Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications.
In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-β-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10(8) M(-1) obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.
School of basic medicine, Weifang medical University, 261053 Weifang, People's Republic of China.
Hong-Ming
HM
Yang
Yong
Y
Chen
Zhi-Qin
ZQ
Gao
Jin-Bao
JB
Tang
D000907:Antibodies, Bacterial / Q000097:blood; D000915:Antibody Affinity; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D009177:Mycoplasma pneumoniae / Q000276:immunology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D000907:Antibodies, Bacterial; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins