[Mouse immune responses elicited by eukaryotic expression plasmid and prokaryotically expressed protein of Brugia malayi myosin 29 epitope].

Abstract

Objective: To determine the immune responses elicited in BALB/c mice by vaccination with eukaryotic expression plasmid pcDNA3.1(+)-BmM29 containing Brugia malayi myosin 29(BmM29) epitode and prokaryotically expressed recombinant BmM2 protein(rBmM29) respectively.

Methods: rBmM29 protein was expressed in E. coli strain BL21, purified as recombinant protein vaccine, and administered via multiple subcutaneous injections. The purified recombinant plasmid pcDNA3.1(+)-BmM29 was used as the nucleic acid vaccine and injected into the tibialis anterior muscle. Sixty BALB/c mice were randomized to receive three immunizations(with intervals of 2 weeks) with PBS (100 μg, group A), pcDNA3.1(+)/CpG (100 μg/30 μg, group B), pcDNA3.1(+)-BmM29/CpG (100 μg/30 μg, group C), rBmM29/CpG(50 μg/30 μg, group D), or pcDNA3.1(+)-BmM29/rBmM29/CpG (two injections of pcDNA3.1(+)-BmM29/CpG 100 μg/30 μg followed by a rBmM29/CpG 50 μg/30 μg). Serum was prepared through ophthalmectomy at week 4, 6, and 8 after primary immunization, and the serum IgG titer was determined by ELISA. The mice were sacrificed at week 8, splenocyte suspension cultured for 48 h, and levels of INF-γ and IL-4 in the supernatant detected by ELISA.

Results: ELISA results showed that the A490 values of serum IgG in groups A-E were 0.038 ± 0.050,0.053 ± 0.009,0.360 ± 0.035,0.456 ± 0.025,0.370 ± 0.025 at week 4,0.045 ± 0.003,0.045 ± 0.005,0.510 ± 0.018,0.548 ± 0.010,0.552 ± 0.018 at week 6, and 0.041 ± 0.004,0.044 ± 0.009,0.606 ± 0.047,0.674 ± 0.042,0.770 ± 0.041 at week 8, significantly higher in groups C, D and E than in groups A and B (P < 0.05) at all time points, and significantly higher in group E than in groups C and D(P < 0.05) at week 8. The IFN-γ levels in splenocyte culture supernatant at week 8 after primary immunization were (47.72 ± 8.94),(50.43 ± 2.81),(304.78 ± 8.42),(242.28 ± 5.99), and(426.52 ± 6.76) pg/ml in groups A-E, respectively, significantly higher in groups C-D than in groups A and B(P < 0.05), and in group E than in groups C and D(P < 0.05). The IL-4 levels in splenocyte culture supernatant were(60.00 ± 11.14),(57.71 ± 15.95),(93.17 ± 12.56),(96.67 ± 11.48), and (101.17 ± 5.81) pg/ml, significantly higher in groups C-D than in groups A and B(P < 0.05).

Conclusion: Both the recombinant plasmid pcDNA3.19(+)-BmM29 and rBmM29 protein could elicit specific humoral and cellular immune responses in mice. Combined immunization with nucleic acid vaccine and protein vaccine is superior to each of the two alone.