoticexpressing plasmid pcDNA3.1(+) to construct pcDNA3.1(+)/mBD2-HA1.The E. coli competent cells were transformed with pcDNA3.1(+)/mBD2-HA1 and selected positive clones through Ampicillin. The correctclone was identified by restriction enzymes digestion, PCR and sequencinganalysis. Then pcDNA3.1(+)/mBD2- HA1 was transfected into HEK293 cellsby PolyFect Transfection Reagent. The expression of mBD2-HA1 gene wasdetected by immunofluorescence. The cells transfected by pcDNA3.1(+)/mBD2-HA1 were incubated with rabbit anti-IAV serum, followed byadding goat anti-rabbit IgG labeled with FITC. In order to assessimmunoprotection effects, we immunized the normal BALB/c mice twice in a3-week interval with plasmids of pcDNA3.1(+)/mBD2-HA1, pcDNA3.1(+)/HA1 and pcDNA3.1(+)separately. Three weeks after the first vaccination, thebloods were taken from the mice tails. Then the mice were vaccinated thesecond time, and seven days later the mice were challenged with influenza Avirus at 10 median lethal dose (10×LD50). 3 days later, the bloods were takenagain. The anti-HA IgG antibodies in the sera were detected by ELISA. Thesurvival rate and weight loss of mice were observed and used for theevaluation of the immunoprotection induced by the recombinant plasmids. ResultsmBD2-HA1 fusion gene was gotten by overlap-PCR. Results ofrestrictive enzyme identification, PCR and sequencing analysis demonstratedthat mBD2-HA1 fusion gene has been successfully inserted intopcDNA3.1(+). The mBD2-HA1 fusion protein could be detected in HEK293cells transfected with pcDNA3.1(+)/mBD2-HA1 through immunoflu-orescenceassay. In animal experiment, the survival rate was 90%, the titer ofanti-HA antibodies was higher and the weight loss was lower than that ofcontrols in the pcDNA3.1(+)/mBD2-HA1 group. While in thepcDNA3.1(+)/HA1 group, the survival rate was only 50%, and the titer ofanti-HA also was lower. C deConclusionThe eukaryotic expression plasmid containing mBD2-HA1 fusion gene,named as pcDNA3.1(+)/mBD2-HA1, was constructed successfully. ThepcDNA3.1(+)/mBD2-HA1 could express the mBD2-HA1 fusion protein inHEK293 cells. It was proved that the mice immunized with pcDNA3.1(+)/mBD2-HA1 DNA were protected effectively through the animal experiment.Thess results suggested that mBD2 could be applied as an effective immuneadjuvant in the influenza DNA vaccine.