Cloning,prokaryotic expression and preparation of polyclonal antibody of Calpain A gene of Echinococcus multilocular
Objective To analyze and clone the open reading frame(ORF)sequence of calpain A gene from the larval stage of Echinococcus multilocularis(Em-Calpain A),then express fusion protein in vitro and produce its polyclonal antibody.Methods The coding sequence of Em-Calpain A was obtained from WormBase database.Some sequence characteristics of Em-Calpain A(physicochemical properties and protein structural domains,etc.)were predicted using online analysis tools,such as ProtParam and SMART.The phylogenetic tree was constructed through the neighbor-joining method in MEGA 7.0 software.Total RNA was extracted fromprotoscolex and then used to synthesize cDNA.Calpain A gene was amplified by PCR.The prokaryotic expression plasmid pET28a-Em-Calpain A was constructed and induced by isopropyl-β-D-thiogalactopyranoside(IPTG)to express the recombinant protein.His-Em-Calpain A proteins were recoveried from PAGE gel and used as the immunogen to produce rabbit polyclonal antibody.The purity of His-Em-Calpain A protein,antibody reactivity and titer were examined by using 10％SDS-PAGE,Western blot and ELISA,respectively.Results Sequence analysis showed that the coding sequence of Em-Calpain A gene was 2 469 bp in length,encoding 822 amino acids,with a predict theoretical molecular weight of about 92.13 ku and an isoelectric point of 5.3.The structure prediction results indicated that Em-Calpain A had a highly conserved calpain-like thiol protease structural domain(CysPc),a calpain Ⅲ domain(Calpain Ⅲ)and two E and F helix-cyclo-helix structures(EF-hand),suggesting that it belongs to the calpain family.Phylogenetic analysis showed that the Em-Calpain A was clustered withthe parasites of the Taeniidae family and more distantly related to Fasciola hepatica,Schistosoma japonicum,human,dog,cattle,and protozoon.The recombinant protein was mainly expressed in the form of inclusion bodies with a band at about 93 ku,which is consistent with the predicted molecular weight.The antiserum titer was 1∶262 144.The antibody could specifically recognize His-Em-Calpain A and the native Calpain A protein in protoscolex.Conclusion The prokaryotic expression system of Em-Calpain A gene was successfullyconstructed and its polyclonal antibody was produced,which provided a basis for the further study of the function of Em-Calpain A gene.
Echinococcus multilocularis larvaeCalpain Asequence analysisgene cloningexpression and purificationpolyclonal antibody
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