Cloning And Expression Of E.granulosus Oncosphere M26 Gene And Screening Of Positive Hybridoma Cells

Echinococcus granulosus parasitized on dogs,wolves and other final hosts.Its eggs and gravid proglottids excret out of bodies with the feces and contaminate forage and drinking water.Cattle,sheep and other intermediate hosts were infected with the disease after ingestion eggs.Oncosphere that is into the digestive tract and invade into the intestinal wall and spread to the body through blood flow or lymph,which its mainly lesions in the liver and lung lead to growth slow.It is a serious threat to human health and the rapid development of animal husbandry,which caused serious losses to the world economy.Therefore,prevention of the disease is very important in all directions.For a long time,although the researchers are trying to explore the effective methods for rapid diagnosis,the control and preventing from this disease,there is no breakthrough in the early of diagnosis.The sheep is the intermediate host of this parasite.If sheep is infected with the disease in the early stages,its oncosphere secreted protein in the blood,diagnosed it and taking effective measures,which is important to control the epidemic of disease.At present,the main diagnosis of hydatid disease were imaging(X-ray radiograph,ultrasound,CT,MR,DSA/SCA),intradermal test,blood detection etc.However,these methods were mostly related to late diagnosis(after the cysts formed).In this study,the oncosphere's secretory protein was used as antigen,and for the early diagnosis of hydatid disease by ELISA method.This is firstly reported about the early diagnosis of hydatid disease.Therefore,it is necessary to choose high specific antigen in order to establish a high sensitive and specific method for the early diagnosis of the disease.This study is for the sake of exploring the antigen which can be detected in the disease in the early stage of infection.We selected differentially expressed proteins on the basis of previous study,namely paramyosin(M26)as the subject investigated.As a result,through prokaryotic expression,preparing polyclonal antibody and screening positive hybridoma cells established ELISA method detecting antigen that oncosphere secreted protein in host serum during the early of infection.In order to find an effective diagnostic antigen and establish an efficient and specific diagnostic method,the following experiments were carried out.1.Screening and prokaryotic expression of Echinococcus oncosphere antigen gene: Differential protein were separated and detected among Echinococcus granulosus,oncosphere,protoscolex as material by iTRAQ in the laboratory.Differentially expressed protein can be used as an antigen in early diagnosis in oncosphere,so the highly effective immune protective paramyosin(M26)was screened out.Its cloning vector and expression vector were constructed successfully in this experiment.The concentration of protein was 0.5mg/mL and the purity was up to 85%.The recombinant protein was 41 KDa by prokaryotic expression.2.Preparation and purification of polyclonal antibody of M26:The Rabbit anti-M26 polyclonal antibody was successfully prepared using M26 protein as antigen.The titer of antibody was 1:12800.Western blot verified that a single band,indicated that the antibody could specifically recognize M26 protein.Using polyclonal antibody detected paramyosin in serum of early each period(4-10 h,24h and 48h)through sheep infected artificially hydatid disease by indirect ELISA method.When multiple of dilution ratio was at 1:3200-1:12800,the ratio of sample holes and negative holes at each time point are greater than or equal to 2.1,which are positive so that each time point has the protein.3.Screening of positive hybridoma cells of M26: M26 protein as antigen immunzed Balb/c mice.Two positive hybridoma cells which can bind specificially to the protein by ELISA analysis,cell fusion and hybridoma screening.Using cell strains detected paramyosin in serum of early each period(4-10 h,24h and 48h)through sheep infected artificially hydatid disease by indirect ELISA method.The results showed that the method was successfully established,which the protein as antigen can be used to diagnose effectively the hydatid disease of sheep by indirect ELSIA in the early of infection.