ABSTRACT
Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine‑elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription‑polymerase chain reaction in muscle tissues. The stimulation index (SI) of T‑lymphocyte proliferation and the levels of interferon‑gamma (INF‑γ) and interleukin‑4 (IL‑4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme‑linked immunosorbent assays. Results: The pcDNA3.1(+)‑BmCPI/ BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF‑γ and IL‑4 of pcDNA3.1(+)‑BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF‑γ of pcDNA3.1(+)‑BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)‑BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.
ABSTRACT
Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.