Preparation And Immunoprotective Study Of Trivalent Subunit Vaccine And Nucleic Vaccine Of Cryptosporidium Parvum CP15-P23-CP15/60in BALB/c Mice

Cryptosporidium parvum is an obligate enteric intracellular protozoan parasite. It infects thegastrointestinal epithelium to cause a self-limited diarrhea in immunocompetent humans or animals butpotentially life threatening in those with compromised immune systems, such as AIDS patients. Atpresent, there is no effective therapeutic agent for the treatment of Cryptosporidium infection. Thus,there have been increasing efforts geared towards development of vaccines to control the disease.The T cell epitopes of the sporozoite surface antigen CP15, P23and CP15/60of Cryptosporidiumparvum were predicted with several antigen epitope prediction software and three immunodominantgene regions were selected. Complete sequences and selected sequences of the three genes wereconnected respectively by overlap extension PCR(SOE PCR) through a flexible linker (GGGGS)encoding gene inserted between each other. The fusion genes named CP15-P23-CP15/60and CpTmwere subcloned into the prokaryotic expression vector pET28a(+). Then the recombinant plasmidpET-CP15-23-60and pET-CpTm were successfully constructed. Subsquencely, the two recombinantplasmids were transformed into E. coli BL21(DE3) for expressing recombinant proteins. The purifiedrecombinant fusion proteins were immunized in BALB/c mice to raise the corresponding polyclonalantibodies. The results showed that the two fusion genes were expressed efficiently in E. coli. Westernblot analysis indicated that the fusion proteins could be specifically recognized by serum from cattleinfected with C. parvum, rabbit immuned with recombinant CP15, P23, CP15/60protein and miceimmuned with the recombinant protein, respectively. The results indicated that the recombinant proteinhad good reactogenicity and immunogenicity.Two subunit vaccines were prepared with recombinant expressed protein rCP15-P23-CP15/60andrCpTm and compared with the vaccines prepared with recombinant expressed proteins rCP15、rP23、rCP15/60. The vaccines were immunized to BALB/c mice and the specific humoral and cellularimmune responses induced by the subunit vaccines were analyzed and the protective effects ofprevention Cryptosporidium infection were observed. The results showed that compared with controlgroups, a series of immune index including the level of specific antibody IgG in sera, the numbers ofCD4~+and CD8~+lymphocyte cells, lymphocyte proliferation, secretion of cytokines IFN-γ and IL-4wereall significantly improved in experimental groups. The five genetic engineer subunit vaccines couldinduce Th1and Th2type cell immune responses. Infection experiments showed that the peaks ofdischarge oocysts were delayed3days and the oocysts product in fecal were also significantly reducedby37.43%~55.06%in experimental groups compared with the negative control, and the reduction rateof the group of rCP15-P23-CP15/60reached to55.06%. The duration time of discharge oocysts becameshorten in the experimental groups of rCP15-P23-CP15/60and rCpTm. The results showed that themice could be induced to produce highly specific immune response after immunizing with the twosubunit vaccines, and partly prevent the infection of Cryptosporidium. The protective effects of the twotrivalent subunit vaccines were better than the three monovalent vaccines.Two eukaryotic expression plasmids, pVAX-CP15-P23-CP15/60and pVAX-CpTm, were also constructed. They were immunized to BALB/c mice comparing with recombinant plasmid pVAX-CP15、pVAX-P23、pVAX-CP15/60. The specific humoral and cellular immune responses induced by the DNAvaccines were detected and the protective effects of prevention Cryptosporidium infection wereobserved. The results showed that there are an evident increase in the level of immune indexes such asthe level of specific antibody IgG in sera, the numbers of CD4~+and CD8~+lymphocyte cells, lymphocyteproliferation, secretion of cytokines (IFN-γ and IL-4) in experimental groups than those of controlgroups. Infection experiments showed that the peaks of discharge oocysts were delayed3days and theoocysts shedding rate is also significantly reduced by35.6%~50.25%in experimental groupscompared with the negative control, and the reduction rate of the group of pVAX-CP15-P23-CP15/60reached to50.25%. The duration time of discharge oocysts became shorten in the experimental groupsof pVAX-CP15-P23-CP15/60and pVAX-CpTm. The results showed that the mice could be induced toproduce highly specific immune response after immunizing with the two DNA vaccines, and partlyprevent the infection of Cryptosporidium. The protective effects of the two trivalent DNA vaccines werebetter than the three monovalent vaccines.In this research, two fusion genes, CP15-P23-CP15/60and CpTm, were constructed. The immuneeffect of subunit vaccines and DNA vaccines produced from the two fusion genes were detected. Thework was helpful to develop the multivalent vaccine of cryptosporidiosis and laid the foundation ofdevelopment efficient prevention vaccines of the disease.