| Cryptosporidium is an important parasite which parasite on the respiratory and digestive tract epithelial cells of human and spinal animals. Cryptosporidiosis is caused by it in the clinical manifestations of severe watery diarrhea disease. In 1907, Tyzzer first reported the case that mice infected with Cryptosporidium. Until 1982, the U.S. Centers for Disease Control reported that the 21 male AIDS patients in six U.S. cities caused the severe diarrhea with cryptosporidiosis. Cryptosporidium was researched over the world from then on. Cryptosporidiosis has been listed as one of the United States six diarrheal disease. In 2003, Cryptosporidiosis was included in the " prevention and application of emerging infectious diseases" that was major scientific and technological projects by China's Ministry of Science, and was one of two parasitic diseases. Cryptosporidium species have been discovered more than 20 strains so far, but most scientists believe that only 19 species are valid species. Cryptosporidium andersoni is one of them. In 2000, it was named as Cryptosporidium andersoni. The adult cattle was mainly infected with Cryptosporidium andersoni. The cattle infected with C. andersoni can cause milk production decreased, weight loss, and it is affected the development of dairy industry. In recent years, C. andersoni was identified by Leoni and Morse in the human body. Although cryptosporidiosis has been studied for many years at home and abroad, but there is no effective preventive measures and drugs. More than 200 kinds of drugs were selected, including all of the antibiotics, sulfa drugs and anticoccidial drugs for the disease, but no drug has effect for Cryptosporidium. So immunization is the main way to control the disease, and the key is to screen effective anti-Cryptosporidium vaccine candidate antigens.Cryptosporidium andersoni is a parasitic protozoa. There was report that it infected human. At present, the reports of C. andersoni has focused on epidemiological surveys. There are no report about Cryptosporidium andersoni vaccine candidated gene screening and cross-protection studies. Cryptosporidiosis is related with the host immune system. So antibodies and vaccines will play an important role for treatment and prevention of the disease. Received Cryptosporidium andersoni vaccine candidate genes and cross-protection study will provide a new research directions of the prevention and treatment of cryptosporidiosis. C. andersoni vaccine candidate genes is important problems to be solved. The method of screening immune-related genes through immune protein gene library is a common technical methods. The phage display technology is an new biotechnology of antigen gene screened. This technique has been widely used in the field of life science research. There is no reported that T7 phage display library screening is used for screening immune-related genes in C. andersoni. In 2003, Andre successfully proved that Human and bovine epithelial cells were infected by Cryptosporidium andersoni in vitro. It provides a theoretical basis for screening of Cryptosporidium andersoni immune-related protein gene in vitro.To screen of C. andersoni immune protein gene and the new vaccine candidate antigen genes, T7 phage display library of Cryptosporidium andersoni has been constructed in this study. Immune-related protein gene was screened from the library using calf intestinal epithelial cells. two genes from the library were used in prokaryotic and eukaryotic expression system. Then the mice were immuned by the recombinant proteins. The level of humoral and cellular immune was detected and then the mice attacked Cryptosporidium parvum to verify the recombinant subunit vaccine and DNA vaccine cross-protection.The results showed that Cryptosporidium andersoni T7 phage display library was successful constructed. The titer of the amplified library was 1.2×108 pfu / mL. The library capacity was 2.4×1010 recons. PCR identification on 50 randomly selected plaques was conducted, the library recombination rate was 90%. The 80% insert segments were greater than 0.3kb. The amplified library was screened and at the fifth round of screening. 17 new immune-related protein gene of Cryptosporidium andersoni were identified. two of these genes named C.andersoni 2 (CA2) and 42 (CA42). They were analysised in NCBI. CA2 was Cryptosporidium andersoni unknown proteins, CA42 was Cryptosporidium andersoni actin.After the amplification of CA2 and CA42, recombinant prokaryotic expression plasmid pET-28a-CA2 and pET28a-CA42 were constructed. The recombinant prokaryotic expression plasmids were transformed into competent E. coli DE3. The expression was induced by IPTG. Western Blotting show that the size of the recombinant protein are about 15ku and 19ku and the protein can be identified by polyclonal antibody from immunized mice of C. andersoni. Then the BALB/c mice were immunized by recombinant protein. ELISA test showed the immune serum antibody titer of experimental group was gradually increased. After the third immunization, antibody titers show significantly increase compared with the control group (P<0.05) . Detection of cellular immunity showed that CD4+ T lymphocytes were significantly increase compared with the control group (P<0.05) and CD8+ T lymphocytes were not significantly increase (P>0.05). CD4+/ CD8+ T lymphocytes were significantly increase compared with the control group (P<0.05). After immunization, mice were inoculated with 1×106 Cryptosporidium parvum oocysts. The results show that the oocysts number of grams in the experimental group fecal were decreased from the third day of inoculation. Compared with the control group. CA2 group reduction rate was 37.5% and CA42 Group 32.2%. There were significantly increased (P<0.05).The results showed that the recombinant protein has immunogenicity and cross-protection.The CA2 and CA42 genes were subcloned into the eukaryotic expression plasmid pVAX1. Recombinant eukaryotic expression plasmid pVAX1-CA2 and pVAX1-CA42 were constructed. The recombinant plasmid was successfully transfected into HeLa cells, then extracted cell protein. Western Blotting show that the recombinant protein was conformed with expectation and was recognized by polyclonal antibody of immunized mice of C.andersoni. Then BALB/c mice were immuned by recombinant plasmid. Compared with the control group, the level of humoral immunity was significantly increas (P<0.05), CD4+ T cells was higher than control group (P<0.05). CD4+/CD8+ T cells were higher than pVAX1 group (P<0.05). Cross-protection experiments prove that CA2 group reduction rate was 34.6%, while CA42 group was 25.8%. Compared with the control group, there were significantly increased (P <0.05). The results show that recombinant DNA vaccine has some cross-protection against Cryptosporidium parvum. This study will be a theoretical basis of the prevention and treatment of cryptosporidiosis andersoni and cross-protective vaccine research. |
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