Preparation And Immunoprotective Study Of Subunit And Nucleic Vaccine Of Cryptosporidium Parvum Rhoptry Protein Cgd8₂530 Sushi Domain Fragment

Apicomplexan parasites, such as Toxoplasma gondii, Cryptosporidium spp., Plasmodium spp., Babesia spp., Eimeria spp. etc., are obligate intracellular protozoa and important pathogens of humans and animals. They have conserved subcellular structures and invasion mechanism. Rhoptry proteins （ROPs）, which were released into the host cell, are considered to be the key molecule of invasion and replication of parasites in host cell.Cryptosporidium parvumcan infects the gastrointestinal epithelium to cause a self-limited diarrhea in competent hosts, but devastating in immune-suppressed individuals, such as AIDS patients. For now, there is no drug therapy has proven to be effective against this disease.Therefore, development ofvaccines is one of the important strategysto prevention and control of this disease.The 708 bp truncated fragments of cgd8₂530 genesharboring the sushi domainwhich logged into EuPathBD was successful amplified from genomicDNA of Cryptosporidium parvumoocystsby polymerase chain reactionPCR.The successful construction of the recombinant plasmid pET-sushi prokaryotic expression system after the amplification of sushi gene was subcloned into the prokaryotic expression vector pET-28a（+）.Subsquencely, High levels of expression are achieved in the pET vector system when were therecombinant plasmid transformed into E. coli BL21（DE3）. The purified recombinant proteins were immunized in New Zealand white rabbits to raise the corresponding polyclonalantibodies. Western blot analysis showed that the serum fromcattle infected with C. parvum and rabbit immuned with the recombinant protein could specifically bindingthe sushi protein, respectively.The results indicated that the recombinant protein had a good antigenicity and immunogenicity.The recombinant plasmids PVAX-sushi was contructed by cloning sushi genes into the eukaryotic expression vector PVAX 1,and immunize ICR mices compared with the immune effect of subunit vaccine. Model rats were randomly divided into 7 groups: healthy group, Blank group, TE group, PVAX 1 group, adjuvant group, PVAX-shushi group, recombinant protein group. Then, we observed the specific humoral and cellular immune responses and analyzed the protective effects of prevention.Compared with control groups, Many related immune indexes including serum specific antibody level, the numbers of CD4+and CD8+lymphocyte cells, lymphocyte proliferation, secretion of cytokines were all significantly improved in recombinant protein groups. The subunit vaccines could induce Thl and Th2 type cell immune responses. Infection experiments showed that the oocysts product in fecal were also significantly reduced by 89% in recombinant protein groups compared with the negative control. The results showed thatthe subunit vaccines could be induced to produce highly specific immune response and protects from the infection of Cryptosporidium in mices.Analyzed on immune system in DNA vaccine groups, many related immune indexes including the numbers of CD4+ and CD8+ lymphocyte cells, lymphocyte proliferation, secretion of cytokines were all significantly improved.The DNA vaccines could induce Thl and Th2 type cell immune responses. But ompared with subunit vaccine, the improvement of DNA vaccine is smaller than subunit vaccine. And the level of specific antibody IgG in seraand the oocysts product in fecal was not significant improvement compared to the other nonimmune groups. The results showed that the mice couldn’t be induced to produce highly specific immune response after immunizing with the DNA vaccines, and could be infected by Cryptosporidium.In this research, therecombinant protein vaccine and nucleic acid vaccineof C. parvum rhoptry protein cgd8₂530 sushi domain fragment, the immune effect of them were constructed, and detected the immune effect of them. The work was beneficial to the exploration of the multivalent vaccine of cryptosporidiosis andlaid the foundation of developing efficient prevention and control methods forthe disease.