[Study on immuno-effect with GRA4 or SAG2 gene recombinant BCG vaccine of Toxoplasma gondii].

OBJECTIVE: To compare the immuno-protection induced by the recombinant BCG vaccine of Toxoplasma gondii GRA4 gene (rBCG-GRA4) and SAG2 gene (rBCG-SAG2) in BALB/c mice. METHODS: 108 SPF BALB/c mice were divided into 6 groups: PBS, BCG, rBCG, rBCG-GRA4, rBCG-SAG2 and rBCG-GRA4+SAG2, each with 18 mice. Each mouse was injected by 100 microl corresponding materials for 2 times. Blood was taken from tail vein before inoculation. 4,6 and 8 weeks after inoculation, spleen was moved and blood was taken from orbit vein of 3 mice from each group for the detection of cytokines, IgG and IgM antibodies, T lymphocyte subgroups and transformation efficiency. 3 weeks after the last inoculation, 9 mice from each group were challenged intraperitoneally with 50 tachyzoites of T. gondii RH strain and their survival time was observed. RESULTS: rBCG vaccine of T. gondii induced immune response. The value of CD3+ CD4+/CD3+CD8+ of group BCG-GRA4+SAG2 was the highest (14.06+/-1.17) in the 4th week; the IgG titer in the BCG-GRA4+SAG2 group was the highest (0.18+/-0.02) in the 6th week and the IgM titer in the BCG-SAG2 group was the highest (0.82+/-0.05) in the 8th week. The average survival time of the mice in BCG-SAG2 group was about 8.61 days after challenged with tachyzoites, and that of the PBS control group, 7.33 days. The average survival time in the 3 immunized groups was one day longer than that of the control. CONCLUSION: The rBCG vaccine of T. gondii shows certain immuno-protection in mice.

[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].

OBJECTIVE: To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. METHODS: The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. RESULTS: The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. CONCLUSION: The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.

[Expression and purification of Toxoplasma gondii GRA4 gene in prokaryotic system].

OBJECTIVE: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity. METHODS: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. RESULTS: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. CONCLUSION: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

[High efficiency expression and antigenicity analysis of the SAG2 gene from Toxoplasma gondii RH strain].

OBJECTIVE: To make high efficiency expression of the SAG2 gene from Toxoplasma gondii RH strain in E. coli and study the antigenicity of the expressed product. METHODS: The SAG2 gene fragment of T. gondii RH strain amplified by PCR method from genome DNA was cloned into the pMD-18T vector and transformed into E. coli DH5alpha. After nucleotide sequencing, the SAG2 gene fragment was subcloned into the expression vector pET23a with correct orientation and transformed into E. coli DH5alpha. The plasmid from the correct clone identified by PCR method and endonuclease digestion was transformed into E. coli BL21 (DE3) and induced for expression. The expressed product was studied by SDS-PAGE and Western blot. RESULTS: 502 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100% homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about Mr 19,000. Western blotting indicated that the antigenicity of the protein was specific. CONCLUSION: The plasmid pET23a-SAG2 was constructed and a high efficiency expression of the SAG2 gene from T. gondii RH strain was made. The expressed product shows a specific antigenicity.