Cloning And Expression Of CP15/60 And CP41 Gene Of Cryptosporidium Parvum Mouse Genotype And Epidemiology Survey Of Pig Cryptosporidiosis In One Hoggery In Shanghai District

Cryptosporidium is a world-wide waterborne protozoan zoonosis and harm severely to humans and animals. It grows and reproduces within epithelial cells of the digestive and respiratory organs of verterbrates with a wide host range, including humans and animals belong to mammals, birds, reptiles and fish. Cryptosporidium infection can lead to persistant, severe and often life-threatening disease in immunocompromized patients and animals. To date, there is still no effective treatment available for cryptosporidiosis in man and animals. It's necessary to develop sensitive and rapid diagnosis technique and effective preventative vaccine.The sporozoite surface antigen CP15/60 and the oocyst wall protein CP41 genes were successful amplified from total RNA of Cryptosporidium parvum mouse genotype by reverse transcription polymerase chain reaction(RT-PCR). The homology of the nucleotide sequences between cloned genes and the genes of C. parvum bovine genotype downloaded from GenBank were 98.66 % and 90.50 %, respectively.The prokaryotic expressed plasmid pET-28b(+)-CP15 and pGEX-5x-3-CP41 were constructed and expressed in E. coli successfully. Western-blot analysis showed that the recombinant expressed proteins could be recognized by the sera of rabbit infected with C. parvum. ELISA results showed that the titer of specific antibody against the recombinant proteins in sera from 3 times immunized SPF rabbits were significantly higher than that of rabbits before immunization, indicating that the recombinant proteins have good immune-reactivity and immunogenicity.Indirect ELISA techniques were developed for detecting specific antibody against Cryptosporidium with the recombinant protein CP15/60 and CP41 as the coating antigen respectively. The techniques were used to detect the specific antibody titre of sera of rabbits infected with C. parvum rabbit genotype or not. The results indicated that the indirect ELISA technique has high sensitivity and specificity.Surveying systematically and extensively of epidemiology of cryptosporidiosis is a basic work for preventing the disease effectively. Infection of Cryptosporidium in pig was surveyed in one hoggery in Shanghai district. 1,629 samples were collected and detected by Sheather's sucrose floatation technique. Results showed that the infection rate of Cryptosporidium in pig was 31.55 %. In one year time, spring and winter are two seasons with the highest infection rate, and then the autumn. The summer has the lowest infection rate. The infection rate also related with the ages of pigs. The pigs at 30-39 and 40-49 days old have the highest infection rate. They were 58.54 % and 43.30 %, respectively. In contrast, the infection rate is very low when pigs smaller than 30-day-old or exceed 90-day-old. The infection intensity of Cryptosporidium in pigs also related with their ages, younger pigs has higher infection intensity.The work was much helpful to know the infection condition about Cryptosporidium in pigs in Shanghai district and to control the disease. The cloning and expression of sporozoite surface antigen CP15/60 and oocyst wall protein CP41 gene and the development of indirect ELISA diagnosis technique for cryptosporidiosis would also be much helpful for next serology survey of the disease and researching of immunological prevention technique with the two recombinant proteins.