Cloning, over-expression and evaluation of a recombinant fusion protein of Wuchereria bancrofti towards its application as a diagnostic agent for bancroftian filariasis.
A low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990). The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level. A recombinant clone (lambda WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies. The 2 kb DNA insert of the clone lambda WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified. The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level. All microfilaraemic individuals were positive by IgG4 assay. However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals. Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection. Among the filarial-IgG4 (assay)-positive 'endemic normals', 14% showed 'microfilariae' during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay.
Theodore
J G
JG
Center for Biotechnology, Anna University, Madras, India.
Kaliraj
P
P
Jayachandran
S
S
Jayaraman
K
K
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood* / Q000276:immunology; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D003429:Cross Reactions; D004605:Elephantiasis, Filarial / Q000175:diagnosis* / Q000276:immunology; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D016678:Genome; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D007194:India / Q000453:epidemiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012680:Sensitivity and Specificity; D017182:Setaria Nematode / Q000235:genetics / Q000276:immunology; D014958:Wuchereria bancrofti / Q000235:genetics* / Q000276:immunology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D000939:Epitopes; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E