Method and system for screening nanobodies
A technology of nanobodies and antibodies, applied in the biological field, can solve the problems of screening nanobodies that need to be improved, long experimental cycle, and technical difficulty, and achieve the effects of shortening the screening preparation time, low cost, and high sequencing throughput
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Embodiment 1
[0154] Screening of nanobodies using immune library technology, the flow chart is shown in Figure 6 ,details as follows:
[0155] 1. Primers designed for enrichment of nanobodies
[0156] Using the antibodies of experimental animal species (dromedary camel, Bactrian camel, llama, etc.) in the IMGT database (http: / / www.imgt.org / ) as reference sequences, primers targeting the C region were designed. Figure 4 A schematic diagram of primer design for VHH, the heavy chain (Common VH) of a common diabody (ie, a traditional antibody) and the light chain (Light Chain) of a common diabody is shown. Among them, for VHH, the primers are designed for the CH2 region, for the heavy chain of the common diabody, the primers are designed for the CH1 region, and for the light chain of the common diabody, the primers are designed for the C region. In this example, the Oligo6 software was used for primer design, and the obtained primer sequences were delivered to a primer synthesis company fo...
Embodiment 2
[0245] In this example, on the basis of Example 1, further combined with protein profiling analysis technology to screen nanobodies, the flow chart of screening nanobodies using immune repertoire technology combined with protein profiling analysis technology is shown in Figure 7 ,details as follows:
[0246] 1. Protein Spectrum Experimental Techniques and Methods
[0247] 1. Affinity Purified Antibody
[0248] (1) IgG enrichment
[0249] The serum (i.e. peripheral blood) of camels immunized with antigens (such as CK18, 863-17 (NDKA)) removes fat, cell debris and small particles, and then enriches IgG with Protein A / G to obtain enrichment product. After the enrichment product is obtained, it is confirmed that the obtained enrichment product can specifically bind to the known antigen polypeptide used by Western blot test or enzyme-linked immunosorbent assay (ELISA).
[0250] (2) Affinity purification
[0251] The antigenic polypeptide used in Example 1 was connected to a c...
Embodiment 3
[0288] Synthesize the antibody gene sequence obtained by screening in Example 1, express the Nanobody through a protein expression system, and identify the Nanobody, as follows:
[0289] 1. Construction of expression vector
[0290] 1. Digestion
[0291] 20 μl plasmid pET28a DNA, 1 μl EcoRI, 1 μl BamHI, 5 μl 10×K buffer, 23 μl ddH 2 O, 37°C for 5h. Using an agarose gel DNA recovery kit, cut the gel and recover the target fragment (ie, the nanobody gene sequence) and the carrier.
[0292] 2. Connection, Transformation
[0293] (1) Connection:
[0294] 1 μl vector, 3 μl target fragment, 1 μl T4 ligase, 5 μl 2× buffer, mix well, and react at room temperature for more than 30 minutes.
[0295] (2) Conversion
[0296] 1) Take out the competent cells (BL21) stored at minus 80°C and thaw slowly on ice.
[0297] 2) Add competent cells to the ligation product or plasmid DNA (1 μl), mix well, and place on ice for 30 minutes.
[0298] 3) Heat shock at 42°C for 90s.
[0299] 4) A...
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