| Cryptosporidiosis is one of the six most common diarrhea diseases in the world,widely distributed and severe,and can cause fatal diarrhea in patients with immunodeficiency(eg HIV patients).Cryptosporidium parvum sporozoite surface adhesion protein gp60 is the major surface antigen of Cryptosporidium parvum,which was found and reported at the same time by Cevallos and Strong in 2000,and is considered as effective target antigen for the prevention and treatment of Cryptosporidum parvum infection.Reveal the related biological function of gp60,which in the process of C.parvum invasive host cell,was of great significence to the reserch of invasion mechanism.Firstly,primers were designed according to the sequence of C.parvum gp60 and gp40,was then amplified by PCR,the target gene fragment was amplified gp60 and gp40 sizes were 885 bp and 570 bp,respectively.After recovering fragment which is connected to the cloning vector p MD18-T,and then the recombinant plasmid was identified by restriction enzyme cloning and sequencing,the correct result will fragment was ligated into an expression vector p GEX-4T-1,and then the recombinant expression vector was identified and sequence comparison.The recombinant prokaryotic expression after the completion of purified gp60-GST-His and gp40-GST-His recombinant proteins by affinity chromatography,and detection and identification by SDS-PAGE and Western blot.The recombinant protein has a high purity after purified by double-label(GST and His tag).Concentrations of gp60-GST-His and gp40-GST-His were 1.96mg/m L and 1.99mg/m L,and can be used for subsequent experiments.Recombinant proteins were used as antigens to the preparation of antiserum by immuned rabbits,and the antiserum titer was 1:32000 detected by ELISA;Antiserum was purified by Protein A,polyclonal antibody heavy chains was around 43-55 KDa,and the light chain was of about 25 KDa.The concentrations anti-gp60 and anti-gp40 Ig G were 2.15 mg / m L and 1.97 mg / m L.Indirect immunofluorescence method was used for the recombinant protein subcellular localization,protein gp60 was found positioned within Cryptosporidium parvum cell development stage,located to the surface of merozoite.gp40 protein was located to the surface of sporozoite.RT-PCR was used to detect the the expression of gp60 and gp40 protein at different time points in the process of C.parvum invasion of host cell,gp60 and gp40 protein expression reaches its maximum at 12 h,which was early developmental stages of C.parvum invasion of host cell;and 24 h and 48 h is maintained at a very high expression level,which was the stage of C.parvum develop into merozoites and form a zygote gametophyte.Thus,we concluded that gp60 may play an important role in the whole developmental stages of Cryptosporidium parvum invade of host cells,particularly the schizogony stage;the specific biological functions still needs further research and demonstration.The purified recombinant gp60 and gp40 antibodies were incubated with C.parvum sporozoites at concentrations of 0?g/ml,1?g/ml,5?g/ml,10?g/ml,20?g/ml,50?g/ml,then detected the effect of different antibody concentrations on C.parvum invasion rate by RT-PCR.Results showed that,compared with the normal invasion group,the low concentration of antibody blocking caused a significant decrease in the invasion rate.Thus,blocking the gp60 and gp40 protein can reduce the rate of invasion of the host cells by C.parvum.The above results showed that gp60 play an important role in C.parvum adhesion and invasion of host cells,polyclonal antibody which can recognize the gp60 have the ability to block C.parvum invasion of host cells. |
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