Abstract:

Objective A polyclonal antibody against Toxoplasma gondii profilin (TgPRF) was prepared in two rabbits and its inhibitory effect on the proliferation of T. gondii in mouse embryonic fibroblasts was observed. Methods The total RNA was extracted from T. gondii RH strain and total cDNA was reversely transcribed. The cDNA encoding for TgPRF gene was amplified from the total cDNA and then cloned into prokaryotic expression vector pET-30a(+). The recombinant pET-30a(+)-TgPRF plasmid was transformed into Escherichia coli BL21 (DE3) and the recombinant TgPRF protein was expressed under IPTG induction. The expressed recombinant protein with His-tag was purified by affinity chromatography with AKTA system and the purified recombinant TgPRF was analyzed by SDS-PAGE and Western blotting. Two New Zealand rabbits were each immunized with 1 mg recombinant TgPRF emulsified with the same volume of Freund’s complete adjuvant by multiple subcutaneous injections on the back, then boosted three times with the same amount of protein formulated with incomplete adjuvant with two weeks interval. Blood was collected from rabbit heart 10 days after the last immunization. The anti-TgPRF specific antibody in the post-immune serum was identified by Western blotting and titrated by ELISA. Mouse embryonic fibroblasts were seeded in 96-well plates (4 × 10³ cells/well) and then infected with 10⁴ T. gondii tachyzoites (RH-GFP). The inhibition of T. gondii tachyzoites in infected embryonic fibroblasts by anti-TgPRF serum was observed when anti-TgPRF serum was added at 1 : 20 (B1), 1 : 80 （B2） and 1 : 200 （B3） dilutions. The pre-immune serum at 1 : 20 was added as negative control (C) and blank medium was used as blank control(A). Each sample was added in triplicate and cultured for 72 h, then observed under fluorescence microscope. Proliferation of tachyzoites was analyzed by Image pro 6.0 software. Results The cDNA encoding for TgPRF was amplified from T. gondii total cDNA by PCR with size of 510 bp, and then subcloned into pET-30a(+) for expression in E. coli. The cloned recombinant pET-30a-TgPRF plasmid contained the correct target sequence confirmed by DNA sequencing. The recombinant TgPRF protein was successfully expressed in E. coli BL21 as size of 25 000 mainly in soluble supernatant fraction. After being purified and concentrated, the concentration of recombinant TgPRF protein was determined by BCA as 4 mg/ml. The expressed recombinant TgPRF was used to immunize two rabbits to obtain antiserum and the recombinant TgPRF was recognized by the rabbit anti-TgPRF serum determined by Western blotting analysis. The rabbit anti-TgPRF serum had no toxic effect on embryonic fibroblasts determined by CCK-8 assay. The anti-TgPRF antibody titer was > 1 : 10⁵ determined by ELISA. After being incubated with anti-TgPRF for 24 h, the T. gondii tachyzoites proliferation was significantly inhibited in group incubated with 1 : 20 antiserum (the relative parasite amount 0.35 ± 0.16) compared with blank control (0.62 ± 0.23) and pre-immune serum control (0.74 ± 0.25) (P < 0.05), however, there was no significant difference among groups incubated with antiserum of 1 : 80, 1 : 200 and blank control and pre-immune serum control (P > 0.05). The tachyzoite proliferation inhibitory rates in group B1-B3 were 43.5%, 11.4%, 3.6% respectively. After being incubated with antiserum for 48 h, the relative tachyzoites amount in group incubated with antiserum of 1 : 20, 1 : 80 and 1 : 200 were 1.09 ± 0.58, 1.92 ± 0.73 and 2.47 ± 0.84, respectively, that were significant lower than blank control group (3.61 ± 0.66) and pre-immune serum group (3.38 ± 0.78) (P < 0.01) with inhibitory rates of 69.9%, 46.9%, and 31.7%, respectively. After being incubated with antiserum for 72 h, the tachyzoites proliferation was significantly inhibited in groups with antiserum at 1 : 20 (5.58 ± 2.43), 1 : 80 (8.06 ± 2.66) (P < 0.01) and 1 : 200 (16.02 ± 6.46) compared with blank control (19.90 ± 3.92) and pre-immune serum control (20.61 ± 4.07), with inhibitory rates of 72.0%, 59.5%, and 19.5%, respectively. Conclusion The E. coli expressed recombinant TgPRF protein was immunogenic and induced strong antibody response in immunized rabbits. The rabbit anti-TgPRF serum could effectively inhibit the proliferation of T. gondii tachyzoites in mouse embryonic fibroblast cells at a dose-dependent pattern in vitro, but could not completely eliminate the parasite.

Key words: Toxoplasma gondii, Profilin, Polyclonal antibody, Prokaryotic expression