| In recent years, using attenuated Salmonella typhimurium as vectors to directly deliver DNA vaccine into antigen presenting cells (APCs) has been reported in many mouse and chicken models and has shown the ability to induce humoral, cellular and local mucosal immune responses. Based on the recombinant bacteria X4550(pVAXl-HA) which had been constructed previously, four DNA vaccines delivered by attenuated Salmonella typhimurium SL7207 were constructed, and evaluated their safety and immune efficacy to chicken.Firstly, a pair of primers were designed and synthesized according to the previously published mRNA sequence in GenBank and used to amplify NP gene by reverse-transcription polymerase chain reaction from the genome RNA of a H5 subtype AIV strain A/Goose/Jiangsu/1/2000. Another two pairs primers were also designed and synthesized according to sequence of the recombinant plasmid pVAXl-HA and to amplify HAl and HA2 gene fragment by polymerase chain reaction. And then the three PCR products were cloned into pGEM-T easy vector for sequencing. It was shown that NP gene is specific for type A influenza virus, and that the sequence of HAl and HA2 gene are 100% identity to that of their corresponding HA template gene. Subsequently, NP gene was cloned into eukaryotic expression vector pVAXl through EcoR V and Xho I double digestion; HAl and HA2 gene were cloned into eukaryotic expression vector pVAXl through HindIII and BamH. I double digestion respectively. The recombinant plasmids pVAXl-NP, pVAXl-HAl and pVAXl-HA2 were transfected COS-7 cell, and the transient expression of NP, HAl and HA2 genewere identified by indirect immunofluorescent assay. Finally, the recombinant plasmids pVAXl-NP, pVAXl-HAl, pVAXl-HA2 and pVAXl-HA were transformed into the attenuated Salmonella typhimurium SL7207, and the recombinants were screened and designated as SL7207(pVAXl-NP), SL7207(pVAXl-HAl), SL7207(pVAXl-HA2) and SL7207(pVAXl-HA), which made a bedrock to evaluate it's safety and immune efficacy.When cultured in vitro or administered into body, the recombinant bacteria will partially lose it's carrying plasmid due to the decreasing or vanished antibiotic selective pressure. It was shown that the percentage of palsmid carried by recombinant bacteria was less than 90%, when the recombinant bacteria cultured in vitro up to 9h. One-day-old SPF chickens were orally innoculated with recombinant attenuated Salmonella typhimurium at dosage of 10~10 CFU, it was observed that these recombinant bacteria sustained in liver and spleen for 24h, and also showed that ther are safe to SPF chicken.Six weeks old BALB/c mice were twice immuninized with SL7207(pVAXl-HA) and SL7207(pVAXl-NP) at dosage of 109 CFU. In two immunized groups, some mice were induced detectable ELISA antibody titers, which showed that SL7207(pVAXl-HA) and SL7207(pVAXl-NP) have the ability to induce humoral immune response.In order to select the recombinant bacteria for vaccine candidate, the immune efficacy of three recombinant bacteria groups was compared with that of negative control groups. The level of protection of SL7207(pVAXl-HA) group and SL7207(pVAXl-HA) plus SL7207(pVAXl-NP) group was significantly higher than that of the negative control groups (p<0.05), and no significant difference was observed between these two groups. It was suggested the recombinant bacteria SL7207(pVAXl-HA) could be a good candidate for the further study.In immune efficacy experiment, both SL7207(pVAXl-HA) and X4550(asd-pVAXl-HA) were used as the vaccine orally administered to chickens. Meanwhile IFN y was used as adjuvant. The results of antibody responses showed that the intestinal mucosal antibody titers in immunized groups were significantly higher than that of chickens injected with killed oil-emulsified vaccine or that of negative control groups (p<0.05). In contrast, the serum antibody titers in immunized groupswere significantly lower than that of the killed oil-emulsified vaccine group. The level of protection of both recombinant bacteria groups and those groups immunized with recombinant...
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