Establishment Of Indirect ELISA Detection Method Employing The Screened And Expressed Recombinant Cryptosporidium Protein

Cryptosporidium sp.,which caused serious clinical disease to the hosts as anorexia,diarrhea,emaciation etc.,affecting their growth and development,can infect approximately 79 different mammal species.C.andersoni is one of the species of genus Cryptosporidium.This species has the highest infection rate in dairy cows,caused different degrees of weight loss,reduced feed reward,at the same time falling cows milk production and has brought great harm to the industry.Some studies have shown that C.andersoni can also infected humans.Therefore,detection and prevention of the parasite have important significance for public health.Two pairs of specific primers were designed based on gene sequences of C.andersoni COWP and HSP70 from GenBank(DQ060431;AB610481).Purpose fragments were amplified by using RT-PCR assay and the cloning vector was constructed following.Double enzyme digestion was performed then the products were subcloned into the prokaryotic expression vector(pET-32a).The recombinant plasmid pET-32a-COWP and pET-32a-HSP70 were successfully constructed respectively and transformed into E.coli.expression strain BL21(DE3).Similar steps were performed for constructing the recombinant plasmid pET-32a-COWP-HSP70 by double enzyme digestion and subcloning.The fragment of HSP70 was successfully ligated into pET-32a-COWP and transformed into E.coli.With the IPTG induced,these three recombinant proteins were expressed.After identifying and analysis by SDS-PAGE and Western blot,products were confirmed as COWP fusion protein(CFP),HSP70 fusion protein(HFP),and COWP-HSP70 combined protein(CHCP).These proteins were purified and then BALB/c mice were experimentally immunized by the three purified proteins for three times,respectively.Serum antibody titers were both higher than control,according to the result of ELISA detection.Mice positive serum against C.andersoni egg was used for Western blotting and the result showed that:reaction could take place between CFP,HFP or CHCP with the serum,which evolved that all these three protein had strong immunogenicity and reactionogenicity.The ELISA method was established by the three recombinant proteins as coating antigen,serum from oocysts antigen immunized BALB/c mice as primary antibody,horseradish peroxidase(HRP)labeled goat-anti-mouse-IgG as secondary antibody.Comparison of these three proteins,found that the ELISA method established by CHCP as coating antigen achieved the best effect.40 clinical samples were detected by the ELISA method which was optimized conditions.The results of detected showed that 30 samples were positive with OD value were higher than 0.215,10 samples were negative with OD value was lower than 0.215.The test results were 100%in comparison with PCR detection.Conclusion:This study successfully constructed three protein gene express vectors,and acquired three purpose recombinant fusion proteins.Screening the different recombinant protein used as the coating antigen,the co-expression protein(CHCP)was considered to be the best one.The indirect ELISA method established by this protein,to detect IgG-antibody in the clinical samples,showed its certain suitability.The study lays a foundation for further research on immune diagnosis,vaccine development,disease preventive control and treatment for cryptosporidiosis.