Construction, identification and expression of recombinant Bifidobacterium bifidum vaccine of Pseudomonas aeruginosa and its protective effect / 中华地方病学杂志

ABSTRACT

Objective To construct a recombinant Bifidobacterium bifidum (Bb) vaccine of Pseudomonas aeruginosa,identify its protective effect in mice after immunization with the vaccine and challenged by Pseudomonas aeruginosa.Methods The OCR-amplified outer membrane protein OprH gene was cloned into vector pGEX-1λT to obtain the plasmid pGEX-OprH.The pGEX-OprH was then electroporated into Bb to obtain the rBb-pGEX-OprH vaccine.After PCR identification,the expression of rBb-pGEX-OprH was induced by Isopropylβ-D-Thiogalactoside (IPTG),and the expression products were analyzed by SDS-PAGE and characterized by Western blotting;21 BALB/c mice were subdivided by random number table into vaccine group,empty vector group and Bb group (7 mice in each group),and immunized by intragastrically administration with rBb-pGEX-OprH,rBb-pGEX-1λT or Bb,respectively.Mice were immunized once a day and three times a week for three consecutive weeks,then the mice were challenged intranasally with Pseudomonas aeruginosa at four weeks after the first immunization,once a day for three consecutive days.Two weeks after the challenge,the mice were sacrificed to detect their lung bacterial loads.Sera were collected before vaccination,four weeks after the first immunization,and two weeks after the challenge.The sera were analyzed by enzyme-linked immunosorbent assay (ELISA) to detect the level of IgG.Results OprH gene of 660 bp was amplified by PCR;PCR results showed that the rBb-pGEX-OprH was constructed successfully;SDS-PAGE confirmed that the recombinant vaccine expressed a relative molecular mass 47 × 103 fusion protein,which was consistent with the expected result;Western blot results showed that the expressed protein could be identified by sera in mice infected with Pseudomonas aeruginosa.Differences of lung bacterial colonies between the three groups were statistically significant (F =1 506.793,P ＜ 0.05).The number of bacterial colonies in the vaccine group [(7.691 ± 0.069) lgCFU/g] was lower than that of the empty vector group [(8.855 ± 0.027) lgCFU/g] or the Bb group [(8.958 ± 0.037) lgCFU/g,P ＜ 0.05].There were statistically significant differences in serum IgG levels between the 3 groups 4 weeks after the first immunization (F =77.216,P ＜ 0.05),the IgG level of the vaccine group (0.078 ± 0.005) was significantly higher than that of the empty vector group (0.054 ± 0.004) and the Bb group (0.052 ± 0.004,P ＜ 0.05).Conclusion The rBb-pGEX-OprH vaccine is constructed successfully,and it could induce certain protective effect in mice.