Screening and identification of novel anti-KPC-2 carbapenemase-specific nanobodies
Objective:KPC-2-producing Klebsiella pneumoniae is able to cause drug resistance to β-lactam antibiotics and car-bapenems antibiotics.Phage display technology was used to screen nanobodies that specifically bind to KPC-2 from the anti-KPC-2 nanobodies library,which provided technical supports for the detection and diagnosis of drug resistance of KPC-2-producing Klebsiella pneumoniae.Methods:A bimodal camel was firstly immunized by the recombinant KPC-2,then,RNA was extracted from peripheral blood lymphocytes of the bimodal camel and reverse transcribed into cDNA.Nanobody fragments were amplified by two rounds of nested PCR,and the antibody library were constructed.The specific nanobodies were screened by the phage display technology.Lastly the epitope analysis and affinity determination were performed by HPLC and OCTET,respectively.Results:A nanobody library was con-structed with a capacity of 5.47×108 cfu/ml and the inserting effective fragments of not less than 81.25%;the immune elutriation method of anti-KPC-2 nanobody was established.Two nanobodies,K2 and K5,with different CDR3 regions were obtained,and their affinities were 6.0 nmol/L and 4.8 nmol/L,respectively.Moreover,K2 and K5 were non-competitive for binding epitopes on KPC-2.Conclu-sion:Two specific nanobodies with different epitopes were successfully panned out,which expect to replace traditional antibodies for detection and diagnosis of Klebsiella pneumoniae drug-resistant diseases.
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