Construction And Protective Immune Research Of Multicomponent Eukaryotic Expression Vector Containing ROP16 And ROP18 Gene Of Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is one of the most common parasites in the world and belongs to the obligate apicomplexan intracellular protozoan.It can live in human and all kinds of vertebrates.It has been paid more attention because pregnant women show high rate of miscarriage,stillbirth,fetal anomalies,or other diseases.Infected immunodeficienct patients can develop encephalitis,retinitis,and even have potentially fatal risk.T.gondii rhoptry proteins(TgROPs)is a key factor in the invasion and virulence of toxoplasma,which play an important role in the process of parasite's invasion and reproduction.It is also used for drug targets and vaccine candidate components for treatment and prevention of toxoplasmosis.In this study,the multicomponent eukaryotic expression plasmid containing ROP16 and ROP18 genes of T.gondii is constructed by the means of molecular biology,and transfected it into Hela cells to verify its expression in eukaryotic cells.After immunization of BALB / c mice with the recombinant plasmid,the immunoprotective effect of the animals is evaluated by measuring the relative immune parameters of the mice.Firstly,the total RNA was extracted from tachyzoites of T.gondii.The primers were designed according to the open reading frames of TgROP16 and TgROP18 genes,ROP16 and ROP18 genes are amplified by Reverse Transcription PCR(RT-PCR)and connected into the eukaryotic expression vector pVAX1 to constructed the recombinant plasmid pVAX1-ROP16 and pVAX1-ROP18.The recombinant plasmid was transferred into E.coli XL1-Blue.The extracted plasmid of positive clones was confirmed by PCR,double restriction enzyme digestion and sequencing.Results shows thatThe RT-PCR products are consistent with the expected sizes,The recombinant plasmid was confirmed by PCR and double restriction enzyme digestion.Sequencing results showed that the TgROP16 gene was 2124 bp and the TgROP18 gene was 1665 bp,with high sequencing consistency(99.8% and 100%)with the TgROP16 gene and TgROP18 gene from GenBank.The results proved that the T.gondii ROP16 gene and ROP18 gene have been successfully cloned.Then,the vectors pVAXA and pVAXB were constructed via modification of the multiple cloning site of vector pVAX1.The PCR of expression unit fragment-PCMV-MCS-BGHpolyA-from vector pVAXA was performed,and product was inserted into vector pVAXB to construct dual-promoter eukaryotic vector pVAXD.Recombinant plasmid pVAX1-ROP16 and pVAX1-ROP18 were digested respectively and ROP16 and ROP18 gene were cloned into the multiple cloning site of the dual-promoter eukaryotic vector pVAXD to construct the recombinant expression vector pVAXD-ROP16-ROP18.After enzyme digesting and PCR amplification,the correct recombinant plasmid and the control group plasmids were transfected into Hela cells.Each gene expression was determined by way of RT-PCR and indirect immunofluorescence.Results shows that the recombinant plasmid pVAXD-ROP16-ROP18 was confirmed by PCR and double restriction enzyme digestion.After transfection,RT-PCR test showed that experimental group and control group beta-actin gene amplification products were consistent with the expected size,the experimental group ROP16 gene,ROP18 gene amplification products were consistent with the expected size,but the control group were not able to amplify ROP16 and ROP18 genes.Indirect immunofluorescence test showed that cells of recombinant plasmid pVAXD-ROP16,pVAXD-ROP18 transfected group can be observed green fluorescence,the empty plasmid transfected group and the control group without green fluorescence.The results demonstrated that the recombinantplasmid pVAXD-ROP16-ROP18 was constructed successfully and it can express ROP16 gene and ROP18 gene successfully in eukaryotic cells.Finally,recombinant expression vector were prepared by alkaline lysis method,the BALB/c mice were immune by recombinant plasmid pVAXD-ROP16-ROP18,pVAXD-ROP18 mixed plasmid,pVAXD-ROP16,pVAXD-ROP18,pVAXD and physiological saline through intramuscular injection.All groups were immune 3 times,at intervals of two weeks.Serum of mice in each group were collected before 1 day before immunization and 2 weeks after last immunization.The level of specific antibody and cytokines in the serum were determined.Each group of mice was challenged with tachyzoites of T.gondii by intraperitoneal injection after immunization for two weeks,and the survival of mice was evaluated.The results showed that recombinant expression vector immunized groups can induce specific anti-T.gondii IgG antibody in mice,and IgG levels rise with the increase of the number of immune,The multicomponent gene vaccine group and hybrid genetic vaccine group have higher levels of antibody,cytokines and survival time compared with other groups.It showed that T.gondii ROP16-ROP18 multicomponent gene expression vector can induce stronger immune response in animal and provides an experimental basis for further research on the development of T.gondii multicomponent gene vaccine.