Expression And Application Of Cryptosporidium Parvum P23 Gene In The Lactobacillus Casei

Cryptosporidiosis is a global zoonotic parasitic protozoal. Cryptosporidium parvum is the major immune self-limiting pathogen. So far, there is no effective vaccine and drug for prevention and control of this disease on the world.In the research of cryptosporidiosis diagnosis and prevention, DNA was extracted from cDNA library of Cryptosporidium parvum and the DNA fragment encoding mature peptide of p23 gene was amplified by PCR method. The fragment was cloned into plasmid pGEX-6p-2 to forming the recombinant prokaryotic expression vector(pGEX-p23) which was identified by EcoR I/Sal I enzymatic digestion and sequence analysis. The E.coli BL21 containing recombinant plasmid was induced by IPTG for expression of recombinant P23 antigen. SDS-PAGE and Western blot was performed to identify the expressed protein. Results of SDS-PAGE analysis showed that the new protein band, which is 47kDa, is the GST-p23 fusion protein. Western blot proves that the fusion protein can be specifically recognized by anti-p23 antibody.Using the same method, the p23 gene was cloned into expression vector pMG36e of lactic acid bacteria to construct the recombinant expression vector pMG-p23 and then transferred into Lactobacillus casei by electric transformation called recombinant L.casei Zhang (including pMG-p23 ). SDS-PAGE, Western blot and IFAT were used to detect the expression product of p23 gene in the components of lysed thallus and viable. The results showed that the expression product of p23 gene was approximately 20kDa, corresponded to the expected theoretical value. The recombinant L.casei Zhang (including pMG-p23 ) reacted with the mouse anti-P23 serum proteins but not with mouse anti-GST serum protein, that showd has a specific.Taking the recombinant P23 and recombinant L.casei Zhang as antigen respectively, indirect ELISA and IFAT test method was established to detect the Cryptosporidiosis. Cryptosporidiosis prevalence was detected in serum samples collected from cattle, goats and sheep in fields by using ELISA and IFAT simultaneously. The results showed that the recombinant P23 is a good antigen. The positive results of these two diagnostic methods are basically identical, showing good repeatability and sensitivity.The test build the basis for research of Cryptosporidiosis early diagnosis.