Protective immunity induced by recombinant surface protein-1 and surface protein-4 against Toxoplasma gondii infection in mice

doi:10.12140/j.issn.1000-7423.2019.02.007

Abstract

Abstract:

Objective To determine the protective immunity induced by the immunization of recombinant surface antigen-1 (rSAG1) and surface antigen-2 (rSAG2) against Toxoplasma gondii infection in mice and compare their potential as vaccine. Methods The DNAs coding for T. gondii SAG1 and SAG4 were amplified by PCR from T. gondii genomic DNA and cloned into expression plasmid pET-28a(+). The success of cloning was confirmed by DNA sequencing, The recombinant plasmid DNAs were transformed into E. coli BL21(DE3) for expression as recombinant proteins under isopropyl-β-D-thiogalactopyranoside(IPTG) induction. The induced bacteria were lysed and the expressed rSAG1 and rSAG4 were purified by Ni-NTA resin under denatured condition. The denatured recombinant proteins were refolded by dialysis method. The refolded recombinant proteins were formulated with Freund’s adjuvant and used to immunize mice (n = 25 for each group) for 3 times. PBS and PBS + adjuvant were used as controls. Sera were collected after each immunization and specific IgG in the sera were measured by ELISA. Each mouse was challenged with 3 000 T. gondii tachyzoite two weeks after the final immunization. The survival time of the infected mice was observed and the data was analyzed using SPSS 16.0. Results The DNAs coding for full length SAG1 (780 bp) and SAG4 (438 bp) were successfully amplified from T. gondii genomic DNA and then cloned into pET-28a(+) to generate recombinant pET-28a(+)-SAG1 and pET-28a(+)-SAG4. The correct sequence and reading frame were confirmed by DNA sequencing. After being transformed into E. coli BL21 (DE3), both rSAG1 and rSAG4 were expressed as inclusion bodies. After being denatured with urea, the rSAG1 and rSAG4 were purified and refolded as soluble proteins. All mice produced high level of specific IgG in sera after being immunized three times with each recombinant protein compared to pre-immunization sera (P < 0.05). The ELISA A₄₅₀ value of rSAG1 group (1.821 ± 0.184) was significantly higher than rSAG4 group (0.695 ± 0.089) when sera were diluted at 1:400 (P < 0.05), but it is less than the combined group (rSAG1 + rSAG4) (1.955 ± 0.097) (P < 0.05). And the A₄₅₀ value of 3 experimental groups was higher than PBS (0.019 ± 0.002) group and PBS + adjuvant group (0.020 ± 0.004). All control mice (without immunization) died 224 h after being infected with 3 000 T. gondii tachyzoite, however, the mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 survived till 296 h, 288 h and 320 h, respectively, after being challenged with the same number of T. gondii tachyzoite, with significant difference compared to the control group (P < 0.05). The increased survival time in the rSAG1 + rSAG4 group was significantly longer than each individual antigen group (P < 0.05). Conclusion Mice immunized with rSAG1, rSAG4 and rSAG1 + rSAG4 produced significant protective immunity against T. gondii infection with prolonged survival time than group without immunization. The co-immunization with both rSAG1 and rSAG4 produced synergic protective immunity compared to the individual antigen.

Key words: Toxoplasma gondii, Surface protein 1, Surface protein 4, Recombinant protein, Combined immunization

CLC Number:

R382.5

Kai LIANG, Yan-wen LI, Bei-bei WANG, Xiao-yin FU, Xiao-quan LIU, Shan-shan HE, Li-li TANG, Deng-yu LIU, Huan-huan SHI. Protective immunity induced by recombinant surface protein-1 and surface protein-4 against Toxoplasma gondii infection in mice[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(2): 155-160.

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组别Groups	血清特异抗体A₄₅₀值 (1:400) Serum specific antibody A₄₅₀ value (1:400)
组别Groups	免疫前 Pre-immunization	首次免疫后第2周 2 weeks after the 1st immunization	首次免疫后第4周 4 weeks after 1st immunization	首次免疫后第6周 6 weeks after 1st immunization
PBS	0.019 ± 0.002	0.019 ± 0.002	0.019 ± 0.002	0.019 ± 0.002
PBS+佐剂PBS + adjuvant	0.020 ± 0.003	0.020 ± 0.001	0.019 ± 0.002	0.020 ± 0.004
rSAG1	0.021 ± 0.002	0.276 ± 0.049	1.649 ± 0.150	1.821 ± 0.184^a
rSAG4	0.020 ± 0.002	0.022 ± 0.002	0.296 ± 0.071	0.695 ± 0.089^a
rSAG1 +rSAG4 SAG1	0.021 ± 0.006	0.226 ± 0.068	1.188 ± 0.097	1.955 ± 0.097^a
SAG4	0.019 ± 0.002	0.020 ± 0.001	0.312 ± 0.071	0.727 ± 0.083^a