Expression Of Toxoplasma Gondii Gene ROP2 In E.coli And BCG And Its Immunogenicity On Mice

Toxoplasma gondii is an obligate intracellular parasite that infects awide range of hosts, including humans and domesticated animalsthroughout the world. and it has been estimated that one-third of thehuman population is infected. Infection of Immuno-competent humans isusually asymptomatic, with clinical disease largely confined to riskgroups. Primary infection during pregnancy can result in severe neonatalmalformations and ocular complications in the fetus. Since the emergenceof AIDS, toxoplasmosis in the immunocompromised host is of greatsignificance, as the recrudescence of a latent infection often results in afatal encephalitis. In addition,toxoplasmosis can cause considerableeconomic loss in the farming industry. Thus, the development of aneffective vaccine against T. gondii would be of great value to both humanand veterinary medicine.,due to ROP2 antigen is secreted by the rhoptryof tachyzoites and bradyzoites.They are highly conservative andimmunogenic,were selected as antigens for diagnosis and vaccination totoxoplasmosis.Mycobacterium bovis BCG, an attenuated mycobacterial strainadministered to protect against tuberculosis and leprosy,is a particularlyattractive vector for the delivery of heterologous antigens. It have severaladvantages:1. low toxicity.2. one dose would be sufficient to conferlong-lasting cellular immunity; 3. the World Health Organizationrecommends that BCG be administered at birth;4. it is the best-knownadjuvant in animals and humans;5. it has low production costs.6.it isthermostable. Stover et al. developed shuttle plasmids that are capable ofautonomous replication in Escherichia coli and BCG. Since then, manyinvestigators have constructed rBCG and expressed a variety ofparasitic,bacterial or virus genes as a vaccine candidate against multiplepathogens,and have examined its use as a live vaccine. in some instances,induce protective immune responses in the mouse model.Genetic systems for the manipulation and expression of heterologousgenes in rBCG have mostly been developed from pAL5000, aMycobacterium fortuitum cryptic plasmid . As five copies are present pertransformed cell, the expression levels of heterologous genes are high inthese rBCG strains and they generally induce good levels of immuneresponses against heterologous antigens. However, some genes might beless stably maintained in rBCG, especially in the hostile macrophageenvironment. This could be detrimental to the obtainment of long-termimmune memory .for the obtainment of long-term immune protection,integration-proficient vector have been developed, Integrative vectors werederived from temperate mycobacterio-phages, such as L5 or Ms6 , Theseintegration-proficient vectors encode integrase functions that allow arecombination event between the phage attP and the bacterial homologousattB sites. Such vectors integrate site specifically into the bacterialchromosome, and when no excision functions are present, the vector can bestably maintained . Integrative rBCG strains have been obtained thanks tosuch vectors and have induced immune responses in mice . However, thedisadvantage of such strains is the reduced expression level of heterologousgenes compared to that of multicopy plasmids, which can becounterbalanced by the persistent synthesis of the foreign antigen in vivo,To enhance the protective efficacy of induction of specific immunity. Thiswork was initiated to see whether the rBCG system is applicable for theprevention of toxoplasmosis in the animal model. The following workshave been done.l The expression of ROP2 in E.coli, and the analysis of immunoreactiv-ity of rROP2 protein. The ROP2 fragment having no introns wasamplified by polymerase chain reaction(PCR) from the genomic DNA ofthe Toxoplasma gondii RH strain, and was cloned into plasmid pMD18-TVector, subsequently it was identified by PCR and restriction enzymecleavage. the BamHI/HindIII digested fragment of pMD18T-ROP2 wasligated to expression vector pET30a,. referred as pET30a-ROP2,it wastransformed into E.coli strain BL21(DE3), which was induced by of IPTG,the fusion protein of ROP2 protein was expressed in the form of inclusionbody , it has been analyzed by SDS-PAGE. the fusion protein of ROP2 waspurifyed, renaturated by glutathione reoxidation to result in a reasonablerenaturation, the immunoreactivity was analyzed by the ELISA assay.2 Construction of the E.Coli-BCG Shuttle Expression vector pMV262-ROP2, using Polymerase chain reactions (PCR) to amplify ROP2 genefragment, The amplified DNA was cloned into plasmid pMD18-T Vector,and digested with EcoRI and HindIII, The plasmid pMV262 was digestedwith the same enzyme. The plasmid pMV262-ROP2 was constructed byligation of this two DNA fragments,it was transferred into E. Coli,thetransforments was screened and identified by PCR and restraction analysis.Subsequently transformed into BCG by electroporation, Recombinant BCGwas grown to mid-log phases in Middlbrook 7H9 broth supplemented with20μg/mL kanamycin at 37°C and then rapidly shifted to 45°C. Afterincubation for 2h, cells were harvested, The cell lysates were analyzed bySDS-PAGE and Western blotting. the ROP2 recombined protein wasdemonstrated to has been expressed.3 Construction of E.coli-BCG integration-proficient vectors pOIP23H-ROP2, and Expression and Identification of a Recombinant BCGVaccine encoding ROP2. due to the integration-proficient vectorspOIP23H has no promoter, pMV262-ROP2 was digested with DraI andHindIII to produce the Phsp60-ROP2 fragment, blunted by treatment withT4 DNA polymerase, was ligated to pOIP23H which was digested by SpeI,phosphatase-dephosphorylated by calf intestine alkaline, the plasmidpOIP23H-ROP2 was constructed, it was transferred into BCG byelectroporation, rBCG,was grown to mid-log phases in 7H9 brothsupplemented with 20μg/mL hygromacin at 37°C and then rapidly shiftedto 45°C. After incubation for 2h,ROP2 was showed to has been expressedby SDS-PAGE and Western blotting hybridization.4 The evaluation of protective efficacy of rBCG against toxoplasmosis.BALB/c mice (6 to 8 weeks old) was vaccinated intravenously. or orallywith rBCG: pMV262-ROP2, rBCG: pOIP23H-ROP2, BCG, PBS-Tween80respectively, the intravenous immunizing dosage is 1×106cfu/time,the oralimmunizing dosage was 1×108cfu/time, Fourteen days after the third...