Preparation And Immunolocalization Of EgAPQ 9 Polypeptide Antibody Of Echinococcus Granulosus

ObjectiveTo establish an economical and effective method for culture of germinal cells released from hydatid cyst walls in laboratory of non-epidemic area and to produce a specific polypeptide antibody against EgAQP 9 to detect the cellular localization in the germinal cells and the tissue distribution of EgAQP 9 in the protoscoleces and hydatid cyst walls.This study laid a foundation for further study on the relationship between aquaporins and cyst fluid formation in the hydatids of Echinococcus granulosus.Methods1.The cyst walls of Echinococcus granulosus secondary hydatids isolated from the abdominal cavities of mice were digested with solution of trypsin-EDTA(0.25%)for 10,20 or 30 minutes,respectively,then the debris were cultured by the debris-adherent culture method,exploring the best time of digestion.2.The debris digested with the best time of digestion were cultured by the debris-adherent culture method comparing to the undigested cyst walls cultured by the simple adherent method.The dynamic features of cell morphology of germinal cells were observed under an inverted microscope,comparing the growth curve.3.The Echinococcus granulosus germinal cells were identified by immunofluorescence assays.4.The germinal cells were inoculated into the abdominal cavities of mice.The new pathological cystic vesicles were identified by HE staining.5.The subcellular localization prediction of the Eg AQP 9 was prepared based on the amino acid sequence in the field of bioinformatics.6.The polypeptide of EgAQP 9 was analysed and designed based on the amino acid sequence in the field of bioinformatics,and the polypeptide antibody was prepared with the synthetic peptide by immunizing New Zealand rabbit.7.SDS-PAGE was used to detect the purification effect of the polypeptide antibody.The antibody titer was detected by ELISA.8.The Eg AQP 9 protein was identified by Western Blot.The cellular localization in the germinal cells of Eg AQP 9 was detected by immunofluorescence assay,and the tissue localizations of EgAQP 9 in the protoscoleces and hydatid cyst walls were detected by immunohistochemical assay.Results1.The best time of digestion with solution of trypsin-EDTA(0.25%)was 10 minutes.Following 2 to 4 days,a plenty of germinal cells released from the edges of debris with intact cytoplasm.2.The growth curves of debris-adherent culture method and disgested adherent culture method were similar to “s” shape.The logarithmic growth intervals of the germinal curve of the two methods were similar,but the level of proliferation of germinal cells released from the digested cyst walls debris in the debris-adherent culture was higher than that in disgested adherent culture.3.The germinal cells cultured with the debris-adherent culture method by Immunofluorescence test,containing antigen components of Echinococcus granulosus.4.Inoculation results of germinal cells cultured with the debris-adherent culture method showed?that new cystic vesicles were spotted in the abdominal cavities of mice.HE staining results showed?that?the developing laminated layer and germinal layer could be observed under light microscope.5.The subcellular localization prediction of the EgAQP 9 showed that EgAQP 9 was distributed on plasma membrane,mitochondria and endoplasmic reticulum.6.The amino acid sequence of EgAQP 9 polypeptide was ASEEHSTDEDEDTEA by means of bioinformatics,and the polypeptide antibody was prepared with the synthetic peptide by immunizing New Zealand rabbit.7.SDS-PAGE showed the purity of Eg AQP 9 polypeptide antibody was good.The purified polypeptide antibody showed high sensitivity,with a titer as 256,000 by ELISA.8.Western blotting showed that the molecular weight of EgAQP9 was consistent with the position of the band.Immunofluorescence tests showed that Eg AQP 9 was distributed in the cytoplasm and membrane in germinal cells of Echinococcus granulosus,which was consistent with the result of preparation of bioinformatics.Immunohistochemistry tests showed that EgAQP 9 was distributed on the surface of protoscoleces and in the germinal layer of hydatid cyst walls in mouse and sheep.Conclusion1.The cyst walls of Echinococcus granulosus secondary hydatids isolated from the abdominal cavities of mice were digested with solution of trypsin-EDTA(0.25%)for 10 minutes,then the debris can be applied to culture vital germinal cells.The method maked full use of the secondary hydatids in infected mice,providing a foundation of the study on the germinal cells of Echinococcus granulosus.2.The polypeptide of EgAQP 9 was designed based on the amino acid sequence,and the polypeptide antibody was prepared with the synthetic peptide by immunizing New Zealand rabbit.The immunolocalization was detected in the germinal cells,the protoscoleces and hydatid cyst walls by immunofluorescence assay and immunohistochemical assay,respectively,providing a foundation to explore the relationship between Eg AQPs and the formation of cyst fluid.