Nano antibody targeting cadherin 17 and application thereof
A technology of cadherin and nano-antibody, which is applied in the field of immunology or molecular biology, and can solve problems such as poor antibody affinity
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Embodiment 1
[0050] Example 1 Expression of cancer cell CDH17
[0051] AGS and IM95 cells were revived, and after three passages, the cells were seeded in 24-well plates for subsequent experiments. Specific steps are as follows:
[0052] a), the cells were placed on ice, washed 3 times with PBS, and fixed with 4% paraformaldehyde for 10 minutes;
[0053] b), washed 3 times with PBS, blocked with 4% donkey serum for 1 hour;
[0054] c) Dilute CDH17 antibody 1:100 with blocking solution and incubate with cells for 1 hour;
[0055] d), wash with pre-cooled PBS for 3 times, dilute the Alexa 488-conjugated secondary antibody with blocking solution at 1:1000, and incubate with the cells for 1 hour;
[0056] e), wash with pre-cooled PBS for 3 times, seal with DAPI-containing mounting medium, detect the expression of CDH17 protein under a fluorescent inverted microscope, and obtain Figure 1A and Figure 1B .
[0057] Depend on Figure 1A It can be seen that the high expression of CDH17 on th...
Embodiment 2
[0058] Example 2 Screening and validation of CDH17 protein extracellular domain nanobody antibody
[0059] 1) Expression and purification of CDH17 domain
[0060] refer to Figure 2A , according to the extracellular structure of CDH17, design and synthesize CDH17 protein 1~3 domain gene and express purified protein for nanobody screening.
[0061] Expression purification steps are as follows:
[0062] a), in order to prevent the formation of inclusion bodies and protein degradation, it is planned to use different concentrations of IPTG to explore the induction conditions at a low temperature of 16°C;
[0063] b) According to the pre-experiment induction conditions, a large amount of induced expression is carried out, and the working condition of the high-pressure bacteriostasis instrument is 1000 W to destruct bacteria;
[0064] c), 17000 g, centrifuge at 4°C for 30 min, take the supernatant and incubate with Ni filler at 4°C for 1 hour;
[0065] d) Molecular sieve separat...
Embodiment 3
[0076] Example 3 Deep Sequencing of Nanobody Library
[0077] On the basis of the nanobody phage display technology screening clones through phage ELISA, more clones with potential binding ability can be mined through deep sequencing.
[0078] The specific steps of deep sequencing are as follows:
[0079] a), extracting the genome of the phages selected by three rounds of enrichment screening, amplifying and enriching the nanobody sequence by PCR to establish a library;
[0080] b), analyzing the enriched sequences in the library by high-throughput sequencing;
[0081] c), align the sequenced sequence to the nanobody sequence library used for phage display, and analyze and rank the enrichment degree of the sequence.
[0082] Through deep sequencing technology, we obtained about 21 trillion nanobody sequence data, as shown in Table 1 below.
[0083] Table 1: Nanobody sequences identified and screened by deep sequencing
[0084]
[0085]It can be seen from Table 1 that th...
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