CN114163526B - A nanobody targeting glucose regulatory protein 78 and its application - Google Patents

Abstract

The present invention provides a nanobody targeting glucose regulatory protein 78 and its application, wherein the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is CDR1 as shown in SEQ ID NO: 2, as shown in CDR2 shown in SEQ ID NO:4, CDR3 shown in SEQ ID NO:6; or CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:13 CDR3 shown. The nanobody targeting glucose-regulated protein 78 of the present invention has a nanomolar affinity with glucose-regulated protein 78, has high affinity, and has specific recognition ability for GRP78 protein on the cell membrane surface.

Description

A nanobody targeting glucose regulatory protein 78 and its application

technical field

The invention belongs to the technical field of immunology or molecular biology, and in particular relates to a nanobody targeting glucose regulatory protein 78 and its application.

Background technique

Glucose regulated protein 78 (glucose regulated protein 78 kDa, GRP78) is a protein with a molecular weight of 78 kDa discovered by IraPastan et al. in 1977 in chicken embryo fibroblasts cultured without glucose. (binding immunoglobulin protein, BIP) is exactly the same, so it is also called immunoglobulin heavy chain binding protein (BiP) is an important endoplasmic reticulum molecular chaperone. , metastasis, and angiogenesis are critical. The full-length GRP78 protein consists of two functional domains, including a nucleotide-binding domain (NBD), which can bind ATP, and a substrate-binding domain (SBD), which can bind polypeptides/proteins . GRP78 is widely present in the cytoplasm, but under stress conditions such as hypoxia, glucose deficiency, and pH reduction in the tumor microenvironment, it will be transported from the cytoplasm to the membrane surface, that is, cell surface GRP78 (csGRP78). Studies have found that csGRP78 is highly expressed in a variety of high-grade malignant tumors (such as TNBC, glioblastoma, gastric cancer); csGRP78 mainly exists in the form of peripheral proteins on the plasma membrane, and interacts with other cell surface proteins (such as glycosylphosphatidyl phosphatidylase). Inositol-anchored protein) interacts to mediate signal transduction in tumor cells.

In addition, the expression profile of the GPRP78 gene showed that among different cancer types, highly malignant and aggressive glioblastomas have the highest expression of csGRP78, as well as lung, breast, gastric, colon and liver cancers and angiogenic endothelial cells. High-expressed reports. These findings confirm that csGRP78 can be present on both malignant and endothelial cells, but is rarely expressed on normal cells, suggesting that csGRP78, a product of dysregulated endoplasmic reticulum protein homeostasis, may serve as a cancer cell-specific biomarker, A target for cancer imaging and therapy, and a potential tool for selective drug delivery.

The high molecular weight and poor tissue penetration of monoclonal antibodies greatly limit the effectiveness of monoclonal antibodies in solid tumors. The variable region (VHH) of the single-chain antibody (HCAb) found in Camelidae is the nanobody (Nb). The relative molecular mass of nanobodies is about 15 kDa, which is about 1/10 of that of traditional antibodies. Nanobodies have strong stability, good solubility, and prokaryotic expression. In addition, the nanobody gene sequence has 80% homology with the human VH gene family III sequence, so the immunogenicity is low. Finally, compared with the unrequited antibody scFv, the nanobody still retains better affinity and specificity. Therefore, nanobodies show unique potential in the fields of new drug development and disease diagnosis. But so far, there has been no related report on the nanobody with good binding to GRP78 protein and high affinity.

SUMMARY OF THE INVENTION

In view of the above technical problems, the present invention discloses a nanobody targeting glucose regulatory protein 78 and its application.

To this, the technical scheme adopted in the present invention is:

A nanobody targeting glucose regulatory protein 78, the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is CDR1 as shown in SEQ ID NO:2, as shown in SEQ ID NO:4 CDR2, CDR3 as shown in SEQ ID NO:6; Specifically, the sequence of CDR1 as shown in SEQ ID NO:2 is INTGNPALV, and the sequence of CDR2 as shown in SEQ ID NO:4 is SISGNTN, as shown in SEQ ID The sequence of CDR3 shown in NO:4 is KKLPFGS;

Or CDR1 as SEQ ID NO:9, CDR2 as SEQ ID NO:11, CDR3 as SEQ ID NO:13. Specifically, the sequence of CDR1 shown in SEQ ID NO: 9 is GFTLDYYAI, the sequence of CDR2 shown in SEQ ID NO: 11 is SSAGVLTN, and the sequence of CDR3 shown in SEQ ID NO: 13 is AAADARQLKVRQCLSSNAYTY.

As a further improvement of the present invention, the framework region of the amino acid sequence of the Nanobody targeting glucose regulatory protein 78 includes FR1 as shown in SEQ ID NO: 1, FR2 as shown in SEQ ID NO: 3, and FR2 as shown in SEQ ID NO: FR3 shown in SEQ ID NO:5, FR4 shown in SEQ ID NO:7.

As a further improvement of the present invention, the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is shown in SEQ ID NO: 15.

As a further improvement of the present invention, the framework region of the amino acid sequence of the Nanobody targeting glucose regulatory protein 78 includes FR1 shown in SEQ ID NO:8, FR2 shown in SEQ ID NO:10, and FR2 shown in SEQ ID NO:10. FR3 shown in SEQ ID NO:12, FR4 shown in SEQ ID NO:14.

As a further improvement of the present invention, the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is shown in SEQ ID NO: 16.

The nanobody of the technical solution of the present invention is an antibody that specifically targets glucose regulatory protein 78, can bind to target glucose regulatory protein 78, and the nanobody has high antigen binding, high affinity, low immunogenicity and relatively Strong tissue penetration.

The present invention also discloses a nucleic acid, which is: a nucleic acid encoding the nanobody targeting glucose regulatory protein 78 according to any one of the above or its complementary sequence.

The present invention also discloses an expression vector comprising the nucleic acid as described above.

The present invention also discloses a host cell comprising the above-mentioned expression vector.

The present invention also discloses the application of the above-mentioned nanobody targeting glucose regulatory protein 78, which is used as a detection reagent, in vivo imaging probe, chimeric immune cell or therapeutic antibody targeting glucose regulatory protein 78.

Compared with the prior art, the beneficial effects of the present invention are:

The nanobody targeting glucose regulatory protein 78 of the present invention has good binding activity to glucose regulatory protein 78, and the affinity with glucose regulatory protein 78 is at the nanomolar level, and has a relatively high affinity. specific recognition ability. Moreover, the nanobody has a smaller molecular weight, better tissue penetration, can be expressed and purified by using the Escherichia coli system, and the preparation cost is low.

Description of drawings

Figure 1 is a graph showing the expression results of GRP78 protein in cancer cell membranes and tumor tissue cancer cell membranes in the example of the present invention, wherein (a) is the expression result of GRP78 protein in 4T1 cell membrane of breast cancer tumor cells, (b) is the expression result of GRP78 protein in 4T1 mice GRP78 staining results of tumor tissue, (c) is the staining results of tumor tissue in gastric cancer cells MKN45 mice.

Figure 2 is an analysis diagram of the purification of GRP78 protein in the embodiment of the present invention; wherein (a) is the electrophoresis image of the full-length GRP78 protein, (b) is the immunoblot of the full-length GRP78 protein; (c) is the purification of the NBD domain protein of GRP78 The electropherogram of , (d) is the electropherogram of the purification of GRP78 SBD domain protein.

Figure 3 is the preliminary result of ELISA verification in the embodiment of the present invention; wherein (a) is the preliminary result of full-length protein phage ELISA verification, (b) is the preliminary result of NBD protein phage ELISA verification, (c) SBD protein phage ELISA verification Preliminary results.

Figure 4 is a diagram of the deep sequencing results of the library after screening of three GRP78 proteins according to the embodiment of the present invention, wherein (a) is the frequency distribution diagram of the deep sequencing sequence of the library obtained by the GRP78 full-length protein, (b) is the GRP78 NBD domain after screening. The frequency distribution of deep sequencing sequences of the library, (c) is the frequency distribution of deep sequencing sequences of the library after GRP78 SBD domain screening.

Figure 5 is the ELISA verification results of the NBD1 Nanobody and SBD C5 Nanobody of the embodiment of the present invention and the comparative Nanobody, wherein (a) is the NBD1 Nanobody, (b) is the SBD C5 Nanobody, (c) is the NBD C1 Nanobody Nanobodies, (d) are SBD2 Nanobodies, (e) are NGS3 Nanobodies.

6 is an analysis diagram of the results of surface plasmon resonance detection of the affinity of NBD1 Nanobody, SBD C5 Nanobody and full-length GRP78 according to the embodiment of the present invention, wherein (a) is NBD1 Nanobody, (b) is SBD C5 Nanobody.

Detailed ways

The preferred embodiments of the present invention will be further described in detail below.

1. In vitro detection of GRP78 membrane expression

Breast cancer cells 4T-1 were seeded on high-content 96-well microplates at the number of 10,000-20,000/well; after the cells adhered, they were washed 3 times with PBS, fixed with 0.25% paraformaldehyde for 10 min; blocked with 5% BSA for 1 h ; Dilute anti-GRP78 antibody (1:200) with PBST, incubate for 1 h at room temperature; wash 3 times with PBST, dilute fluorescent secondary antibody (1:1000) with PBST, incubate for 1 h at room temperature; wash 3 times with PBST, with DAPI-containing Mount the slides with a mounting medium, and detect the membrane expression of GRP78 by a high-content cell imager.

2. In vivo detection of GRP78 membrane expression

The tumor models of breast cancer 4T1 and gastric cancer MKN45 were constructed. When the tumor volume reached 300 mm ³ , the tumor tissues were collected, and 10 μm frozen sections were prepared. The expression of GRP78 protein in the membrane of tumor tissues was detected by immunofluorescence.

In this example, the membrane expression of GRP78 on cancer cells and frozen cancer tissues was detected. After fixation of breast cancer cells 4T1 and 4T1, and MKN45 mouse tumor tissues with low-concentration paraformaldehyde, GRP78 protein staining was performed. The results are shown in Figure 1. Show. As can be seen in Figure 1(a), the 4T1 cell membrane of breast cancer tumor cells highly expressed GRP78; as seen in Figure 1(b), GRP78 staining in the tumor tissue of 4T1 mice showed that the tumor cell membrane highly expressed GRP78; as seen in Figure 1(c), gastric cancer cells GRP78 staining in tumor tissue of MKN45 mice also indicated that GRP78 was highly expressed in the cancer cell membrane. Overall, GRP78 is highly expressed in cancer cell membranes, suggesting that GRP78 can be a potential therapeutic target for tumors.

3. Expression and purification of GRP78 full-length and NBD and SBD domains

The full-length, NBD and SBD gene sequences of GRP78 were cloned into the prokaryotic expression vector PET-14b. The expression purification steps are as follows:

a) Transformation: Add the plasmid to BL21 (DE3) competent cells, place on ice for 20 min, heat shock for 90 s, add LB medium and shake for 1 h, spread the bacterial solution on an ampicillin plate, and invert culture;

b) Mass induction: pick a single clone for amplification, add a certain concentration of isopropyl-β-D-thiogalactoside (IPTG) to induce overnight at 16 °C;

c) Collect bacteria by centrifugation and break bacteria by high pressure sterilizer;

d) Centrifuge at 12000 g for 1 h at 4 °C, take the supernatant and incubate with Ni-NTA resin for 1 h at 4 °C;

e) Ni column purification; f) Molecular sieve (AKTA purifier) for further separation and purification;

g) Identification of protein purity by SDS-PAGE electrophoresis.

GRP78 contains two domains, NBD and SBD, as shown in Figure 2. In this example, the full-length (Figure 2(a), Figure 2(b)) and NBD domains (Figure 2(c) were expressed and purified ), SBD domain (Fig. 2(d)) three proteins with molecular weights of about 80KD, 45KD and 35KD, respectively.

4. Nanobody library screening

A natural phage nanobody library constructed from the peripheral blood of 103 alpaca was used, and the capacity of the phage display library was 2×10 ⁹ . Screening using immunotube method. The filtering steps are as follows:

a) Coat the target protein on the immunotube at a concentration of 50 μg/mL, and perform 3 rounds of panning enrichment;

b) The third round of phage eluate was used to plate, and 96 single clones were randomly selected for ELISA verification. The positive standard was ELISA readings greater than 3 times that of BSA and readings greater than 0.5;

c) The positive monoclonal identified by Phase-ELISA will be sent to the company for sequencing.

Using the above three proteins, the nanobody phage library constructed from 103 alpaca peripheral blood was screened by immune tube method. After three rounds of screening, a total of 96 proteins were picked from the full-length protein library, the NBD protein library, and the SBD protein library. Cloning and preliminary verification by phage ELISA, the results are shown in Figure 3. As shown in Figure 3(a), the full-length protein phage ELISA verification found 7 potential positive clones with low affinity. As shown in Figure 3(b) and Figure 3(c), the phage ELISA of NBD and SBD respectively obtained one potential affinities. Good clone.

The verification experiment based on phage ELISA can only detect a limited number of clones, and cannot reflect the frequency distribution of clone sequences in the library after the overall screening. The results were analyzed, and a total of more than 20 million independent Nanobody sequences were obtained, and the obtained Nanobody sequences were sorted according to the frequency of occurrence, as shown in FIG. 4 . Sequences with higher frequencies were selected for the next protein expression and purification comparison experiments. Among them, there were five sequences with a frequency of more than 1% of the full length of GRP78, one sequence with a frequency of nearly 1% of the NBD domain, and one with a frequency of SBD exceeding 1%. The sequence is four.

Through a lot of experiments, NBD1 nanobody and SBD C5 nanobody were finally selected.

The framework region of the amino acid sequence of the NBD1 Nanobody is FR1 shown in SEQ ID NO:1, FR2 shown in SEQ ID NO:3, FR3 shown in SEQ ID NO:5, shown in SEQ ID NO:7 FR4; the complementarity determining region is CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:4, CDR3 as shown in SEQ ID NO:6; the amino acid sequence of the antibody is as shown in SEQ ID NO:15 shown. That is, the sequence of the NBD1 Nanobody is: MAVQLVESGGGSVQAGGSLTLSCAASINTGNPALVGWYRQAPGKQREMVAMISISGNTNYAPSVKGRFTISRDNANKTIFLQMNSLTPEDTAVYYCKKLPFGSWGQGTQVTVSS.

The framework regions of the amino acid sequence of the SBD C5 Nanobody are FR1 shown in SEQ ID NO:8, FR2 shown in SEQ ID NO:10, FR3 shown in SEQ ID NO:12, and FR3 shown in SEQ ID NO:14 The FR4 shown; the complementarity determining region is the CDR1 shown in SEQ ID NO: 9, the CDR2 shown in SEQ ID NO: 11, the CDR3 shown in SEQ ID NO: 13; the amino acid sequence of the antibody is shown in SEQ ID NO: 16 shown. That is, the sequence of the SBD C5 Nanobody is: MAVQLVESGGGLVQAGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSAGVLTNYVDSVKGRFTISRDNAKGAVYLQMNDLKPEDAALYFCAAADARQLKVRQCLSSNAYTYWGQGTQVTVSS.

5. Nanobody expression and purification:

The sequence of the nanobody coding region obtained by sequencing was constructed into a plasmid (with HA tag and His tag added), and expressed and purified in E. coli. The expression purification steps are similar to the antigen protein purification steps. An HA tag was added to the nanobody sequence for the detection of subsequent experiments. The obtained nanobody was about 15KD to 18KD in size, and the purity was over 90%.

6. ELISA Affinity Assay

The ELISA plate was coated with human full-length GRP78 and BSA protein was used as the control antigen. Nanobodies of different concentrations were incubated with the antigen, and then the HRP-labeled HA secondary antibody was used to amplify the signal and observe the color development. The nanobodies include NBD1 Nanobodies and SBD C5 Nanobodies, and also NBD C1 Nanobodies, SBD2 Nanobodies, NGS3 Nanobodies as comparisons. Specific steps include:

a) GRP78 protein was coated on Elisa plate at a concentration of 10 μg/mL, overnight at 4 °C;

b) Block with 5% BSA at room temperature for 2 h;

c) Wash twice with PBST, add doubling-diluted nanobody solution, and incubate at room temperature for 1 h;

d) Wash three times with PBST, add anti-HA HRP (1:3000) to each well, and incubate at room temperature for 1 h;

e) Wash three times with PBST, add TMB reader, observe the color change;

f) The reaction was terminated by adding 1 M HCl in time, and the light absorption value was read at 450 nm with a microplate reader.

Using human GRP78 full-length protein and bovine serum albumin as controls, the above nanobodies were verified by ELISA. The results are shown in Figure 3. It can be seen that SBD C5, NBD1, NBD C1, SBD2, and NGS3 nanobodies all bind to GRP78 to varying degrees, among which The two antibodies, SBD C5 and NBD1, had the best binding activity.

The following experiments will further use surface plasmon resonance (SPR) to detect the protein affinity of NBD1 Nanobody, SBD C5 Nanobody and GRP78.

7. Surface Plasmon Resonance (SPR) Identification of Affinity Constants of Nanobodies with GRP78

Using a Biacore T200 system, the GRP78 protein was adjusted to 1 mg/mL, dissolved in sodium acetate buffer at pH 5.5, and programmed to flow the protein solution through the chip, thereby immobilizing it by aminocarboxy coupling. on a negatively charged chip. Then the doubling-diluted nanobody solution flows through the chip, and the mutual binding affinity is calculated from its response value. The affinity KD is obtained by dividing the kinetic constants ka and kd.

Using the above steps, the affinity of NBD1 nanobody and SBD C5 nanobody to GRP78 full-length protein was determined by surface plasmon resonance technology. The affinity of the antibody to GRP78 was 92.45 nM, both reaching the nanomolar level.

The embodiment of the present invention also discloses a nucleic acid, which is: a nucleic acid encoding an NBD1 nanobody, an SBD C5 nanobody, or a complementary sequence thereof.

The embodiment of the present invention also discloses an expression vector, which comprises the nucleic acid as described above.

The embodiment of the present invention also discloses a host cell comprising the above-mentioned expression vector.

The embodiment of the present invention also discloses the application of the above-mentioned nanobody targeting glucose regulatory protein 78, which is used as a detection reagent, in vivo imaging probe, chimeric immune cell or therapeutic antibody targeting glucose regulatory protein 78 .

The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be considered that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, such as adding, reducing or replacing the original sequences of NBD1 Nanobody and SBDC5 Nanobody Such modified derivative antibodies should be regarded as belonging to the protection scope of the present invention.

sequence listing

<110> Shenzhen People's Hospital

<120> Nanobody targeting glucose-regulated protein 78 and its application

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 1

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser

20 25

<210> 2

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 2

Ile Asn Thr Gly Asn Pro Ala Leu Val

1 5

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 3

Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Met Val Ala Met

1 5 10 15

Ile

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 4

Ser Ile Ser Gly Asn Thr Asn

1 5

<210> 5

<211> 37

<212> PRT

<213> Artificial Sequence

<400> 5

Tyr Ala Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Asn Lys Thr Ile Phe Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys

35

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 6

Lys Lys Leu Pro Phe Gly Ser

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 7

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 8

<211> 26

<212> PRT

<213> Artificial Sequence

<400> 8

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 9

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 9

Gly Phe Thr Leu Asp Tyr Tyr Ala Ile

1 5

<210> 10

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 10

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Cys

1 5 10 15

Ile

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 11

Ser Ser Ala Gly Val Leu Thr Asn

1 5

<210> 12

<211> 37

<212> PRT

<213> Artificial Sequence

<400> 12

Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Lys Gly Ala Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Ala

20 25 30

Ala Leu Tyr Phe Cys

35

<210> 13

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 13

Ala Ala Ala Asp Ala Arg Gln Leu Lys Val Arg Gln Cys Leu Ser Ser

1 5 10 15

Asn Ala Tyr Thr Tyr

20

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 14

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 15

<211> 114

<212> PRT

<213> Artificial Sequence

<400> 15

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Ile Asn Thr Gly Asn Pro

20 25 30

Ala Leu Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Met

35 40 45

Val Ala Met Ile Ser Ile Ser Gly Asn Thr Asn Tyr Ala Pro Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Lys Thr Ile Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Lys Lys Leu Pro Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val

100 105 110

Ser Ser

<210> 16

<211> 129

<212> PRT

<213> Artificial Sequence

<400> 16

Met Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr

20 25 30

Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly

35 40 45

Val Ser Cys Ile Ser Ser Ala Gly Val Leu Thr Asn Tyr Val Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Gly Ala Val

65 70 75 80

Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Ala Ala Leu Tyr Phe

85 90 95

Cys Ala Ala Ala Asp Ala Arg Gln Leu Lys Val Arg Gln Cys Leu Ser

100 105 110

Ser Asn Ala Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

Claims (10)

1. The nanometer antibody of target glucose regulatory protein 78, characterized in that: the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is composed of CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 4 and CDR3 shown in SEQ ID NO. 6.

2. The nanobody targeting glucose regulatory protein 78 according to claim 1, characterized in that: the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 comprises FR1 shown in SEQ ID NO. 1, FR2 shown in SEQ ID NO. 3, FR3 shown in SEQ ID NO. 5 and FR4 shown in SEQ ID NO. 7.

3. The nanobody targeting glucose regulatory protein 78 according to claim 2, characterized in that: the amino acid sequence of the nano antibody of the targeted glucose regulatory protein 78 is shown in SEQ ID NO. 15.

4. Nanobodies targeting glucose regulatory protein 78, characterized in that: the complementarity determining region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 is composed of CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 11 and CDR3 shown in SEQ ID NO. 13.

5. The nanobody targeting glucose regulatory protein 78 according to claim 4, characterized in that: the framework region of the amino acid sequence of the nanobody targeting glucose regulatory protein 78 comprises FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 10, FR3 shown in SEQ ID NO. 12 and FR4 shown in SEQ ID NO. 14.

6. The nanobody targeting glucose regulatory protein 78 according to claim 5, characterized in that: the amino acid sequence of the nano antibody of the targeted glucose regulatory protein 78 is shown in SEQ ID NO 16.

7. A nucleic acid, wherein said nucleic acid is: nucleic acid encoding the nanobody targeting glucose regulatory protein 78 of any of claims 1 to 6, or a complementary sequence thereof.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell comprising the expression vector of claim 8.

10. Use of the nanobody targeting glucose regulatory protein 78 of any one of claims 1 to 6 in the preparation of a detection reagent targeting glucose regulatory protein 78, a biopsy probe, a chimeric immune cell or a therapeutic antibody.

Images