| Riemerella anatipestifer(RA)disease has been a global epidemic disease,which causes serious effect to the duck industry.The outer membrane protein(OmpA)has a good immunogenicity in different serotypes of RA and can be made into a RA vaccine against multiple serotypes.IL-2 has the functions of immune enhancement,immunotherapy and immune prevention,and therefoe has good application prospect in the construction of new genetic engineering vaccine.Combined with the advantages of the OmpA and IL-2,the RA OmpA gene and duck IL-2 gene were fused to construct the recombinant plasmid pcDNA3.1(+)-d IL-2-OmpA.The immune effects of d IL-2-OmpA fusion protein that induced ducklings to produce antibody was investigated to evaluate the immune enhancement effect of duck IL-2 on RA nucleic acid vaccine.This study may provide the foundation for further developing the safe,cheap and efficient new RA vaccine.1.The Sequence Analysis of OmpA and 16 S rRNA Genes of Riemerella an atipestifer Isolates in Guizhou ProvinceTo understand the correlationship between Omp A and 16 S rRNA genes of Riemerella anatipestifer(RA)isolates in Guizhou Province and explore the relationship of the two genes with RA serotypes,the OmpA and 16 S rRNA genes were amplified by PCR,respectively,to construct the phylogenetic trees.Results showed that the OmpA genes of 12 RA strains were situated in two groups and the homology of nucleotide and amino acid sequences of the isolates were 96.9% to 100.0% and 98.4% to 100.0%,respectively.The regular variation sites in OmpA proteins were located at 100(R/G),143(S/P),145(T/I)and 268(S/P).The 16 S rRNA genes of 12 RA isolates belonged to one group and their nucleotide homology was 99.4% to 100.0%.These results demonstrated that no apparent correlation was found between OmpA and 16 S rRNA gene cluster an d RA serotype among all the RA isolates.2.Cloning and Sequence Analysis of Interleukin-2 Gene of Sansui Sheldrake DuckOne specific pair of primers were designed and synthesized according to d uck IL-2 gene sequences published in Gen Bank.Total RNA was extracted from Sansui Sheldrake Duck Peripheral blood lymphocytes stimulated by ConA.The IL-2 gene was amplified by RT-PCR method using the primers and then sequenced and analyzed Sequencing results showed that the full length of the coding region sequence of Sansui sheldrake duck IL-2 gene was 423 bp and encoded 140 amino acids.The predicted molecular formula was C695H1125N175O226S13.The IL-2 gene of Sansui Sheldrake duck shared the highest homology with that of the mallard duck,Muscovy duck and Shaoxing duck,which reached 98.3%.Compared with the amino acids of IL-2 of other waterfowls,there were eight amino acid variation sites located at 29(N/D),32(K/T),49(D/N),58(T/K),90(K/N),105(M/T),122(N/K),and 129(Q/R).Only four amino acids(L66,C68,E72 and F127)in IL-2 were conserved among Sansui sheldrake duck and human and other animals.This research laid the foundation for the further study on the biological function of Sansui sheldrake duck IL-2 and the development of vaccine adjuvant.3.Construction and Identification of Recombinant Plasmids co-expressing RA OmpA and dIL-2 genesThe OmpA gene of RA serotype2 Guizhou isolates and the dIL-2 gene of Sansui sheldrake duck were fused together by the SOE-PCR method using two pair of linker primers.The fused gene d IL-2-OmpA was inserted into eukaryotic expression vector pcDNA3.1(+)to construct pcDNA3.1(+)-d IL-2-OmpA by Enzyme digestion and connection.The recombinant eukaryotic expression plasmid pcDNA3.1(+)-d IL-2-Omp A was transfected into avian DF-1 cells by lipofection transfection.The expression of dIL-2-OmpA fusion protein was identified by SDS-PAGE and Western-blot analysis.The results showed that d IL-2-Omp A fusion protein with molecular weight of 57 kDa was detected by anti-OmpA polyclonal antibody detection.These results showed that the recombinant plasmid pcDNA3.1(+)-d IL-2-OmpA could be correctly expressed in eukaryotic cells,which provided foundation for further animal immune experiment.4、Immune Protection effect of Recombinant Plasmid pcDNA3.1(+)-dIL-2-O mpA in Ducklings against RA70 Sansui Sheldrake ducklings were randomly divided into five groups,in cluding pcDNA3.1(+)-dIL-2-Omp A group,pcDNA3.1(+)-OmpA group,pcDNA3.1(+)group,inactivated vaccine group and blank group.The serum samples were collected at different times after immunization and the RA antibodies were d etected by ELISA method.The immunity protective test was performed using s erotype 1 and 2 RA to study the immunization effect.The results showed tha t the recombinant plasmid pcDNA3.1(+)-d IL-2-OmpA group、pcDNA3.1(+)-Omp A group and inactivated vaccine group could induce the ducklings to produce RA antibody.The antibody level of plasmid pcDNA3.1(+)-d IL-2-OmpA group was significantly higher than pcDNA3.1(+)-Omp A group(P<0.01).3.14×106CF U/mL and 1×106CFU/m L of serotype 1 and serotype 2 of RA strains were used to infect ducks,and the immune protection rates of recombinant plasmid pcDNA3.1(+)-dIL-2-Omp A group,pcDNA3.1(+)-OmpA and inactivated vaccine group were 66.67% and 66.67%,33.33% and 50%,83.33% and 100%,respectively.The results showed that d IL-2 in fusion gene dIL-2-OmpA could enhance the immunogenicity of recombinant plasmid pcDNA3.1(+)-dIL-2-OmpA in ducklings.Recombinant plasmid of construction immunized ducklings had a weak cross-immunoprotective effect on serotype 1 and serotype 2 of RA.This study laid the foundation for the development of a safe,cheap and efficient new RA vaccine in the future.In summary,we successfully cloned the OmpA gene of serotype 2 RA strain and IL-2 gene of Sanshui sheldrake duck.Ducks immunized with the recombinant plasmids pcDNA3.1(+)-dIL-2-OmpA and pcDNA3.1(+)-OmpA could induce OmpA specific antibody in ducks and had certain cross-immune protective effect on serotype 1 and serotype 2 RA isolates.d IL-2 as immune adjuvant could enhance the immunoprotective effect of recombinant plasmid pcDNA3.1(+)-dIL-2-OmpA. |
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