Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

China ; Communicable Diseases ; epidemiology ; Humans ; Population Surveillance ; Respiratory Tract Infections ; epidemiology

We designed the primers based on the sequence of the follistatin-related protein from Haemaphysalis longicornis Okayama strain accessed in GenBank. We cloned a gene encoding follistatin-related protein by RT-PCR, and the length cDNA is 814 bp, encoding a deduced protein of 289 amino acids. The alignment with the sequence of follistatin-related protein from the H. longicornis Okayama strain showed that the percent of nucleotide sequence and amino acid sequence is 97.8% and 99%, respectively. The expected size of GST-fused recombinant protein was 57 kD. We purified the recombinant protein through MagneGST protein purification system. Western blotting revealed that stronger reaction happened with the antiserum against eggs, but not clear with antisera against other developmental stages.
Amino Acid Sequence ; Animals ; Cloning, Molecular ; Follistatin-Related Proteins ; genetics ; immunology ; Ixodidae ; chemistry ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Alignment

To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P<0.05) and the significant lymphoproliferation of splenocytes (P<0.05). The neutralizing antibody titer induced by the prMEIII protein was close to that induced by the commercial attenuated vaccine SA14-14-2 (P>0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Encephalitis Virus, Japanese ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; Immunogenicity, Vaccine ; Mice ; Mice, Inbred BALB C ; Vaccines, Subunit ; immunology ; Viral Vaccines ; immunology

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-E^rns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 10⁶ TCID₅₀ virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.
Animals ; Antibodies, Viral ; Diarrhea ; Diarrhea Virus 1, Bovine Viral ; Guinea Pigs ; Mineral Oil ; Viral Envelope Proteins ; Viral Vaccines

In order to evaluate the immune effect of the genotype Ⅰ Japanese encephalitis virus prM-E DNA vaccine and the prM-EⅢ fusion protein subunit vaccine on mice using DNA prime-protein boost strategy, the prM-E gene was inserted into the pVAX1 eukaryotic expression vector. The recombinant expression vector prM-E-pVAX1 was constructed as a DNA vaccine for initial immunity, and the recombinant prM-EⅢ fusion protein was obtained using a prokaryotic expression system as a subunit vaccine for enhanced immunity. Thirty two female BALB/c mice aged 4-6 weeks were randomly divided into four groups, and a prM-E-pVAX1 DNA vaccine group, a DNA prime-protein boost immune group, a prM-EⅢ subunit vaccine group, and a pVAX1 vector control group were set up. The specific antibody level in serum was monitored by ELISA, the neutralizing antibody titer was detected by plaque reduction neutralization, and the cellular immune responses induced by different vaccine immune groups were analyzed by cytokine expression abundance and lymphocyte proliferation experiments. The results showed that the neutralizing antibody titers induced by mice immunized with the DNA prime-protein boost strategy were close to that of the group immunized with the single prM-EⅢ subunit vaccine, but significantly higher than that of the group immunized with the single prM-E-pVAX1 DNA vaccine. DNA prime-protein boost strategies induced effective Th1/Th2 immune responses in mouse models, in particular the Th1 cell-mediated immune responses. This study provides a new immune strategy that may facilitate the prevention of Japanese encephalitis.
Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; DNA ; Disease Models, Animal ; Encephalitis Virus, Japanese/genetics* ; Female ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA/genetics* ; Vaccines, Subunit

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.
African Swine Fever/diagnosis* ; African Swine Fever Virus/genetics* ; Animals ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins/genetics* ; Swine

Humans ; Staphylococcus lugdunensis/genetics* ; Peptides, Cyclic ; Chromosomes ; Operon/genetics* ; Staphylococcal Infections/genetics* ; Bacterial Proteins/genetics* ; Anti-Bacterial Agents