Proteome Analysis Of Echinococcus Multilocularis Under Different Parasitic Conditions

The larva of Echinococcus multilocularis was usually parasitic in the liver and lungs of animals or humans.It can cause Echinococcosis multilocularis disease,and also known as alveolar echinococcosis.The clinical study showed that,echinococcus can be parasitic in different locations.There are two types of cysts: one is a fertile cysts with a large number of protoscolex,which is attached to germinal layer or free in the cyst fluid,and the other is the sterile cysts with no or only a small amount of protoscolex.Currently,the research of cystic infertility is still in the preliminary stage,and its molecular mechanism is still unknown.The protein components in cysts play a key role in the growth and development of parasites.Therefore,parasitized on subcutaneous parasites and liver parasites were used as experimental materials to study the cysts under two parasitic conditions for mass spectrometry analysis,mass spectrometry analysis,sequence analysis of gene,prokaryotic expression and preparation of polyclonal antibodies,tissue localization of proteins,and morphological observation of cysts.The main results were as follows:1.Each BALB/c mouse was vaccinated subcutaneously with about 600 protoscolex,and the subcutaneously parasitic cyst was isolated after one month,two months and three months after vaccination.Two and three months later,the liver's parasitic cysts were isolated.The isolated cysts were extracted and analyzed by mass spectrometry.The results showed that there were 203 proteins in the cyst of subcutaneous parasitism for one month,538 proteins in the cyst of subcutaneous parasitism for two months,939 proteins in the cyst of subcutaneous parasitism for three months,135 proteins in the cyst of liver parasitism for two months,and 217 proteins in the cyst of liver parasitism for three months.2.The cDNA of Echinococcus multilocularis was used as the template.The fructose 1,6-diphosphate aldolase(EmFru)gene of Echinococcus multilocularis was amplified by PCR,and the gene was analyzed for related bioinformatics.The results showed that the ORF sequence size of EmFru gene was 1092 bp,which can encode 363 amino acids,and its molecular weight was about 39 ku.3.The prokaryotic expression recombinant plasmid pET28a-EmFru was constructed,and the soluble recombinant EmFru(rEmFru)with the molecular size of about 39 ku was obtained.The New Zealand white rabbits were immunized with purified rEmFru and obtained rEmFru specific IgG antibody.Indirect ELISA analysis showed that rEmFru had a good immune effect and the antibody titer reached 1:204800.Western Blotting analysis showed that the expression level of rEmFru in subcutaneous parasitic cyst was higher than that in liver parasitic cyst.4.EmFru was located by immunofluorescence staining.The results showed that EmFru was mainly located in the germinal layer of cysts,the epidermis of protoniae and the jelly of cysts.Conclusion: Echinococcus multilocularis has different proteome cysts under different parasitic conditions,and EmFru may play a key role in this process.