Immunology comparision of composite gene recombinant plasmid and expressed protein of periodical Brugia malayi

Field: Immunology and Microbiology (Life Sciences)

Objective To construct a plasmid DNA vector expressing cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase of periodic Brugia malayi（BmCPI/BmGAPDH）, and purify the recombinant protein after transfecting the vector into human cervical carcinoma cells（Hela） for expression. To make a comparison of immunity efficacy between the recombinant plasmid and the homologous protein and to a lay theoretic andexperimental basis for developing novel anti-filarial genetic engineering vaccines. Methods The amplified genes BmCPI and BmGAPDH and a plasmid vector were double enzymes digested and ligated to construct a recombinant plasmid pcDNA3.1（＋）-BmCPI/BmGAPDH, and this plasmid was transfected to Hela cells after being identified. G418 was used for screening transfectants, and the monoclonal resistant cell strain was determined by RT-PCR and SDS-PAGE. The recombinant protein was purified by affnity chromatography and identified by Western blotting. Sixty BALB/c mice were divided into 5 groups, 12 per group, and they were immunized at 2, 4, and 6 weeks. Mice in control groups were injected with PBS 100 μl or pcDNA3.1 100 μg/CpG 30 μg, and mice in experimental groups were injected with＇ pcDNA3.1-BmCPI/BmGAPDH 100 μg/CpG 30 μg, the recombinant protein 50 μg/CpG 30 μg, or pcDNA3.1-BmCPI/BmGAPDH 100 μg/CpG 30 μg for the first two times and the recombinant protein 50 μg/CpG 30 μg for the third one. Target genes in muscular tissue were detected by RT-PCR. Stimulation index （SI） of the spleen lymphocytes of immunized mice was measured by specific proliferation test （MTr assay）. Serum antibody titer and the level of secreted IL-4 and INF-γ in mice were detected by ELISA. Results The gene BmCPI/BmGAPDH was detected in the injected muscle of mice after injection with pcDNA3.1 （＋）-BmCPI/ BmGAPDH. Serum antibody titers of mice in plasmid/protein/CpG group （1 695.12 ± 1.43, 3 199.63 ± 1.34） were higher than those in plasmid/CpG group（712.69 ± 1.08, 1 510.08 ± 1.43, P 〈 0.05） and those in protein/CpG group （800.02± 1.34, 1 510.08 ±1.43, P 〈 0.05） 4 and 6 weeks after immunization. The proliferation of spleen T lymphocytes seen on MTr assay was higher in plasmid/protein/CpG group （1.629 ±0.235） than that in protein group （1.248 ± 0.110, P 〈 0.05）. The levels of serum INF-γ were higher in plasmid/protein/CpG group[ （101.660 ± 5.101）, （178.265 ±7.139）mg/L] and plasmid/CpG group [ （102.067 ± 3.722）, （115.148 ±6.031）mg/L] than those in protein/CpG group [ （75.438 ± 2.102 ）, （82.004 ± 3.777 ） mg/L, P 〈 0.05 ] 4 and 6 weeks after immunization. The level of serum IL-4 from plasmid/protein/CpG group [ （75.385± 3.318）mg/L3 and protein/CpG group [ （ 46.363 ±3.672 ） mg/L ] was significantly higher than that from plasmid/CpG group [ （36.691 ± 3.443）mg/L, P 〈 0.05 ] 6 weeks after immunization. Conclusions Both the DNA vaccine pcDNA3.1 （＋）-BmCPI/BmGAPDH and its homologous protein could elicit specific humoral and cellular immune responses in immunized BALB/c mice. Efficacy of DNA/protein vaccine has obvious advantages.

Key words:
Brugia malayi ; Cysteine protease inhibitor; Glyceraldehydes-3-phosphate dehydrogenase ; Composite gene ; Expressed protein; Immunization