A nanobody nb2-37 specifically recognizing paraquat and its application
A nanobody, paraquat technology, applied in specific peptides, recombinant DNA technology, DNA/RNA fragments, etc., to achieve the effect of short time consumption, simple operation and high sensitivity
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Embodiment 2
[0076] Example 2 Affinity panning and identification of nanobodies
[0077] (1) Affinity panning of nanobodies
[0078] First, use PH-B-OVA as the coating source, dilute the PH-B-OVA coating source to a final concentration of 10 μg / mL with the coating solution, and coat overnight at 37°C. The next day, after washing twice with PBST (0.01M PBS, 0.06% Tween-20 (v / v)), 1% fish collagen was added to block for 2 hours at 37°C. Shake and pat dry the liquid in the well, add 100 μL phage library to each well (library titer is 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 (pH7.0), add Gly-HCl (0.2M, pH 2.2) After elution at 37°C for 10 min, it was immediately neutralized 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...
Embodiment 3
[0088] Example 3 Sequencing of the gene encoding Nanobody Nb2-37 and determination of its amino acid sequence
[0089] 1. Experimental method
[0090] The strain of Nanobody Nb2-37 obtained through indirect competitive ELISA identification was sent to a sequencing company for sequencing to obtain the nucleotide sequence of Nanobody Nb2-37; according to the DNA sequencing results and codon table, the nucleotide sequence of Nanobody Nb2-37 was obtained. amino acid sequence.
[0091] 2. Experimental results
[0092] The amino acid sequence of the VHH of Nanobody Nb2-37 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the Nanobody Nb2-37 is shown in SEQ ID NO.2.
[0093] The amino acid numbering and structural domain diagram of Nanobody Nb2-37 are as follows figure 1 As shown, it can be seen that the Nanobody Nb2-37 includes 4 framework regions (Framework region, FR) and 3 complementarity-determining regions (Complementarity-determining region, CDR); th...
Embodiment 4
[0095] Example 4 Mass Preparation of Nanobody Nb2-37
[0096] Prepare the nanobody Nb2-37 in the form of protein expression, the specific method is:
[0097] The obtained nanobody Nb2-37 strain was extracted with a kit for its plasmid, and the plasmid was transformed into E.coil BL21 by chemical transformation. Pick a single colony from the transformation plate and inoculate it in 10 mL LB (Amp) medium, and culture overnight at 37° C. and 250 rpm. Inoculate the overnight culture into 1000mL LB(Amp) medium at a ratio of 1:100, and cultivate to OD at 37°C and 250rpm 600 At about 0.4-0.6, add IPTG (1:1000 ratio, v / v), and culture overnight at 37°C and 250rpm. The next day, centrifuge at 12000rpm for 5min at 4°C, collect the bacterial pellet, use sucrose osmotic pressure freeze-thaw method, centrifuge at 12000rpm for 10min, take the supernatant, and purify the supernatant by affinity chromatography to obtain the expressed nanobody Nb2-37 .
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