Stable expression of Hantavirus H8205 strain G1/IL-2 gene and immune protection of the fusion gene

Abstract

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20–1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

Keywords

Hantavirus / fusion gene / stable expression / immune effect

References

[1] HooperJ. W., CusterD. M., ThompsonE., et al.. DNA vaccination with the Hantaan virus M gene protects Hamsters against three of four HFRS Hantaviruses and elicits a high-titer neutralizing antibody response in Rhesus monkeys. J Virol, 2001, 75: 8469

[2] SunH., GuanW. X., HuangH. J.. Cloning and Expression of the Fusion Gene of G1 Segment of Hantavirus H8205 Strain and IL-2 Gene of Human. J Huazhong Univer Sci Technol [Health Science] (Chinese), 2003, 32(6): 578-580

[3] SambrookJ., FritscbE. F., ManiatisT., et al.. Molecular Cloning: A Laboratory Manual, 19892New York, Cold Spring Harbor Laboratory Press: 24-34

[4] Sun W M, Wang H Q. Methodology of Cytokine Study. People’s Medical Publishing House (Chinese), 1999. 387–408

[5] LuoH. Y., YangX., SuX. S.. Study of optimization HBV DNA vaccine by insertion of IL-2 gene. Chin J Immunol (Chinese), 2002, 18: 301-304

[6] GoodM. F., BerzofskyJ. A., MalloyW. L., et al.. Genetic control of the immune response in mice to aplasmodium falciparum sporozoite vaccine. Widespread nonresponsiveness to single malaria T epitope in highly repetitive vaccine. J Exp Med, 1986, 164(2): 655-660

[7] DavisH. L., Brazolot MillanC. L., ManciniM., et al.. DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgentic mice. Vaccine, 1997, 16(8): 849-852

[8] GeisslerM., GesienA., WandsJ. R.. Inhibitory effects of chronic ethanol consumption on cellular immune responses to hepatitis C virus core protein are reversed by genetic immunizations augmented with cytokine-expressing plasmids. J Immunol, 1997, 159(10): 5107-5113