| Mycoplasma pneumoniae is the causative agent of atypical pneumonia and is also responsible for other respiratory tract infections such as tracheobronchitis, bronchiolitis, croup, and less severe upper respiratory tract infections in older children and young adults. Cardiovascular disease and neurological diseases are serious nonrespiratory symptoms associated with M. pneumoniae infection. It has been estimated that between10and30%of pneumonia cases that occur in the endemic period and that up to30and50%of all cases that occur in the epidemic period are caused by M. pneumoniae. Traditionally, M. pneumoniae infection is controlled by the use of antibiotics. However, the increased prevalence of antimicrobial-resistant strains of M. pneumo-niae increases the risk of reinfection. Accordingly, the prevention of atypical pneumonia through vaccination is needed.The P1protein is considered to be the major ligand mediating attachment of virulent M. pneumoniae to the host cell membrane. An earlier study showed that pretreatment of M. pneumoniae with antiserum directed against P1blocked cytadherence to hamster tracheal rings by up to80%. As the polymorphism regions RepMP4and RepMP2/3of p1gene are variablility and the P1gene contains an open reading frame of4,881nucleo-tides and contains21UGA codons that code for tryptophan, it is very difficulted to express the P1protein in Escherichia coli (E.coli) and mammalian cells as UGA codes for a stop codon. In this study, we studied the p1gene genotyping and the variation of p1gene to determined the conserved sequence of p1gene, chose the biological characteristics of M.pneumoniae P1immundominant region (1125-1395aa, P1C protein) and explored the use of a DNA vaccine encoding P1C. ObjectivesTo determine the conserved sequence of p1gene, the p1gene genotyping and variation was studied with the159M. pneumoniae clinical samples which were isolated from pediatric patients with respiratory tract infection. The prokaryotic expression vector pGEX6p-2/plc with M. pneumoniae p1adhesin immundominant region gene (3374-4185nucleotide, plc gene) was constructed and the recombinant protein was expressed in E.coli BL21(DE3). The purified P1C-GST protein was used to immunize the BALB/c mice and obtain polyclonal antibody. Antibody was used for adhesion and inhibition assay. The immunogenicity or antigenicity of P1C was analyzed. The pcDNA3.1(+)/plc (named pP1C) monogenic nucleic acid vaccine and pcDNA3.1(+)/plc/IL-2(named pP1C-IL-2) fused gene vaccine were contructed and immunogenicity were analyzed in BALB/c mice immunized by intramuscular injection and intranasal innoculation. pcDNA3.1(+)//LTB-plc (named pLTB-P1C) fused gene vaccine was innoculated by intranasal route. An animal model for M.pneumoniae infection was established using BALB/c mice, the humoral and cellular immune response in mice induced by the vaccines and the pathological changes of lung tissue were investigated. This study may provide experimental evidence for the development of high performance and new-style M.pneumoniae nucleic acid vaccine.Methods1. M. pneumoniae clinical isolates were revived and passaged in broth medium, and then cultured in agar medium for single colony which was further characterized by PCR. p1genotyping and the variation of p1gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).2. P1adhesin immundominant region gene (3374-4185nucleotide, plc gene) was amplified by PCR from the genome of M. pneumoniae and cloned into pGEX6p-2after digestion with BamH I and EcoR I. A codon TGA encoding Tryptophan was changed into TGG by site-directed mutagenesis. The mutagenized recombinant expression vector pGEX6p-2/plc was transformed into E.coli BL21and induced to express PIC-GST with IPTG. The obtained fusion protein, which was purified by high affinity GST Resin, was analyzed by SDS-PAGE and Western blot. The purified protein was used to immune BALB/c mice. Then the polyclonal antibody against the purified protein was acquired and used for adhesion and inhibition assay. ELISA was used to analyze the immunogenicity and antigenicity of P1C.3. Plc gene was subcloned to pcDNA3.1(+)(named pcD) eukaryotic plasmid. The plc gene and interleukin2(IL-2) gene or B subunit of E.coli heat-labile enterotoxin (LTB) gene was fused with a linker by recombination PCR to construct pP1C-IL-2or pLTB-P1C eukaryotic expression vectors. After verifying that the P1C and P1C-IL-2antigens could be expressed in HeLa cells by Western-blot,6-week-old BALB/c mice were immunized with pP1C-IL-2or pP1C or pcDNA3.1(+)/IL-2(named pIL-2) or pcD or PBS buffer intramuscularly and nasally at2-week interval for three times, while pLTB-P1C was vaccined nasally. ELISA was used for the quantitative detection of the P1C-specific total antibodies and IgG1, IgG2a isotypes, IgA the cytokines IFN-y and IL-4in the sera and bronchoalveolar lavage fluids of BALB/c mice. The proliferation response of spleen cells was detected by MTT assay.4. Mice were inoculated by plc gene or its fusion gene respectively. Mice were exsanguinated and lungs were removed for histological examination after M.pneumoniae challenge inoculation to analyze the immune. Serial10-fold dilutions of BAL fluids were immediately cultured on SP4agar plates, quantification was performed by counting colonies on plated specimens and expressed as log10CFU per milliliter.Results1.159cases of clinical samples in PCR-RFLP indicated that142samples (89.31%) were classified as typel,17samples (10.69%)were classified as type2.21clinical variarable samples were identified by the PCR-RFLP. Among the M.pneumoniae mutants,10were V2c strains,3were V2a strains and7isolates of the point mutants of type1. A new V2d mutant strain was found in the study, which contained a novel variable region of142bp between nucleotides (nt)2772-2913and these variable regions showed sequence homology with the RepMP2/3-j (606517-606658) elements beyond the p1gene.2. The, p1c gene fragment (813bp) was successfully amplified. The prokaryotic expression recombinant pGEX-6P-2/plc was successfully constructed. The codon TGA encoding Tryptophan was changed into TGG by site-directed mutagenesis. A soluble fusion protein with molecular weight about66kDa was obtained after expression and purification. The result of Western blot showed that P1C-GST could be recognized by the monoclonal mouse anti-M.pneumoniae sera.3. The polyclonal antibody against the purified protein was acquired, the titers of the specific antibodies were above1:4000. ELISA and western blot showed the PIC-GST protein could react with the M.pneumoniae infective sera.4. P1C protein could attach to the HeLa cells and the polyclonal antibody against the P1C protein could inhibit the binding of M. pneumoniae to the HeLa cells.5. Immunization with pP1C by the intramuscular route induced a moderate antibody response, and the response was better than those obtained by nasal immunization (P<0.01). IgG1and IgG2a were the dominant subclasses. While antibody titers of mice immunized with pPlC-IL-2were markly increased and mainly of the IgG2a subclass. Nasal-immunized animals exhibited significantly higher levels (P<0.05) of anti-P1C IgA in bronchoalveolar lavage (BAL) fluids than intramuscular-immunized animals, and there was no specific antibody in the BAL fluids of mice immunized with pcD or PBS. The pP1C and pPlC-IL-2groups immunized by intramuscular injection produced significant levels (P<0.05) of IFN-y and IL-4in BAL fluids and sera than those inoculated by nasal route, and only small numbers of nonspecific IFN-y and IL-4secreting cells were detected in the control groups. The stimulation index of pP1C group or pP1C-IL-2group was higher than that of control group (P<0.05).6. M.pneumoniae inoculated Mock-vaccinated or pcDNA3.1-treated mice consisted of peribronchial and perivascular mononuclear infiltrates, but there was no intrabronchial exudate or parenchymal pneumonia (neutrophilic alveolar infiltrate). The histopathologic scores of pP1C and pP1C-IL-2-vaccinated animals were significantly decreased. The infective control mice, which inoculated with sterile SP4broth, had an HPS of0-1. There was no significant difference in the number of M. pneumoniae in BAL Fluids cultures grown from the pP1C-IL-2immunized mice compared with cultures from the pP1C inoculated group.7. The pLTB-P1C immunizations elicited high levels of IgA and IFN-y, while the proliferation response of spleen cells had no difference from pP1C vaccinated mice. When the mice were challenged intranasally with107CFU M.pneumoniae strain (M129), the LTB-plc fusion DNA vaccine conferred significantly better protection than controls (P<0.05), as characterized by lighter inflammation, lower HPS values and lower detectable number of M.pneumoniae strain. The M.pneumoniae strains in BAL fluids of pLTB-P1C immunized mice is lower than pP1C immunized mice. These results indicate that the pLTB-P1C DNA vaccine can efficiently improve protective efficacy against M.pneumoniae infection, and effectively attenuate development of Mycoplasmal pneumomae in mice.Conclusions1.159M.pneumoniae clinical isolates maily for type1strains and21mutants were detected, the mutatuon rate of type2is higher than type1. A new p1mutant strain V2d was discovered.2. P1C protein was shown to be cytadherent, strong immunogenicity and antigenicity.3. Strong cellular immunity and humoral immunity responses could be induced by all pP1C, pP1C-IL-2and pLTB-P1C DNA vaccines in BALB/c mice.4. Both pP1C-IL-2vaccine and pLTB-P1C vaccines could induce more powerful immune response and immune protection than pP1C vaccine.5. Intranasal route of pP1C vaccine Induced higher sera IgG, lower BAL fluids IgA in BALB/c mice than intramuscular immuned mice, and effected mainly in Late stage of M.pneumoniae infection. While the latter was opposite. |
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