Cloning,Prokaryotic Expression,Immunolocalization Of Cofilin Gene From Sarcoptes Scabiei And Evaluation On Indirect-ELISA

Sarcoptes scabiei,as an ectoparasite,cause a disease named scabies which spreads worldwide not only in humans,but also in companion animals,livestock and wildlife,such as dogs,sheep,goats,foxes,raccoons,camels,wombat.etc.In animals,pruritus and hypersensitive dermatitis may lead to economic loss caused by depression in growth and feed conversion rates.Many countries and international organizations realize the importance of scabies,which is listed among the top 50 most prevalent diseases worldwide and treated as "Neglected tropical diseases".But no commercial immunodiagnostic or molecular based tests for human scabies is currently available.According to the EST of S.Scabiei cofilin gene from NCBI database,a pair of primers was designed to amplify 447bp targeted DNA fragment by polymerase chain reactions(PCR).After blasting the sequenced targeted DNA fragment in GenBank database,the gene showed highly homology to the DermatopHagoides farinae allergen Der f31(90%identity).The molecular mass of the recombinant protein was 16.8 kDa,of which the isoelectric point was 5.59.C751H1175N191O231S8 is the deduced chemical formula,with a negative charge.The S.Scabiei cofilin protein is an extracellular protein,which doesn't have signal peptide cleavage site and transmembrane domain.Targeted DNA fragment was linked to the pET32a(+)expression vector,and positive clones were picked and sequenced.After transforming the recombinant plasmid into E.coli BL21(DE3),the S.Scabiei cofilin recombinant proteins were expressed and purified by passing the Ni+ affinity chromatograpH collumn.The purified recombinant proteins could be recognized by rabbit scabies positive serum in the immunoblot analysis,which showed its antigenicity.After vaccinating rabbit with S.Scabiei cofilin protein,IgG from the corresponding antiserum was purified to be the primary antibody in the Fluorescent immunohistochemistry.The result demonstrated S.Scabiei cofilin distributes widely in the body of S.Scabiei but not in the epidermis.Using the purified recombinant protein as the antigen,a procedure of indirect-ELISA was established after optimizing the expeeimental condition.The optimal coating concentration of antigen is 5?g/mL,and the optimal working concentration of serum and secondary antibody is 1:100.1:3000,respectively.The cut-off value between negative and positive results was calculated as the average result of the mange negative animals plus three times the standard deviation(S.D.).Finally,if OD450>0.188,the serum is sentenced as positive;if OD450<0.188,the serum is sentenced as negative.Besides,there is no cross-reactivity in this study.The coefficient of variation(CV)and the concordance correlation coefficient(CCC)were calculated by estimating the repeatability of same samples measuring within and between plates.CV is 1.28%?4.08%and CCC is 1.81%?5.30%.The sensitivity and specificity for measuring the assay-were calculated,sensitivity:83.33%;specificity 87.9%.It could be demonstrated that recombinant S.Scabiei cofilin protein has the value to be an antigen used in the indirect-ELISA for the diagnosis of scabiei.