ObjectiveTo construct an Enterococcus faecium （Ef）-based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa（Pa）and to study its protective immune mechanism in mice. MethodsThe PA0057 gene was cloned from the genomic DNA of PA01 strain ATCC9027 by PCR and inserted into pGEX-1λT to construct pGEX-PA0057. The recombinant plasmid was electroporated into TX0016 strain to construct rEf-PA0057 vaccine. The plasmid was extracted from rEf for PCR. The rEf vaccine was expressed through IPTG induction， and the expression of protein was analyzed by SDS-PAGE and Western blot. BALB/c mice were immunized intragastrically with 5×10⁸ CFU rEf-PA0057 vaccine 3 times per week for 3 weeks. 4 weeks after the first immunization， mice were challenged intranasally with 5×10⁷ CFU of PA01 strain. 2 weeks after challenge， mice were sacrificed， and their lungs were separated. Bacteria in lungs were incubated and colonies were counted. Sera were collected at 0， 4， and 6 weeks after the first immunization. The IgG and its subclasses， and IgE were detected by ELISA. ResultsThe 900 bp PA0057 gene was successfully cloned by PCR. PCR showed that PA0057 gene was amplified when the extracted plasmid from rEf as template； the relative molecular mass （Mr） of the expressed PA0057-GST fusion protein was approximately 58 ku， detected by SDS-PAGE. The amount of the expressed protein was 18% of the total bacterial proteins. Western blot showed that the target protein could be recognized by Pa sera. The colony numbers of lung tissue in rEf-PA0057 vaccine group， blank vector group and Ef control group were （0.297±0.011）×10⁸ CFU， （7.576±0.206）×10⁸ CFU and （7.551±0.185）×10⁸ CFU， respectively， the difference was statistically significant （P0.01）. The levels of IgG， IgG1， IgG2b， IgG3 and IgE increased. At the same time point， there was a significant difference compared with the two control groups （P0.01）. ConclusionThe rEf-PA0057 vaccine is successfully constructed. It may induce mice to produce humoral response against challenge with PA01.

Objective:To construct Lactococcus lactis (LL) based EmⅡ/3 vaccine of Echinococcus multilocularis and observe its expression efficiency. Methods:EmⅡ/3 gene was obtained through PCR amplification using the pCD-EmⅡ/3 as a template, the gene was cloned into pMG36e to construct pMG36e-EmⅡ/3 and transformed into Escherichia coli BL21 (DE3) competent cell, the recombinant plasmid was identified by double restriction endonuclease digestion, then was electroporated into LL MG1363 to construct recombinant (r) LL-EmⅡ/3 vaccine. After screening for roxithromycin resistance, the plasmid was extracted for PCR identification, and the expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results:The pMG36e-EmⅡ/3 recombinant plasmid was identified by double restriction endonuclease digestion, and there was an approximately 3 600 bp carrier band and a 1 759 bp EmⅡ/3 gene band. The plasmid extracted from roxithromycin resistant rLL strain was used as a template, and 1 759 bp EmⅡ/3 gene was amplified by PCR; SDS-PAGE demonstrated that the rLL-EmⅡ/3 vaccine could express EmⅡ/3 protein with a relative molecular weight of 66 × 10 3, and the expressed protein accounted for about 20% of the total bacteria protein after 3 days of culture; Western blot showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:We have successfully constructed the rLL-EmⅡ/3 vaccine of Echinococcus multilocularis, which expresses Em Ⅱ/3 protein with specific antigenicity.

Objective:To construct a recombinant Efs-EmⅡ/3-Em14-3-3 vaccine against Echinococcus multilocularis (Em) using Enterococcus faecalis (Efs) as a vector, and investigate its antigenicity. Methods:The recombinant plasmid pGEX-EmⅡ/3-Em14-3-3 was transformed into Efs ATCC47077 strain using electroporation method, and the recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was constructed. The plasmid was extracted for PCR amplification and identification. The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine was expressed through isopropyl-β-D-thiogalactoside (IPTG) induction, and the recombinant protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the proportion of expressed proteins in the total proteins of the bacteria was analyzed by thin layer scanning technology.Results:Using the plasmid extracted from recombinant Efs bacteria as a template, the EmⅡ/3-Em14-3-3 fusion gene with a size of about 2 554 bp could be amplified by PCR. The relative molecular mass ( Mr) of the expressed EmⅡ/3-Em14-3-3 fusion protein was approximately 119 × 10 3 by SDS-PAGE; after 5 h induction by IPTG, the expression level of target protein was high, accounting for about 9% of the total bacterial protein. Western blotting showed that the expressed protein could be recognized by mouse serum infected with alveolar hydatid cyst. Conclusion:The recombinant Efs-EmⅡ/3-Em14-3-3 vaccine is successfully constructed, and the expressed fusion protein shows specific antigenicity.

Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.

Objective To construct Lactococcus lactis(LL)-based recombinant LL-Eg95(rLL-Eg95)vaccine for Echinococcus granulosus(Eg)and to examine its expression efficiency.Methods Eg95 gene was obtained by PCR from the template of pCD-Eg95.Then,pMG36e was inserted in the Eg95 gene after double cleaving with restriction endonucleases Xba Ⅰ and Hind Ⅲ to construct recombinant plasmid pMG36e-Eg95,which was transformed into E.coli BL2(DE3)competent cells.The recombinant plasmid was extracted and identified by double restriction endonuclease digestion and was then electroporated into LL MG1363 to construct rLL-Eg95 vaccine.Then,the plamid was extracted and identified by PCR.Results Examination of the recombinant plasmid by double restriction endonuclease digestion showed that the segment was of the expected length.PCR showed that 471 base pairs of Eg95 gene were amplified when the plasmid extracted from roxithromycin-resistant recombinant LL was used as the template.Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the relative molecular mass of the Eg95 protein expressed was approximately 16.5×103 and that the amount of the expressed protein was 17％of the total bacterial proteins.Western blot findings suggested that the expressed protein could be recognized by mice serum infected with hydatid cyst.Conclusion The rLL-Eg95 vaccine was successfully constructed,expressing Eg95 protein that has specific antigenicity.