Research On CP2/P30 Bivalent DNA Vaccine And Subunit Vaccine Against Cryptosporidium

Cryptosporidium is a kind of protozoa parasitic diseases which can infect people and animal. Cause many mammals and people diarrhea and cause diarrhea of a variety of mammals. There is no effective therapy. Today, many scholars believe that the vaccine is one of the ways more suitable can control of cryptosporidiosis.National scholars and researchers so far have been studied a variety of Cryptosporidium antigen genes for vaccine candidate genes. And found that certain protection, but the protective effect is not satisfactory. In this experiment, CP2 genes are found in international in 2004, This gene was associated with the invasion that initially identified by the university of Iowa in U.S.A. South Korea's Konkuk College of Medicine using real-time quantitative PCR of the CP2 gene whether it is viable Cryptosporidium parvum has been studied sign in 2008.Determine the minimum detectable number of CP2 gene is 10 sporulated oocysts. CP2 gene was identified as the most sensitive, reliable and accurate candidate genes. The trial of another gene P30 is a Newly identified gene in 2007. Currently the two genes have not done the DNA vaccine in international. Studied of these two genes are relatively small. Use the two genes and tandem cloned into the eukaryotic and prokaryotic expression vector,and construct the DNA vaccine and subunit vaccine. Use the two genes and two series of recombinant protein and gene eukaryotic expression vector were separate immunized animals, Both can induce a specific immune response, have some protective effect.Clone the CP2 and P30 genes,construct the eukaryotic expression vector. According to reported of CP2, P30 gene in Genebank designed primers and extraction of total Cryptosporidium parvum genome. The two genes open reading frame were amplified by PCR. The original sequence by Blast alignment homologies were 99.91% and 99.41%. The purified fragment was connected to the pMD18-T vector plasmid and double digestion analysis and sequenceing.Construct prokaryotic expression vector and expression. Cloned the two genes and two genes tandem into eukaryotic expression vector pVAX-1. Construction of reorganization eukaryotic expression vector Use the recombinant plasmid transiently transfected into Hela cells and using indirect immunofluorescence method to detect specific proteins in the transfected cells. The expressed of protein was Confirmed by SDS-PAGE and protein reactogenicity confirmed from Western blot. Construct prokaryotic expression vector and expression. Cloned the two genes and tandem genes into pET-28a (+) expression vector with T4 ligase and then transformed into Rosetta (DE3). Positive bacteria induced with IPTG and results identified by SDS-PAGE, induced 33.9 KDa,79.7 KDa and 122 KDa fusion protein. The recombinant protein with a final concentration of lmM of IPTG can induce 5h of expression available to the largest amount of protein at 37℃. The protein was expressed in soluble form and good immunoreactivity confirmed from Western blot..The immune protection experiments of use DNA vaccine against Cryptosporidium parvum. Use the recombinant plasmid immune BALB/c mice and pregnant goats,and detect the dynamic changes of humoral and cellular immunity. The results was that mice and goat immunized with recombinant plasmid,CD4+and CD8 + and immune parameters such as specific antibody titers were higher. The significantly different by compared with the control group(P<0.01). CD4+/CD8+ ratio was not significant among the groups (P> 0.05). After oocysts attack test shows that the test group mice and goats offspring Cryptosporidium parvum oocysts excretion decreased than in the control group. oocyst discharge time is reduced 2 days. Result was not significant compare With the vector control group (P> 0.05). The experimental results in mice and goats display that the DNA vaccine has some immune protection.The immune protection experiment of CP2, P30 and the two gene tandem recombinant antigen against the Cryptosporidium parvum. Use the recombinant protein immune of mice and pregnant goats three times by intraperitoneal immunization and each time interval of two weeks. After three weeks the test to avoid the humoral and cellular immunity and humoral immunity in goats measured the level of offspring. Detection of humoral and cellular immunity and humoral immunity of goat offspring. The results showed that the test group the number of specific antibody titer increased gradually with the increased immune than the control group, and with the significant difference (P<0.01).,CD4+and CD8+and immune parameters such as specific antibody titers were higher. The significantly different by compared with the control group(P<0.01). CD4+/CD8+ratio was not significant among the groups (P> 0.05). After oocysts attack test shows that the test group mice and goats offspring Cryptosporidium parvum oocysts excretion decreased than in the control group. oocyst discharge time is reduced. The experimental results in mice and goats display that the subunit vaccine has a certain immune protection.