| ObjectivesTo improve the effect of a single Mtb8.4 DNA vaccine, we constructed a chimeric Mtb8.4/hIL-12 eukaryotic plasmid by linkage of Mycobacterium tuberculosis Mtb8.4 gene to human IL12 gene with a simple linker (Gly4-Ser)3. We analyzed the immunogenicity of chimeric DNA vaccine and investigated the immune responses elicited when Mtb8.4/hIL12 was presented as endogenous Ag. Methods 1. Construction of eukaryotic expression plasmid pcDNA3.1 (+)-Mtb8.4The Mtb8.4 (without signal peptide) gene and MS gene (with signal peptide) were PCR amplified by using the genome of M. tuberculosis H37Rv as template and subcloned into pcDNA3.1(+) vector. Restriction Endonuclease digestion, PCR and DNA sequencing confirmed that the recombinant eukaryotic expression plasmid containing M. tuberculosis Mtb8.4 gene (pcDNA3.1(+)-Mtb8.4 and pcDNA3.1(+)-MS) had been constructed correctly.2. Construction of chimeric Mtb8.4/hIL-12 eukaryotic expression plasmid(1) Construction of pCI-neo-Mtb8.4-linker (pML) and pCI-neo-MS-linker (pMSL)Mtb8.4-linker and MS-linker gene (without stop codon) were PCR amplified by using two oligonucleotides designed to generate Nhe I and Mlu I restriction sites at the 5' and 3' ends of the amplified fragments, respectively. Then the amplified Mtb8.4 and MS gene were subcloned into the unique Nhe I and Mlu I cloning sites of pCI-neo expression vector.(2) Construction of the chimeric eukaryotic plasmid pCI-neo-Mtb8.4/hIL12 (pMI) and pCI-neo-MS/hIL12 (pMSI)hIL-12 gene (without initiation codon) was PCR amplified by using pORF-hIL12 as template and using two oligonucleotides designed to make a PCR product that would begin 5' to the secretory sequence of human IL-12 gene and would continue to the stop codon. The 5 ' primer included sequence for Mlu I restriction enzyme sites, and the 3' primer included sequence for Sal /restriction enzyme sites. The resulting PCR fragment was ligated with T4 DNA ligase in Mlu I/ Sal I-digested pML or pMSL plasmid vector to construct chimeric plasmid pMI or pMSI.3. Expression In vitroCOS-7 cells were transfected with pM, pMS, pMI and pMSI constructs by cationic liposom, respectively. 48 hours later, mRNA of targets gene were detected by RT-PCR and hIL-12 protein in culture supernatnant and cell lysates were detected by Western blotting. p815 cells were transfected with pM and pMS constructs and selected by G418. mRNA of targets gene were detected by RT-PCR.4. DNA immunizationMice were immunized between 6-8 weeks of age. The DNA vaccines were injected intramuscularly into the quadriceps of eight C57BL/6N mice per group three times at 3 weeks intervals. The vaccine dose used was 100 g of plasmid DNA per injection for the single vaccines and 2 50 g per constructs for the combination vaccines. Four weeks after the final inoculation, three mice were sacrificed to assess cytokine response and CTL induction. 5. Animal challenge studiesThe other five mice were challenged intravenously in a lateral tail vein with 1 106 CFU of virulent M. tuberculosis H37Rv. Control animals were injected with PBS, control plasmids (pCI-neo and pcDNA3.1), or once subcutaneously with 1 106 CFU of BCG at the time of the first DNA immunization. Spleen and the left lung were harvested from each mouse at 4 weeks after infection and homogenized in sterile saline. Serial dilutions of organ homogenates were plated on L-J agar and incubated 37 C until colonies were visible 4 weeks later. Protective efficacies in each experiment were expressed as reduced CFU and were compared with the negative control group by using student's t test. The right lung was obtained from each mouse and immediately inflated with and stored in 10% formalin saline. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Results1. Recombinant plasmids pM, pMS, pMI and pMSI were constructed and their accuracies were confirmed by a series of molecular biology techniques.2. Recombinant plasmid pM, pMS, pMI and pMSI all allow proteinstransient expression...
|
PDF Full Text Request