Recombinant Expression,purification Identification Of Protomyosin And Analysis Of The Ability Of Binding SIgE

Purpose:Shrimps are one of the leading causes of food allergies,with an prevalence of2.8% to 8% in food allergies.With increasing shellfish consumption in recent years,immunoglobulin E(Ig E)-mediated shellfish allergy poses a growing health threat to children and adults.The tropomyosin is the main sensitization component of crustacean seafood,the component diagnosis based on the tropomyosin can significantly improve the diagnostic accuracy of shrimp allergy.At present,most of the studies of allergenic proteins are directly extracted from organisms,but the natural extraction of allergenic proteins is not only cumbersome and complex,but also easy to be doped with other non-allergen components in the extraction process,and some important allergen proteins have less content and are not easy to extract.Recombinant proteins can avoid these defects,but need to determine their immunological activity.The aim of this study was to recombinant expression,purification of shrimp major allergen-tropomyosin Pen a1 and evaluation of its immune activity by means of light-initiated chemiluminescent assay(LICA).Method:1.Tropomyosin Pen a1 gene synthesis.Synthesized the Pen a1 gene artificially according to the known pena1 sequence,designed and synthesized specific primers,and performed PCR amplification to identify the PCR amplification results.2.Construction of recombinant expression plasmid p ET-28a(+)-Pen a1.Firstly,the target gene pena1 and vector p ET-28a(+)were double-cut;then,nucleic acid electrophoresis,gel recovery;finally,the recombinant expression plasmid p ET-28a(+)-Pen a1 was constructed under the action of DNA ligase.The constructed recombinant plasmids were transformed into receptive Top10 for culture and PCR identification,and the positive clonal bacterial solution was selected for sequencing.Sequencing aligned the correct plasmid for further Bamhi-Hind III double enzymatic verification.3.Expression of the recombinant allergen protein Pen a1,purified recombinant Plasmid into the expression host E.coli BL 21(DE3).IPTG induced expression afterovernight culture.the recombinant allergic protein pena1 was obtained and the nickel column purified expression product.the protein was further verified by mass spectrometry.4.Immunoactivity assessment of recombinant allergen protein Pen a1 for serum allergen-specific Ig E(s Ig E)detection using light-initiated chemiluminescent assay,LICA.First,25μL was added to the 96-well plate in turn to dilute the serum of shellfish allergy patients with PBS containing 1% serum albumin(HSA)(p H 7.4,0.01mol/L),50μL(1:1000,dilution:PBS/HSA)anti-human Ig E antibody-labeled luminescent spheres and 1μg/l of biotinylated Pen a1 solution incubated at 37°c for30 min.Subsequently,150μl of streptavidin-labeled photoreceptor spheres were added and the reaction mixture was shaken and incubated for 10 min at 37°c to avoid light.Finally,the chemiluminescence signal(chemiluminescence,cl)was detected on a LICA instrument.At the same time,serum parallel treatment was used as negative control.Double hole detection of each serum sample.Results:1.PCR Amplification and Identification of Promyosin(Pen a1)Gene after the synthesized target gene was amplified by PCR and Agarose Gel Electrophoresis,the size of the obtained target fragment was about 930bp(plus protective base and restriction site),which was consistent with the theoretical predicted value.2.Verification of recombinant expression plasmid p ET-28a(+)-Pen a1The results of colony PCR amplification of some samples were about 930 bp,which was consistent with the predicted size of 930 bp,indicating that the target gene was successfully attached to the plasmid vector.The positive and reverse sequencing results showed that the sequence was consistent with the Pen a1 sequence published on NCBI.moreover,the correctly sequenced colony shaking bacteria were cultured,the plasmids were extracted and double digested.the obtained bands were about 867bp、5400bp consistent with the size of the expected 867 bp、5400bp.The results of enzyme digestion showed that the recombinant plasmid containing the Pen a1 gene was obtained.3.Target protein expression,purification,and identification of IPTG-induced culture.SDS-PAGE Electrophoresis showed a significantly increased protein band at36 k Da.the molecular weight was consistent with the predicted value,and no obvious band was found in the uninduced bacteria.The target protein is mainly produced in the supernatant after ultrasonic treatment,so it is soluble expression.After purification of the nickel column,the SDS-PAGE electrophoresis results showed that a single purified target protein was obtained.Furthermore,the target protein was identified by further mass spectrometry as protomyosin.4.Binding ability of serum Ig E antibody to prokaryotic expression product Pen a1 using LICA technique to detect binding ability of serum Ig E antibody in recombinant Pen a1 allergen-bound shellfish allergy patients.71.4%(25/35)of patients with serum s Ig E antibodies were sensitive to recombinant Pen a1 allergens,similar to those reported in the literature,indicating that the recombinant allergen has good allergenicity.Conclusion:By exploring the experimental methods and optimizing the experimental conditions,a method for the expression and evaluation of the immune activity of the protomyosin Pen a1 recombinant was established.At the same time,the successful expression and purification of protomyosin Pen a1 laid the foundation for further better study of the role of Pen a1 in the diagnosis and treatment of shellfish(e.g.shrimp)allergies.