A kind of anti-his tag heavy chain antibody and application thereof

Abstract

The invention relates to the field of molecular immunology, and particularly provides an anti-His label heavy chain antibody. Sequences of three complementary decision regions CDR and four frame regions FR are CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, CDR3 shown in SEQ ID NO:3, FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:5, FR3 shown in SEQ ID NO:6 and FR4 shown in SEQ ID NO:7. The invention further provides a preparation method and application of the anti-His label heavy chain antibody. The affinity of the heavy chain antibody and the anti-His label protein reach 7.11*10<-8>,and the antibody has the advantages of being low in molecular weight, easy to express, low in cost, good in stability and the like.

Description

technical field

[0001] The invention belongs to the field of molecular immunology, and specifically relates to a phage display library and a nanobody recombinant expression technology, in particular to a heavy chain antibody specifically recognizing a His tag and an application thereof. Background technique

[0002] Heavy chain antibody (heavy chain antibody, HcAb) is found in camels and sharks. It is an antibody that naturally lacks light chains and only consists of heavy chains. Cloning its variable region can obtain a single-domain antibody composed of only the variable region of the heavy chain, called VHH (Variable domain of heavy chain of heavy chain antibody), also known as nanobody (nanobody), which is the smallest functional antigen Combine fragments. Unlike ordinary antibodies, nanobodies are a peptide chain containing about 110 amino acids, and their molecular weight is about 1 / 10 of that of ordinary antibodies. Compared with ordinary antibodies and recombinant s...

Examples

Embodiment 1

[0027] Example 1 Panning of Anti-His Tag Nanobodies

[0028] The single domain heavy chain antibody targeting His tag was panned from the single domain heavy chain antibody phage display library by solid phase affinity panning. Dilute the His-tagged protein to 30-100 μg / μL with 1×PBS solution, coat it on an ELISA plate, add 100 μL to each well, and coat overnight at 4°C; suck out the coating solution, wash the plate 3 times with PBST, and wash each well 3 times. Add 300 μL of 4% skimmed milk, block at 37°C for 2 hours; wash the plate with PBST three times, add the phage display library (containing about 1×10 12 CFU), incubated at 37°C for 1 h; aspirate the unbound phage, wash the plate 5 times with PBST (increase the number of washes for each round, and the number of washes for each round is shown in Table 1), wash the plate with PBST for 3 times, and then add 100 μL wash Remove the liquid (glycine-hydrochloric acid solution, pH 2.2) to elute the phage adsorbed in the plate w...

Embodiment 2

[0037] Example 2 Expression and purification of anti-His tag nanobody

[0038] The gene of the nanobody obtained in Example 1 was cloned into the expression vector pET-25b (cloned by Qingke Biological Co., Ltd.), and an anti-His tag nanobody expression plasmid was constructed. The constructed expression plasmid was transformed into Escherichia coli BL21, and a single clone was picked for induced expression. Inoculate the single clone into 1L LB liquid medium (containing 100 μg / mL ampicillin) for culture, shake culture at 37°C, 220rmp / min until the OD600 of the bacterial solution reaches 0.5, add IPTG with a final concentration of 0.1mM, and grow at 16°C, 80rmp / min min overnight induction culture. After the culture was completed, the cells were collected by centrifugation, resuspended in 50 mL PBS solution, and then ultrasonically disrupted on ice at 200w for 3 seconds with an interval of 4 seconds for a total of 30 minutes. The supernatant was collected by centrifugation at 8...

Embodiment 3

[0040] Example 3 Affinity Determination of Anti-His Tag Nanobody

[0041] Use Octet @ The RED96 intermolecular interaction detection system (ForteBio Company) was used to measure the affinity of the Nanobodies prepared in Example 2. Octet @ RED96 intermolecular interaction detection system is based on biofilm laminography interferometry (BLI) technology, which can measure the interaction between proteins and biomolecules with only a small amount of sample and without labeling.

[0042] The His-tagged protein was diluted to 10 μg / mL, and the nanobody obtained in Example 2 was diluted to 50 μg / mL. The diluent used was PBS+0.1% Tween 20+0.1% BSA, and the samples were loaded according to Table 3. Affinity detection chart see Figure 4 .

[0043] Table 3 Affinity detection sample loading table

[0044]

[0045] Equilibrium dissociation constant (affinity) K D (M)=k dis (1 / s) / k on (1 / Ms)

[0046] It was detected that:

[0047] k dis (1 / s) = 0.009213;

[0048] k on (1...

technical field

[0001] The invention belongs to the field of molecular immunology, and specifically relates to a phage display library and a nanobody recombinant expression technology, in particular to a heavy chain antibody specifically recognizing a His tag and an application thereof. Background technique

[0002] Heavy chain antibody (heavy chain antibody, HcAb) is found in camels and sharks. It is an antibody that naturally lacks light chains and only consists of heavy chains. Cloning its variable region can obtain a single-domain antibody composed of only the variable region of the heavy chain, called VHH (Variable domain of heavy chain of heavy chain antibody), also known as nanobody (nanobody), which is the smallest functional antigen Combine fragments. Unlike ordinary antibodies, nanobodies are a peptide chain containing about 110 amino acids, and their molecular weight is about 1 / 10 of that of ordinary antibodies. Compared with ordinary antibodies and recombinant s...

Examples

Embodiment 1

[0027] Example 1 Panning of Anti-His Tag Nanobodies

[0028] The single domain heavy chain antibody targeting His tag was panned from the single domain heavy chain antibody phage display library by solid phase affinity panning. Dilute the His-tagged protein to 30-100 μg / μL with 1×PBS solution, coat it on an ELISA plate, add 100 μL to each well, and coat overnight at 4°C; suck out the coating solution, wash the plate 3 times with PBST, and wash each well 3 times. Add 300 μL of 4% skimmed milk, block at 37°C for 2 hours; wash the plate with PBST three times, add the phage display library (containing about 1×10 12 CFU), incubated at 37°C for 1 h; aspirate the unbound phage, wash the plate 5 times with PBST (increase the number of washes for each round, and the number of washes for each round is shown in Table 1), wash the plate with PBST for 3 times, and then add 100 μL wash Remove the liquid (glycine-hydrochloric acid solution, pH 2.2) to elute the phage adsorbed in the plate w...

Embodiment 2

[0037] Example 2 Expression and purification of anti-His tag nanobody

[0038] The gene of the nanobody obtained in Example 1 was cloned into the expression vector pET-25b (cloned by Qingke Biological Co., Ltd.), and an anti-His tag nanobody expression plasmid was constructed. The constructed expression plasmid was transformed into Escherichia coli BL21, and a single clone was picked for induced expression. Inoculate the single clone into 1L LB liquid medium (containing 100 μg / mL ampicillin) for culture, shake culture at 37°C, 220rmp / min until the OD600 of the bacterial solution reaches 0.5, add IPTG with a final concentration of 0.1mM, and grow at 16°C, 80rmp / min min overnight induction culture. After the culture was completed, the cells were collected by centrifugation, resuspended in 50 mL PBS solution, and then ultrasonically disrupted on ice at 200w for 3 seconds with an interval of 4 seconds for a total of 30 minutes. The supernatant was collected by centrifugation at 8...

Embodiment 3

[0040] Example 3 Affinity Determination of Anti-His Tag Nanobody

[0041] Use Octet @ The RED96 intermolecular interaction detection system (ForteBio Company) was used to measure the affinity of the Nanobodies prepared in Example 2. Octet @ RED96 intermolecular interaction detection system is based on biofilm laminography interferometry (BLI) technology, which can measure the interaction between proteins and biomolecules with only a small amount of sample and without labeling.

[0042] The His-tagged protein was diluted to 10 μg / mL, and the nanobody obtained in Example 2 was diluted to 50 μg / mL. The diluent used was PBS+0.1% Tween 20+0.1% BSA, and the samples were loaded according to Table 3. Affinity detection chart see Figure 4 .

[0043] Table 3 Affinity detection sample loading table

[0044]

[0045] Equilibrium dissociation constant (affinity) K D (M)=k dis (1 / s) / k on (1 / Ms)

[0046] It was detected that:

[0047] k dis (1 / s) = 0.009213;

[0048] k on (1...

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