| Toxopasma gondii(T.gondii)is an extremely harmful opportunistic pathogen that can infect all warm-blooded animals,including humans.T.gondii is parasitized in a specialized vacuole(parasitophorous vacuole,PV)within the host nucleated cells.Dense granule antigens(GRAs),the important secretory metabolic antigens of T.gondii,play the key roles in the PV formation,access to nutrients and immune evasion.When T.gondii invades host cells,the dense granules continously release their content into the PV.GRA7 protein is an important member of the GRA family and is expressed at all developmental stages of T.gondii.This protein released by tachyzoites is mainly located on either PV or PV membrane formed after host cell invasion,whereas it released by bradyzoites was found to be located in the cytoplasm of the host cells.GRA7 protein can stimulate the host to produce humoral and cellular immunity.Therefore,this gene may be a candidate antigen gene for T.gondii vaccine.Although T.gondii is the only recognized species in the genus Toxoplasma,the isolates from animals vary worldwide in terms of their genotype and virulence.And the slight differences among genomes may be responsible for the different virulence and infectivity of T.gondii strains.China is an important center for pig farming.Recently,the epidemiological data on T.gondii in pigs mainly come from the serological tests of pig farms and the PCR detection of slaughter pig tissues.However,limited information is available concerning T.gondii infection in sick pigs.T.gondii strains used for analyses of biological characterazations are mainly RH strain or Chinese I strains isolated from cats.Therefore,the present study performed PCR detection of T.gondii in sick pigs from the veterinary hospital of Yangzhou University.GRA7 gene sequences of four strains,including two Chinese I genotype strains(YZ-1 and YZ-2)from dead pigs and two strains(RH and KS)from humans,were cloned and then were compared and analyzed,respectively.And GRA7 proteins of YZ-2 and RH strains were prokaryotically expressed,and afterwards,the immunoprotection and cross-immunity protection of the two recombinant proteins were also evaluated,respectively.This study was conducted to provide theoretical information for the clinical diagnosis of toxoplasmosis,the molecular mechanism of T.gondii virulence and the development of T.gondii vaccine.1.Molecular epidemiological investigation and genetic diversity of T.gondii in sick pigsA total of 295 tissues of 59 sick pigs collected from the veterinary hospital of Yangzhou University were detected by a PCR method targeting the 529-bp repetitive element of T.gondii.The results showed that the positive rate for T.gondii was 86.44%(51/59)in the sick pigs,in which the weaned pigs had the highest rate of infection(96.15%,25/26),followed by fattening pigs(86.36%,19/22),and the lowest in the suckling piglets(63.64%,7/11)that was significantly different from the infection rate of the weaned pigs(P<0.05).The infection rate of T.gondii was highest in spring(96.15%,25/26),followed by in summer(92.85%,13/14)and winter(75.00%,6/8),and lowest in autumn(66.64%,4/7)that was significantly different from that in spring(P<0.05).The total rate of T.gondii infection was 73.22%in the 295 tissue samples,in which the prevalence was significantly higher(P<0.05)in the lung and lymph nodes compared with the lowest prevalence of the spleen(61.02%,36/59).2.Cloning and sequence analysis of GRA7 genes of different T.gondii strainsThe primer was designed based on the sequence of GRA7 gene of T.gondii in GenBank.The whole genome DNA samples of tachyzoites of Chinese I genotype strains(YZ-1 and YZ-2)and type I strains(RH and KS)were extracted and used as the templates to clone the full-length sequences of GRA7 gene,respectively.And then the phylogenetic analysis was performed by combining the related sequences in GenBank.The results showed that the complete open reading frame of the GRA7 gene of the KS strain was 711 bp,which was completely identical to that of the RH standard strain;the GRA7 gene sequences of the YZ-1 and YZ-2 strains were 708 bp with a deletion of three bases at the positions 290?292 bp compared with the RH strain.In amino acid sequence and epitope analysis,compared to RH or KS strain,the deletions of one glutamic acid were respectively found in the GRA7 protein sequences of YZ-1 and YZ-2 strains,which resulted in the occurrence of another epitope.In addition,this study also suggested that the Chinese I genotype was more closely related to type ? strains.3.Cloning and prokaryotic expression of GRA7 gene of type I and Chinese I genotype strains of T.gondiiSpecific primers were designed based on the sequence characteristics of the expression vector pET-30a(+)and sequence of the target gene after removing the signal peptide.A PCR were performed with the recombinant plasmids RH-pGEM-T-Easy-GRA7 and YZ-2-pGEM-T-Easy-GRA7 used as the templates,respectively.Then,the amplified fragments(approximately 633 bp)were inserted into the vector pET-30a(+)to construct the recombinant expression vectors RH-pET30a(+)-GRA7 and YZ-2-pET30a(+)-GRA7,respectively.After that,the recombinant vector was transformed into Escherichia coli competent cells BL21(DE3)and the soluble proteins(RH-rGRA7,YZ-2-rGRA7)with a molecular weight of 37 kDa was induced to express.Western-blot assay showed that both RH-rGRA7 and YZ-2-rGRA7 proteins can be recognized by anti-6xHis-tagged mAbs and mouse anti-recombinant GRA7 protein polyclonal antibodies,and also by the sera of mice immunized with soluble proteins of tachyzoites.Furthermore,a murine anti-recombinant GRA7 protein polyclonal antibody were used to detect the soluble antigens of tachyzoites by western-blot,and an endogenous protein with a molecular weight of 29 kDa was found,namely the natural GRA7 protein.4.Evaluation of immune protective effects of the recombinant protein RH-rGRA7 and YZ-2-rGRA7BALB/c mice were used as the experimental animals.After immunization for three times using the recombinant GRA7 protein,mice were intraperitoneally inoculated with 100 tachyzoites of T.gondii.The levels of serum antibodies and cytokines(IFN-?,IL-4,IL-10 and IL-12)were detected,and the survival time of mice was simultaneously observed to evaluate the immunoprotective effects of the recombinant proteins RH-rGRA7 and YZ-2-rGRA7 and their cross-protective immunity.The results showed that compared with the controls,the both of recombinant proteins can significantly increase the levels of serum antibodies and IFN-? in the immunized mice,and the survival time of mice was significantly greater(P<0.05),and the greatest is the high-dose immunized group.Also,both recombinant proteins displayed a good cross-immunoprotective effects. |
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