Construction of naïve camelids VHH repertoire in phage display-based library.
Camelids have unique antibodies, namely HCAbs (VHH) or commercially named Nanobodies(®) (Nb) that are composed only of a heavy-chain homodimer. As libraries based on immunized camelids are time-consuming, costly and likely redundant for certain antigens, we describe the construction of a naïve camelid VHHs library from blood serum of non-immunized camelids with affinity in the subnanomolar range and suitable for standard immune applications. This approach is rapid and recovers VHH repertoire with the advantages of being more diverse, non-specific and devoid of subpopulations of specific antibodies, which allows the identification of binders for any potential antigen (or pathogen). RNAs from a number of camelids from Saudi Arabia were isolated and cDNAs of the diverse vhh gene were amplified; the resulting amplicons were cloned in the phage display pSEX81 vector. The size of the library was found to be within the required range (10(7)) suitable for subsequent applications in disease diagnosis and treatment. Two hundred clones were randomly selected and the inserted gene library was either estimated for redundancy or sequenced and aligned to the reference camelid vhh gene (acc. No. ADE99145). Results indicated complete non-specificity of this small library in which no single event of redundancy was detected. These results indicate the efficacy of following this approach in order to yield a large and diverse enough gene library to secure the presence of the required version encoding the required antibodies for any target antigen. This work is a first step towards the construction of phage display-based biosensors useful in disease (e.g., TB or tuberculosis) diagnosis and treatment.
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia.
Jamal S M
JS
Sabir
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia.
Ahmed
A
Atef
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia; Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.
Fotouh M
FM
El-Domyati
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia; Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt; Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders (PACER-HD), Faculty of Medicine, King Abdulaziz University (KAU), Jeddah, Saudi Arabia.
Sherif
S
Edris
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia.
Nahid
N
Hajrah
Genetics Department, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egypt.
Ahmed M
AM
Alzohairy
Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), PO Box 80141, 21589 Jeddah, Saudi Arabia; Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Electronic address: bahieldin55@gmail.com.
Ahmed
A
Bahieldin
D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000276:immunology*; D001419:Bacteria / Q000276:immunology; D017104:Bacteriophage M13 / Q000276:immunology; D002162:Camelus / Q000276:immunology*; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000276:immunology; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D018076:DNA, Complementary; D007074:Immunoglobulin G; D019151:Peptide Library; D012313:RNA
Biosensor; Diagnosis; Nanoparticle; Non-immunized; Treatment