A nanobody nt8 specifically recognizing 19-nortestosterone and its application
A technology of nortestosterone and nano-antibody, applied in the biological field
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Embodiment 1
[0033] Example 1 Construction of Camel Immune Antibody Library
[0034] (1) Preparation of complete antigen 19-NT-KLH and 19-NT-OVA
[0035] The 19-nortestosterone hapten with the structural formula shown in formula (I) is coupled with ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) by an active ester method to prepare complete antigens 19-NT-KLH and 19-NT-OVA.
[0036] The structural formula of 19-nortestosterone hapten is as shown in formula (I):
[0037]
[0038] (2) Immunity of camels
[0039] Healthy camels were used as experimental animals, and 19-NT-KLH was used as the immunogen, and subcutaneously injected in the back and neck of the camels, with a dose of 0.5 mL (containing 0.5 mg of the immunogen) for each immunization. For the first immunization, 0.5 mL of complete Freund's adjuvant was emulsified with the immunogen for immunization, and for subsequent booster immunizations, 0.5 mL of incomplete Freund's adjuvant was used for immunization after emulsificat...
Embodiment 2
[0077] Example 2 Affinity panning and identification of nanobodies
[0078] (1) Affinity panning of nanobodies
[0079] First, 19-NT-OVA was used as the coating source, and the 19-NT-OVA coating source was diluted to a final concentration of 12 μg / mL with the coating solution, and coated overnight at 37°C. The next day, after washing twice with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), 1% fish collagen was added to block for 2 hours at 37°C. Shake the liquid in the well and pat dry, add 100 μL phage library (titer about 10 12 cfu / mL), incubated at 37°C for 1h. Discard unbound phage, wash 5 times with PBST (0.01M PBS, 0.05% Tween-20 (v / v)), wash 15 times with PBS (pH 7.0), add Gly-HCl (0.2M, pH 2.2)37 After elution at ℃ for 10 min, immediately neutralize with 10 μL Tris-HCl (1M, pH 9.0). Take 10 μL of the eluted phage to measure the titer, and the rest is used to infect 4 mL of E.coil TG1 strain grown to the logarithmic phase for amplification. On the third day, the amplifie...
Embodiment 3
[0089] Example 3 Sequencing of the gene encoding the specific Nanobody NT8 and determination of its amino acid sequence
[0090] 1. Experimental method
[0091] Send the specific nanobody NT8 strain identified by indirect competitive ELISA to a sequencing company for sequencing to obtain the nucleotide sequence of the specific nanobody NT8; according to the DNA sequencing results and the codon table, obtain the specific nanobody NT8 strain. amino acid sequence.
[0092] 2. Experimental results
[0093] The amino acid sequence of the VHH of the specific nanobody NT8 is shown in SEQ ID NO.1, and the amino acid sequence and structural domain division of the specific nanobody NT8 are shown in figure 1 As shown, it can be seen that the specific Nanobody NT8 includes 4 framework regions (Framework region, FR) and 3 complementarity-determining regions (Complementarity-determining region, CDR); the framework regions (FR1-FR4) are selected from SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6,...
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