Construction And Neutralizing Humoral Response Of A Fusion DNA Vaccine Against Duck Hepatisis B Virus PreS/S Protein And C Protein

The genome of duck hepatitis B virus (DHBV). a complete minus and incomplete plus strand, is relaxed-circular DNA and approximately 3.0 kb in length. Genomic DNA is maintained in circular conformation by a short cohesive overlap between the two DNA strands. In this study, the complete genome sequence of DHBV was amplified through PCR and sequenced using classical dideoxy Sanger sequencing, and then constructed a fusion DNA vaccine against DHBV preS/S protein and C protein using eukaryotic expression plasmids pVAXl and investigated its neutralizing humoral response in ducks.1. Construction and analysis of complete genome sequence of duck hepatitis B virusThe DHBV strain, designated SCPOl. was isolated from cherry valley duck suspected DHBV infection in Sichuan Province. Subsequently, the nucleotide sequence of DHBV was amplified by PCR and cloned into the pGM-T vector and then sequenced using classical dideoxy Sanger sequencing. Then the sequences were assembled using the Chromas software package to produce the final genome sequence of 3021 bp in length (GenBank accession number KM676220). Sequence analysis revealed that the genome of DHBV SCP01 had a size of 3.021 bp and had three open reading frames (ORFs). designated ORF C. S. and P. which were on the minus strand and predicted to encode the viral surface, core protein, and polymerase protein. Sequence comparisons based on complete genome sequences of the 13 reference strains available in the GenBank of NCBI showed that complete genome sequence similarity was approximately 89.47%-99.64%. The DHBV strain SCP01 had the highest similarity (99.64%) with the sequence of strain DHBV-XY (GenBank accession number HQ214130). but had the lowest similarity (89.47%) with the sequence of strain DHBV (GenBank accession number M32991). Phylogenetic analysis and signature amino acids of polymerase protein revealed that the DHBV strain SCP01 belonged to "Western" isolates.2. Prokaryotic expression of duck hepatisis B virus preS gene and preparation of mouse anti-preS polyclonal antibodyDHBV SCP01 envelope protein (preS/S protein) was analyzed by bioinformatic softwares and online prediction tools. The results showed that the preS/S protein was composed of 328 amino acids, its molecular weight was approximately 36.23 kDa; this protein had no signal peptide sequence and had two transmembrane domains, located in 162-184 aa and 235-257 aa, respectively; antigen regions of the preS/S protein were mainly concentrated in the first 160 amino acids at the N terminal. Therefore, the preS gene was selected and amplified by PCR, and then constructed the prokaryotic expression plasmid and transferred it into expression bacteria of Rosetta. The expression plasmid correctly was substantially induced by IPTG, and the expression protein was purified and analyzed by SDS-PAGE and Western blot. The female Kunming mouses were immunized with purified preS protein for preparation of polyclonal antibody, mouse sera were analyzed by indirect ELISA. The results indicated that the preS gene was 483 bp, and prokaryotic expression plasmid designed pET32a(+)-preS was constructed successfully; recombinant preS protein, as the expected size (theoretical value 38.6 kDa), was soluble protein and largely obtained when recombinant bacteria were induced by IPTG with the concentration of 0.6mg/mL at 37? for 4 h; the titer of polyclonal antibody was 1:25600.3. Construction and expression of a fusion DNA vaccine against duck hepatisis B virus preS/S protein and C proteinThe DHBV SCP01 preS/S gene and C gene were amplified by PCR, while the preS/S-C fusion gene was amplified by overlapping PCR, and then the PCR products were cloned into pGEM-T vector, respectively. The eukaryotic expression plasmids pVAXl-preS/S, pVAX1-C, and pVAX1-preS/S-C were constructed through restriction enzyme digestion and ligation using the vector pVAX1, respectively. Forty-eight hours after transfection, the cells were collected and lysed. Enzyme identification and DNA sequence analysis showed that the sequence length of amplified preS/S gene, C gene, and preS/S-C gene were 987 bp, 789 bp, and 1818 bp, respectively, which was corresponding to the preS/S gene and C gene of DHBV strain SCP01 published in the GenBank of NCBI (100%), indicating that the three eukaryotic expression plasmids had been constructed successfully. Western blot analysis showed that the recombinant plasmids was shown to express those proteins (preS/S protein,36.2 kDa; C protein,30.3 kDa; preS/S-C protein,67.5 kDa), which were consistent with the expected sizes after transfection in COS 7 cells.4. Immunogenicity of a fusion DNA vaccine against duck hepatisis B virus preS/S protein and C proteinTwenty.1-day-old ducklings were randomly divided into five groups, and then the ducklings were primed intramuscularly (IM) with 300?g of the DHBV DNA vaccines. pVAX1-C (Group B). pVAX1-preS/S (Group C). pVAX1-preS/S and pVAX1-C (Group D). pVAX1-preS/S-C (Group E) or the pVAXl vector only (Group A), injected into the anterior quadriceps muscle at day 4 of age. At day 14 of age. the ducks were boosted IM with a second dose of 300?g of the Groups A-E after ten days. Serum samples, obtained from ducklings venous blood at days 2.4.6.8.10.12.14.21.28.35.42.49. and 56 after the first immunization, were used to monitor the production of anti-preS antibody and anti-C antibody by indirect ELISA. The results showed that the antibody levels of Group B (1.5395±0.0599). Group C (1.5096±0.0452). Group D (1.4784±0.0462.1.4788± 0.0571). and Group E (1.6394±0.0462,1.6555±0.0421). had reached the highest and extremely significantly higher than Group A (0.2569±0.0673.0.2750±0.0687) at days 21 after the first immunization (P<0.001). and the Group E induced antibody levels were the best and significantly higher than Groups B-D (P<0.05). which provides a foundation for studying the effective protection of DHBV DNA vaccination.