Screening And Identification Of Immunoactive Flab Protein Fragments Of Treponema Pallidum For The Serodiagnosis Of Syphllis

Objective: To screening out the immunoactive Fla B protein fragment,which is highly sensitive and specific,of Treponema pallidum,nine fragments were constructed and expressed and identified and purified,and the serodiagnostic value of each fragment was evaluated using the indirect Ig G ELISA.Methods: The amino acid sequences of three complete Fla B proteins of Treponema pallidum,namely Fla B1,Fla B2 and Fla B3,were obtained from Gen Bank.The amino acid sequences of each Fla B protein were aligned with amino acid sequences of that from other pathogens utilizing BLAST.Each full-length Fla B protein was segmented into continuous three portions,i.e.,both the conserved N-terminal and C-terminal,and the middle variable part.The specific primers of nine fragments were synthesized and the regions encoding each fragment were amplified by PCR utilizing the respective recombinant plasmids of whole Fla B as template.The resulting amplicons were cloned in frame into the p ET28a(+)plasmid.After being identified by DNA sequencing,the corresponding recombinant plasmids were re-transformed into E.coli BL21(DE3).The expressions of nine Fla B fragments were induced by isopropyl-?-D-thiogalactopyranoside(IPTG).The recombinant nine fragments were idenitified by Western Blot and purified by affinity chromatography using Ni2+-nitrilotriacetic acid(NTA)beads.All fragments were then subjected to dialyzed process for renaturation,the concertrations of which were measured by bicinchoninic acid(BCA)method.The recombinant Fla B fragments-based ELISA were established for detecting Immunoglobulin G antibody in the sera from healthy individuals,syphilitic patients,and those with potentially cross-reactive infection,the results of which were compared to that of TPPA assay,and the serodiagnostic value of each fragment was preliminarily evaluated.Results: The gene fragments were amplified by PCR and the recombinant plasmids were idenitified by DNA sequencing.The nine Fla B fragments were expressed in E.coli as insoluble inclusion bodies and subsequently purified by immobilized metal affinity chromatography method.Each purified fragment with high purity and appeared as a single band.The data showed that all the fragments based Ig G ELISA exhibited excellent sensitivities(91.1%-95.0%)and the value of B3 M was 95%.There are no significant differences among sensitivities of nine fragments(P>0.05).The conserved N-terminal and the C-terminal based Ig G ELISA demonstrated poor specificities(64.1%-78.4%),while three middle regions exhibited higher overall specificities for detecting Immunoglobulin G antibody,with 96.1% for B1 M,94.8% for B2 M,and 100% for B3 M,respectively.There are significant differences among specificities of nine fragments(P<0.05).These finding identified B3 M as the immunoactive Fla B protein fragment.Conclusion: The recombinant Fla B3 fragment,B3 M,which showed high sentivity(95%)and specificity(100%)in detection of syphilis.B3 M was the immunoactive one among the nine fragments screened.