Double Antigen Sandwich Method Of Genetic Engineering Em18Alveolar Echinococcosis Rapid Diagnostic Kit Developed

Objective:(1)Multiple clones by genetic engineering techniques Echinococcus Em18antigen.(2)The HRP labeled on Em18antigen preparation of genetic engineering,the use of double antigen sandwich method,the preparation of bubbly hydatid rapid diagnostic kits.Methods:(1)From the multiuse room hydatid in RNA extraction kit to extract RNA,reverse transcribed into cDNA,by cloning,construction,sequencing,the use of genetic characteristics DNA man software Em18Eml8analyze and construct nucleic acid sequences.With DNA man software design primers were5’end and3’end in added EcoRI and Xhol restriction sites in pET28a/Eml8prokaryotic expression plasmid as a template,PCR amplification of gene fragments Em18,after digestion,transformed into Escherichia bacterial expression vector BL21,digestion,PCR and sequencing of its insertion sequence is correct.Em18antigen was amplified and sequenced to identify the correct sequence.(2)Induced by IPTG rEml8,eluting sucrose concentration by SDS-PAGE electropHoresis and Western blot tests to identify the expression level of the recombinant protein.(3)HRP labeled using recombinant antigens.(4)Screening checkerboard coating concentration of antigen,ELISA antigen dilution,dilution of antibody to be tested,the use of double antigen sandwich prepared Em18bubbly hydatid rapid diagnostic kits. Results:(1Successfully cloned and constructed prokaryotic expression vector pET28a/Em18induced by IPTG,SDS-PAGE electropHoresis showed pET28a/Em18recombinant protein was successfully expressed in relative expression at a molecular weight of50kD band.(2)Western Blot analysis showed that the protein can be multiroom rEm18hydatid sera to identify patients with good antigenicity.(3)By combining the enzyme substance evaluation to determine the success of HRP labeled antigen.The enzyme-linked recombinant protein coated plate in accordance with the filtered assembled kit good condition,select a test sample,the kit is detected with high sensitivity and specificity.(4)Through the use of single-blind test to compare paired double antigen sandwich and dot immune colloidal gold with the gold standard (surgery) sensitivity and specificity..P<0.05,the difference was statistically significant.Conclusion:The use of cloning technology to Em18antigens cloned and expressed successfully,identification, Concentration and purification,the successful use of HRP labeled recombinant antigen screening kit of various factors checkerboard titration optimal conditions for use,by the principle of double antigen sandwich ELISA reagent assembly boxes,using a paired comparison test double antigen sandwich and dot immune colloidal gold with the gold standard,the sensitivity and specificity. x2=5.1, P<0.05, the difference was statistically significant.