High-level Expression Of A Recombinant Microneme Protein 3 Of Toxoplasma Gondii And Its Potential For Serodiagnosis

Toxoplasma gondii is a protozoan parasiting in cells of avian and mammal animals, causing congenital and acquired toxoplasmosis, which are harmful to human health and affect the development of animal husbandry.Microneme protein 3 (MIC3) of toxoplasma parasites is a secretary protein from their micronemes. To modify the serodiagnotic methods for the detection of toxoplasma infection in animals and analyse the function of MIC3 in the adhere and invasion of the parasite to host cells, a DNA fragment encoding mature MIC3 protein was amplified from genomic DNA of T. gondii by PCR. The PCR product was then inserted into an bacterial expression vector pGEX-4T-3 and transformed to Eschericlia coli (E.coli) DH5a strain, resulted in the recombinant plasmid pGEX-4T/MIC3. Sequencing analysis showed that the sequence of the amplified MIC3 corresponded with the sequence of the fragment of gene numbered AF509564 in GenBank, with the homology of 100%. E. coli with the recombinant plasmid was induced with isopropylthio-β-D-galactoside to express the recombinant MIC3 (rTgMIC3) as a fusion protein with glutathione S transferase. The recombinant protein was purified by using glutathione sepharose 4B. Sodium dodeoysulfate-Polyarylamide gel electrophoresis and Western blotting analysis were used to analyse the recombinant MIC3. The results showed that the molecular mass of rTgMIC3 was 61.2 kDa, consistent with the theoretical prediction; the sera from BALB/ C mice infected with T. gondii ME49 strain reacted with rTgMIC3. On the other hand, the sera from the mice immunized with the rTgMIC3 recognized the native MIC3. Indirect immunofluorecent antibody test showed that the native MIC3 located at the frontier end of the parasites. An enzyme-linked immunosorbent assay (ELISA) to detect specific antibody against toxoplasma infection was established by using rTgMIC3 as an antigen, The result in the detection of specific IgG antibodies in the mice infected with T. gondii ME49 strain by the ELISA demonstrated that the antibody produced on the seventh day, reached a peak in the third week, and maintained at a high level.ELISA with rTgMIC3 was used to detect specific IgG antibody in the sera of pigs and goats from slaughterhouse in Fuzhou. And the result was compared with that by using ELISA with recombinant truncated surface antigen 2. The results showed that the two ELISAs are consistent. The positive rates of the specific antibody to toxoplasma were 0.6% in swine and 2.2% in the goats from the slaughterhouse, respectively.To observe the possible role of MIC3 in adhere on and invasion to their host cells, toxoplasma parasites incubated with antibody against rTgMIC3 were used to infect the monolayer of vero cells, and then compared with those untreated parasites. The result showed that anti-rTgMIC3 treated parasites had no difference from those untreated parasites, which suggested that the antibody can not effectively prevent the invasion of T. gondii to host cell.In a conclusion, the results above suggested that a recombinant MIC3 without signal peptide was expressed successfully in E. coli at a high level, which is suitable for use in the modification of serodiagnostic methods. However, the antibody against rTgMIC3 could not inhibit the invasion of the parasite into vero cells. The mechanism of the parasite invasion to host cells remains to be studed.