Screening And Polyvalent Biosynthesis Of Procalcitonin Nanobody

Nanobody is the smallest antibody molecule that can bind to antigen,because of its advantages of small size,high environmental tolerance and large amount of biosynthesis,which has a broad application prospect in the field of food safety.Foodborne pathogenic bacteria are one of the main factors affecting food safety in China.It has been reported that Procalcitonin（PCT）can be used as a marker of sensitivity to food-borne bacterial infection,and the rapid,accurate and sensitive detection of PCT is of great value and significance for the early determination of foodborne pathogenic bacteria infection.This study was mainly based on the phage display nanobody library technology and prokaryotic expression system,prepared nanobodies that specifically bind to PCT,and analyzed their immunoassay performance in ELISA detection method.On this basis,in order to improve the binding ability of nanobodies,the construction of bivalent nanobodies and their biosynthesis were carried out,with a view to provide a new type of nanobody immunoassay element for the establishment of PCT immunoassay methods.The main research progress of this study is as follows:1)The recombinant PCT protein was passively immunized with alpaca.After six rounds of animal immunization and ELISA verification,the antibody titer in the peripheral blood of the alpaca met the construction of phage display library,and the nanobody encoding gene contained in lymphocytes in alpaca’s peripheral blood was successfully amplified.Finally,the phagemid display library of PCT nanobodies（p Comb3XSS）with a library capacity of 5.0×10⁸ pfu and a phage display library with a titer of 1.15×10¹³pfu/m L were constructed to provide a basis for biopanning PCT nanobodies.2)The recombinant PCT protein was used as the target antigen for four rounds of solid-phase panning.After strictly controlling the biopanning conditions,the positive rate of phages in the fourth round of screening was 89.58%.Through Phage-ELISA identification and verification,23 kinds of Phage-displayed PCT nanobodies with different gene sequences were obtained,of which 7 phage-displayed PCT nanobodies（P-4-1,P-4-6,P-4-11,P-4-14,P-4-24,P-4-35 and P-4-38）showed strong binding ability to recombinant PCT protein,and two phage-displayed PCT nanobodies（P-4-24 and P-4-35）could specifically recognize and bind natural PCT antigens contained in serum samples.3)The prokaryotic expression vectors of 7 kinds of PCT nanobodies（P-4-1,P-4-6,P-4-11,P-4-14,P-4-24,P-4-35,P-4-38）were constructed,and the above-mentioned 7 kinds of PCT nanobodies were biosynthesized by induction expression and affinity purification.With Western blot and ELISA identification,the seven PCT nanobodies had good binding ability and specificity with recombinant PCT protein.BLI technology analysis showed that the PCT nanobody（P-4-6）have the highest affinity,The affinity constant was 1.559×10^-7 M.In addition,two functionalized PCT nanobodies（P-4-24-HRP complex and P-4-24-Nluc fusion protein）were prepared by chemical labeling and fusion protein expression methods,which were verified by indirect ELISA and sandwich ELISA,Both Both of them retained the immunoassay properties of specific binding to PCT protein and catalytic substrate luminescence.It provided a basis for the later application of PCT nanobodies as labeled antibodies in immunoassays.4)Based on PCT nanobodies（P-4-6）,four bivalent PCT nanobody prokaryotic expression vectors（N-C-Flexible Linker-N-C、N-C-Flexible Linker-C-N、N-C-Rigid Linker-N-C and N-C-Rigid Linker-C-N）were designed with rigid and flexible linking peptides,and different linking directions.The above four bivalent PCT nanobodies were biosynthesized by inductive expression and affinity purification.With indirect ELISA identification,the four biosynthetic bivalent PCT nanobodies all retained the binding specificity to the PCT recombinant protein,among which the bivalent PCT nanobodies（N-C-Rigid Linker-C-N）had the highest binding constant,which was 2.39×10⁷ L/mo L.These results indicate that the combination of the rigid linking peptide and the double N-terminal exposure can effectively improve the binding ability of the bivalent PCT nanobody to the PCT recombinant protein.In addition,combined with the strategy of rigid linking peptide and N-terminal exposure,the PCT nanobody-Fc fusion protein was biosynthesized.With ELISA identification,the PCT nanobody-Fc fusion protein not only retained the performance of specific binding to the recombinant PCT protein,but also improved its immune performance as a capturing antibody on solid phase carrier.These results indicate that biosynthesis of nanobodies with increased its molecular weight,such as multivalent nanobodies and fusion antibody fragments,may be an effective strategy to improve the immunoassay performance of nanobody,and provide immunoassay components for the detection and application of PCT nano-antibody in the future.