A kind of anti-her-2 heavy chain antibody and its application
A HER-2 and heavy chain antibody technology, applied in the field of molecular immunology, can solve the problems of cumbersome monoclonal antibody development and production process, low specificity and instability of polyclonal antibody, and achieve excellent genetic resources and antibody resources. , strong affinity, reliable source effect
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Embodiment 1
[0027] Example 1 Panning of anti-HER-2 Nanobodies
[0028] Single-domain heavy-chain antibodies against HER-2 were panned from a single-domain heavy-chain antibody phage display library by solid-phase affinity panning. Dilute HER-2 protein with 1×PBS solution to 30-100μg / μL, coat on ELISA plate, add 100μL to each well, and coat overnight at 4°C; aspirate the coating solution, wash the plate 3 times with PBS, add to each well 300 μL of 4% skim milk, blocked at 37°C for 2 h; after washing the plate 3 times with PBS, add the phage display library (about 1×10 12 CFU), incubate at 37°C for 1 h; aspirate the unbound phage, wash the plate 5 times with PBS (increase the number of washes round by round, and the number of washes in each round is shown in Table 1), and then wash the plate with PBST for 3 times and then add 100 μL to wash the plate Desaturate (glycine-hydrochloric acid solution, pH 2.2) to elute the phages adsorbed in the plate wells, incubate at 37°C for 5 min, gently p...
Embodiment 2
[0036] Example 2 Expression and purification of anti-HER-2 nanobodies
[0037] The gene of the nanobody obtained in Example 1 was cloned into the expression vector pET-25b (cloned by Qingke Biotechnology Co., Ltd.), and the anti-HER-2 nanobody expression plasmid was constructed. The constructed expression plasmid was transformed into Escherichia coli BL21, and single clone was picked for induced expression. The single clone was inoculated into 1L LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C, 220rmp / min shaking until the bacterial liquid OD600 reached 0.5, adding IPTG with a final concentration of 0.1mM, 16°C, 80rmp / min min overnight induction culture. After the cultivation, the cells were collected by centrifugation, resuspended in 50 mL of PBS solution, and then sonicated on ice. The conditions were 200 w, crushed for 3 s, intermittent for 4 s, for a total of 30 min, and the supernatant was collected by centrifugation at 8000 g at 4 °C.
[0038] T...
Embodiment 3
[0039] Example 3 Affinity determination of anti-HER-2 nanobodies
[0040] The nanobody prepared in Example 2 was tested for affinity using the Octet@RED96 intermolecular interaction detection system (ForteBio Company). Octet@RED96 Intermolecular Interaction Detection System is based on biofilm layer interferometry (BLI) technology, which can measure the interaction between proteins and biomolecules without labeling with only a small amount of sample.
[0041] The human HER-2 protein was diluted to 10 μg / mL, and the Nanobody obtained in Example 2 was diluted to 50 μg / mL. The dilution used was PBS+0.1% Tween 20+0.1% BSA, and the samples were added according to Table 3. Affinity test chart see Figure 4 .
[0042] Table 3 Affinity detection sample loading table
[0043]
[0044] Equilibrium dissociation constant (affinity) K D (M)=k dis (1 / s) / k on (1 / Ms)
[0045] It has been detected:
[0046] k dis (1 / s)=0.0004768;
[0047] k on (1 / Ms)=567.7;
[0048] K D (M)=k ...
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