Establishment Of Dot-ELISA Test Method Based On Cryptosporidium Parvum Virus Capsid Protein

Cryptosporidium, mainly parasitizing in the gastrointestinal tract of humans andanimals, is a serious parasitic zoonosis protozoa, which causes cryptosporidiosis. Themain clinical symptoms of cryptosporidiosis is persistent diarrhea. Feces of humansand animals suffering from cryptosporidiosis contains oocysts, which causes waterpollution so that cryptosporidiosis waterborne exploding.When humans and animals were infected by Cryptosporidium, Cryptosporidiumstimulated the bodies to produce immune response so that the bodies can produceanti-Cryptosporidium antibodies. So we can detect whether the serums of humansand animals had anti-Cryptosporidium antibodies to determine whether humans andanimals were infected by Cryptosporidium. Molecular epidemiology investigationsdemonstrated that most human Cryptosporidiosis is caused by C.parvum andC.hominis. C.parvum virus has been found in many strains of C.parvum andC.hominis, while the virus has not been found in other species of Cryptosporidium.Therefore, this virus is expected as a molecular marker to distinguishCryptosporidium infecting humans from the other species of Cryptosporidium， alsoit can be as a basis for the detection of human cryptosporidiosis. When C.parvuminvaded humans and animals，C.parvum virus capsid protein was released and thebodies produced immune response so that the bodies can produce anti-C.parvum virusantibodies. So we can detect whether the serums of humans and animals hadanti-C.parvum virus antibodies to determine whether humans and animals wereinfected by C.parvum. At present，conventional ELISA is the most widely usedimmunological detection method to detect Cryptosporidium, but this process is time and energy consuming and requires matched equipment. In this study, to develop theDot-ELISA for detecting the antibody against C.parvum, C.parvum virus capsidprotein was used as coating antigen. The Dot-ELISA method is simple, rapid and easyto observe with eyes, so this method can be used for the investigation ofCryptosporidium well.The recombinant E.coli BL21constructed early was used to express theC.parvum virus capsid protein plentifully, the recombinant protein exists mainly in theform of inclusion bodies. Purify the recombinant protein by Ni-NTA affinity columnand SDS-PAGE showed a single band in37kDa, which is consistent with expectation.To develop the Dot-ELISA method for detecting the antibody against C.parvum,C.parvum virus capsid protein was used as coating antigen. In this study, the optimalantigen concentration for coating was0.25μg each well; the optimal dilutions of thesera of C.parvum was1:200；the optimal dilutions of the anti-bovine antibodyHRP-labeled was1:2000; the optimal reaction temperature is37℃；the optimal serumaction time is60min；the optimal action time of enzyme labelled antibody is30min；the optimal substrate action time is20min； the sensitivity, reproducibility andspecificity of this method is pretty good; compared to the microscopy method， thecoincidence was100%. Furthermore, we detected the564cattle srea samples from aChangchun slaughter house with established Dot-ELISA method, the result showedthat the infection rate of Cryptosporidium was6.03%. This study provided atheoretical basis for epidemiological investigation and prevention of Cryptosporidiumin Changchun environ area, also it provided a theoretical basis for detecting humanCryptosporidiosis.