ABSTRACT
Objective To select the immunodominant epitope of human serum albumin (HSA) and provide the basis for setting up a special and rapid detecting method of HSA. Methods Bioinformatic method was used to compare protein sequences of human, pig, horse, ox, and ovine, and the immunodominant epitopes of HSA were predicted. The E.coli preferred codons were used todesign the DNA sequences of the selected epitopes. The genes of the epitopes were expressed after they were inserted into the PGEX-4T-2 vector. The recombinant antigens were identified and valued by HSA antibody with indirect ELISA. Results The length of the selected epitopes was H1 [126-162 amino acid (aa)], H2 (314-355aa), and H3 (373-424aa) and the relative molecular weight was 3.01×104, 3.06×104 and 3.17×104, respectively. The epitope of 373-424 aa were more active and its cross-reactivity IC50 of enzymelabeled antibody was 1.635 mg/L, which was higher than HSA (P0.05). Conlusion The immunodominant epitope of HSA is obtained, which is significant for developing a rapid and special reagent of HSA.
ABSTRACT
Objective:To investigate the immunogenicity of pORF5 plasmid protein,and further to screen and identify its im-munodominant domian.Methods: 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamHⅠand NotⅠrestriction endonucleases.After identification by PCR and sequencing,the recombinant plasmids were transformed into XL1 Blue E.coli to express the GST fusion proteins.ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein.10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method.Results: pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human.The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de-stroyed.F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was.However,no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies.Conclusion:pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope,and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation.The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.
ABSTRACT
Objective To select the immunodominant epitope of human serum albumin(HSA)and provide the basis for set?ting up a special and rapid detecting method of HSA. Methods Bioinformatic method was used to compare protein sequences of hu?man,pig,horse,ox,and ovine,and the immunodominant epitopes of HSA were predicted. The E.coli preferred codons were used to design the DNA sequences of the selected epitopes. The genes of the epitopes were expressed after they were inserted into the PGEX-4T-2 vector. The recombinant antigens were identified and valued by HSA antibody with indirect ELISA. Results The length of the selected epitopes was H1〔126-162 amino acid(aa)〕,H2(314-355aa),and H3(373-424aa)and the relative molecular weight was 3.01×104,3.06×104 and 3.17×104,respectively. The epitope of 373-424 aa were more active and its cross-reactivity IC50 of enzyme-labeled antibody was 1.635 mg/L,which was higher than HSA(P<0.05). Conlusion The immunodominant epitope of HSA is obtained, which is significant for developing a rapid and special reagent of HSA.
ABSTRACT
Objective To search for the neutralizing epitopos on hepatitis E virus (HEV) capsid besides the known neutralizing epitope (aa459-606). Methods By analysis of several strains of monoclonal antibodies against HEV capsid and their recognized epitopes, the neutralizing activity of epitope (aa394-458) at N-terminus was compared with that of an immunodominant neutralizing epitope (an459-606). Re-suits The research showed a novel potential neutralizing epitope in aa423-437 of HEV ORF2 though detec-ting and comparing the characteristics of several antibodies and corresponding determinations. The epitope is a linear non-immunodominant epitope which is different from the other neutralizing epitope in aa459-606.And the amino acids sequence of this novel epitope is conservative. Conclusion ORF2 aa423-437 is a no-vel potential neutralizing leaner epitope of HEV. It is believed that the present work adds fundamental knowl-edge to our understanding of HEV capsid domain and contributes to the prevention and control of this dis-ease.