Cloning And Expression Of Toxoplasma Gondii CDPK1-EF And The Research Of Its Immunological Features

Toxoplasmosis is a zoonotic protozoal disease by parasiting in humans and animals cells. With wide popularation in the word, it causes great economic losses to animal husbandry as well as poses serious threats to human health. However, chemical treatment is one of the main measure to contol it currently, but its side effects were obvious, such as drug resistance, residues. Therefore, developing the safe and effective vaccine to prevent it become the tendency. The gene of CDPKs is one kind of calcium dependent calmodulin independent protein kinase, which plays the important reole in invasion, egress, gamete formation transition and other key physiological activities to Toxoplsam gondii. The current data on it was little, especially its protection against T. gondii was not reported. Based on the facts, the gene clone of CDPKs and its immunity was determined to explore if it can become the candidate antigen to resist T. gondii.Firstly, RT-PCR was applied to amplify the target gene, then the obtained gene was sequenced; the gene was cloned into the vector of pET-32a to construct the recombinant plasmid, after identification, the expression was made by IPTG induction, the expressed protein was purified by Ni-NTA affinity chromatography.Secondly, the purified protein, mixed with Freund’s complete adjuvant, was administration by hypodermic inoculation; in the first immunization, every 15 days, the second and third immunization was made by mixing with Freund’s incomplete adjuvant, the dosage was 0.1 mg every nice, after immunized, blood serum were obtained at 7d, Western-blot and indirect ELISA was apply to detection the induced antibody and its level, and the,mRNA of IFN-y, IL-4, TLR4 from spleen tissue were quantified by fluorescence quantitative PCR technology. Protective efficacy of recombinant protein were also tested by compared the survival time after infected mice.The experimental results showed that:（1）The CDPK1-EF gene sequence alignment of T. gondii strains are highly conservative, in which the homology between RH strain and ME（XM₀02371625.1） strain was 100%. However, the difference existed between T.gondii RH strain and Eimeria tenella（FJ873128.1）,only was 70.0%; the divergence among T.gondii, Plasmodium and Cryptosporidium in their amino acid sequence was notable, in which RH strain has three varied amino acid in the last EF motif region compared with TgCDPK4. （2）The constructed pET32a-TgCDPK1-EF was able to be expressed as soluble protein in E.coli Rosseta steadly. （3）After immunized, the specific antibody could be induced, and the average titer of antibody is 1:14933. The cytokines of immunized mice with recombinant protein were up-regulation, in which IL-4 increased by 9.2 times and IFN-y increased 5.7 times respectively, at the transcriptional level, the difference was significant compared with that of the conrol （P<0.01）; after infected with T. gondii at 3d and 5d, IL-4 could increased by 11.5 times and 11 times respectively, meanwhile IFN-y increased by 5.7 times and 5.5 times respectively. And at the transcriptional level, the difference also was significant compared with that of the conrol （P < 0.01）.However, the change of TLR-4 mRNA was poor, there was no significant difference compared with control groups（P>0.05）.The immune protection test showed that the average survival time of mice immunized with recombinant protein was 12d, the average duration of symptom was 3d, the time for appearance of the first symptom was 8.8d. The average survival time was 7.4d to 7.8d, the average duration of symptom was 2,8d to 3.Id, the time for appearance of first symptom was 4.5d to 4.8d in the control. There was a great significant difference between recombinant protein group and the control group at the time for appearance of first symptom（P< 0.01）. Alike, there was significant difference between recombinant protein group at survival time and the other groups （P< 0.01）, and the difference between them in each group at duration of symptom were not significant （P> 0.05）.In conclusion, in this test, the gene of T. gondii RH strain CDPKl-EF motif sequence was amplified in vitro by RT-PCR technology. The recombinant protein of CDPK1-EF can be expressed steadly by IPTG induction. Furthermore, the protein has great immunogenicity and is able to induce mice produce highly specific antibody protective immunity. In addition, TgCDPKl-EF has potential application value in the development of vaccine against toxoplasma.