Paraquat hapten PH-A, artificial antigen, antibody and preparation methods and applications thereof
A PH-A, artificial antigen technology, applied in the biological field, achieves the effect of simple preparation method, broad application prospect and strong specificity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0042] (1) Preparation of Paraquat Hapten PH-A
[0043] Since paraquat is a small molecule and has no immunogenicity, it cannot stimulate the alpaca to produce an immune response and then produce antibodies. Therefore, it is necessary to couple the paraquat hapten to the carrier protein through protein linkage technology to obtain immunogenicity. Active groups commonly used in protein coupling techniques include amino, carboxyl, hydroxyl, sulfhydryl, etc. In view of the fact that there are no such active groups in the molecular structural formula and it is not easy to connect arms; therefore, 4'-(4-methylphenyl )-benzaldehyde and carboxymethylhydroxylamine hemihydrochloride undergo oximation condensation derivation reaction to obtain hapten, which is denoted as hapten PH-A. The preparation method of paraquat hapten PH-A is as follows:
[0044] 100mg of 4'-(4-methylphenyl)-benzaldehyde, 74mg of carboxymethylhydroxylamine hemihydrochloride in 5-10mL ethanol solution, reflux ove...
Embodiment 2
[0090] Example 2 Affinity panning and identification of nanobodies
[0091] (1) Affinity panning of nanobodies
[0092] First, use PH-A-OVA as the coating source, dilute the PH-A-OVA coating source with the coating solution to a final concentration of 10 μg / mL, 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 13 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
[0102] Example 3 Sequencing of the gene encoding Nanobody Nb2-23 and determination of its amino acid sequence
[0103] 1. Experimental method
[0104] The strain of Nanobody Nb2-23 obtained through indirect competitive ELISA identification was sent to a sequencing company for sequencing to obtain the nucleotide sequence of Nanobody Nb2-23; according to the DNA sequencing results and codon table, the nucleotide sequence of Nanobody Nb2-23 was obtained. amino acid sequence.
[0105] 2. Experimental results
[0106] The amino acid sequence of the VHH of Nanobody Nb2-23 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the Nanobody Nb2-23 is shown in SEQ ID NO.2.
[0107] The amino acid numbering and structural domain diagram of Nanobody Nb2-23 are as follows figure 2 As shown, it can be seen that the Nanobody Nb2-23 includes 4 framework regions (Framework region, FR) and 3 complementarity-determining regions (Complementarity-determining region, CDR); t...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More