Preparation Of Three Nucleic Vaccines And Three Subunit Vaccines Of Cryptosporidium Mouse Genotype And Their Immunoprotective Study

Cryptosporidiosis which caused by the world-wide protozoan parasite, Cryptosporidium spp., is a zoonosis with clinical feature of persistent diarrhea. Cryptosporidiosis has a wide range of host types. The pathogen can grow in digestive tract and respiratory passage of human being and more than 170 kinds of animals, including mammals, birds, reptiles, amphibians and fish. Cryptosporidiosis has a severe economic impact on husbandry and threatens the lives of the immunocompromised individuals. Currently, research of cryptosporidiosis has become one of global hot spots. Untill now there is no specific medicines to cure the disease, so the immune prevention research of cryptosporidiosis becomes increasingly important.Three major protective antigen gene of Cryptosporidium mouse genotype, sporozoite surface protein P23, CP15/60 and the oocyst wall protein CP41 were selected in this study. The nucleic vaccines were prepared by cloning three genes into the eukaryotic expression vector pVAX1 using recombinant DNA technology. BALB/c mice were immunized with the three prepared nucleic vaccines of Cryptosporidium mouse genotype, the level of IgG induced by vaccines and the numbers of oocyst produce were detected. The results showed that the titers of specific IgG antibody in the serum of immuned mice increased accompany with immunization times, and presented a gradual increase trend. The titers level reached the highest after the third immunization and was extremly higher than the control group(P <0.01), while there was no significantly different among the experimental groups and control groups (P> 0.05), respectivly. The number of oocysts discharged in experimental group were significantly lower than the saline control and pVAX1 control groups, respectively, while there was no significantly different among the experimental groups and control groups(P> 0.05), respectively. The oocysts reduction rates are 85%~100% in experimental groups. The time of begining to discharge Cryptosporidium oocysts of mice in the experimental groups was delayed and the duration time became shorten compared with saline, pVAX1 control groups, respectively. The results showed that the mice could be induced to produce highly specific immunization response after immunizing with the three nucleic vaccines, and partly prevent the infection of Cryptosporidium mouse genotype.The recombinant plasmid pGEX-4T-1-P23, pGEX-5x-3-CP41, pET-28b(+)-CP15/60 were transformed into Escherichia coli BL21(DE3) and induced by IPTG. Western blot analysis showed that the expressed three recombinant proteins had good immunogenicity. They all could be recognized specifically by the sesa from mice infected with Cryptosporidium mouse genotype. Subunit vaccines were prepared by emulsifying the three recombinant proteins with Freund's adjuvant, and BALB / c mice were immunized 3 times with the three prepared subunit vaccines. The level of IgG induced by subunit vaccines and the numbers of oocyst production of the infected mice were detected. The result of indirect ELISA showed that good humoral immune responses of mice were induced by the recombinant protein rP23, rCP41, rCP15/60, respectively. The titers of specific IgG antibody in the serum of immunized mice increased followed with immunization times, presenting a gradual increase trend. After three times immunization the titers level reached the highest. The IgG titer levels of the experimental group was extremly higher than the control group(P <0.01), respectively. The number of oocysts discharged in experimental group were significantly lower than control group (P <0.05), including PBS, adjuvant, rGST(4T)and rGST(5x)control groups, but there was no significantly different among the experimental groups and control groups(P> 0.05), respectively. The oocysts reduction rate are 73%~80% in experimental groups. The time of begining to discharge Cryptosporidium oocysts of mice in the experimental groups was delayed and the duration time became shorten compared with PBS, adjuvant, rGST(4T)and rGST(5x)control groups, respectively. The results showed that the mice could be induced highly specific immunization response after immunizing with the three subunit vaccines, and partly prevent the infection of Cryptosporidium mouse genotype.This study does a primary observation about the immune prevention of cryptosporidiosis. The prepared three nucleic vaccines and three subunit vaccines are useful supplement to the vaccine research of the disease. The study provides the basis for further research and development of higher protective vaccines of cryptosporidiosis.