CN114591436A

CN114591436A - Specific antibody of pepsinogen I as well as preparation method and application thereof

Info

Publication number: CN114591436A
Application number: CN202011418752.1A
Authority: CN
Inventors: 刘剑峰; 张胜蓝
Original assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Current assignee: Huazhong University of Science and Technology; Bioisland Laboratory
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-06-07
Anticipated expiration: 2040-12-07
Also published as: CN114591436B

Abstract

The present invention relates to an antigen binding protein, antibody or antibody active fragment obtained by immunizing camelids with pepsinogen I. The invention relies on the immune system of camelid to screen, identify and prepare the antibody which is specifically identified and combined with pepsinogen I, and the obtained antibody has strong specificity, can be used for stomach lesion detection and has potential clinical diagnosis and treatment values; the antibody provided by the invention has the advantages of simple structure, easiness in genetic engineering transformation, easiness in humanization realization, high stability, low mass production cost and contribution to large-scale production.

Description

Specific antibody of pepsinogen I and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a specific antibody of pepsinogen I, a preparation method and application thereof.

Background

Antibodies are proteins secreted primarily by plasma cells and used by the immune system to identify and neutralize foreign substances, such as bacteria, viruses, etc., called antigens. The binding of antibodies to antigens relies entirely on noncovalent interactions, and this specific binding mechanism allows the antibodies to capture foreign microorganisms as well as infected cells, further induce other immune mechanisms to attack them, or directly neutralize their targets. Antibodies and antibody-related products have been widely used in the research fields of life science and medicine, and many experimental techniques derived based on antigen-antibody specific binding lay important foundations for scientific research and clinical treatment, such as immunodiagnosis, immunoblotting, enzyme-linked immunosorbent, flow cytometry analysis, and the like.

Pepsinogen I (PGI) is one of the two pepsin precursors secreted by the stomach and involved in digestion. PGI is a pointer for detecting the function of the cells of the gastric acid gland, and the PGI is increased due to the increase of gastric acid secretion, and the PGI is decreased due to the reduction of secretion or the atrophy of gastric mucosa glands. Therefore, monitoring the concentration of PG I in serum can be used as a means to monitor the status of gastric mucosa: (1) and (3) helicobacter pylori infection screening: the PGI content in the blood of the infected patient is obviously increased, and the content is reduced after recovery; (2) gastric ulcer: the increase of the serum PGI content indicates that gastric ulcer possibly exists in the human body, and particularly the serum PGI content of a patient in an active period or with the patient is obviously increased; (3) gastric cancer: high-risk signals of gastric cancer when the content of PGI in serum is reduced, and the early diagnosis of gastric cancer is facilitated.

At present, the PGI detected by scientific research or clinical diagnosis mainly depends on antibodies, various PGI monoclonal antibodies are disclosed or sold on the market, and the technical scheme for preparing the antibodies is as follows: (1) extracting or recombining and expressing PGI protein antigen from the tissue; (2) injecting the antigen into the bodies of animals such as mice, rabbits, monkeys and the like, and generating antibodies by depending on the immune system of the animals; (3) collecting animal blood, separating, extracting serum, and further separating to obtain components containing specific antibody.

However, the above-mentioned technology of generating antibodies against PGI based on the immune system of animals such as mice and isolating and extracting them has the following disadvantages: (1) the stability of the antibody is poor, the characteristics of the concentration, the specificity and the like of the antibody can be maintained only by storing, transporting, testing, verifying and the like at low temperature (4 ℃), and the long-distance transportation, the popularization and the detection are not facilitated; (2) the scale batch production of finished products is high. The monoclonal antibody is a full-length immunoglobulin (IgG), needs repeated immunization and injection of animals and separation and purification of the antibody from animal fluid, or separation, purification and extraction after recombinant expression in expensive expression systems such as mammalian cells, and the like, has complex operation and high cost, and is not beneficial to large-scale batch production.

Disclosure of Invention

The invention overcomes the defects of the prior art, designs and implements an effective and feasible antibody screening and preparation technical scheme, obtains the antibody which can specifically recognize and combine with pepsinogen I, and performs batch production preparation and application thereof.

The above object of the present invention is achieved by the following embodiments.

In a first aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment obtained by immunising a camelid with pepsinogen I.

In some embodiments, the camelid is selected from dromedary, bactrian, llama, alpaca and llama, preferably alpaca.

In some embodiments, the antibody is a nanobody and the antibody-active fragment is a nanobody-active fragment.

In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

In some embodiments, the antigen binding protein, antibody or antibody active fragment binds to the pepsinogen I with a kd value below 1200nM, preferably below 500nM, more preferably below 200nM, more preferably below 150 nM.

In some embodiments, the pepsinogen I has an amino acid sequence as shown in SEQ ID NO 1. Further preferably, the pepsinogen I is prepared by a method comprising the following steps: constructing a nucleotide sequence encoding the pepsinogen I into a vector plasmid; and (3) transfecting the vector plasmid into a eukaryotic cell line for expression and purification.

In a second aspect, the present invention provides a method of constructing an antibody library, the method comprising the steps of:

(1) immunizing camelidae animals by taking pepsinogen I as an antigen, collecting venous peripheral blood of the immunized animals, and separating to obtain lymphocytes;

(2) extracting total mRNA of the lymphocytes, performing reverse transcription on the total mRNA into cDNA, and amplifying the cDNA;

(3) and inserting the amplified DNA into a virus expression vector, transforming the virus expression vector into bacteria, and collecting bacterial colonies to obtain an antibody library.

In some embodiments, the immunizing of step (1) is performed by subcutaneous injection. The frequency of immunization is preferably 3-5 times. The venous peripheral blood is preferably collected before and after the last immunization, respectively.

In some embodiments, the viral expression vector of step (3) is a phage expression vector.

In some embodiments, the bacterium of step (3) is escherichia coli.

In a third aspect, the present invention provides an antibody library obtained by the above method for constructing an antibody library, or a polyclonal antibody produced by expression of the antibody library.

In a fourth aspect, the present invention provides a method of constructing an antigen-specific antibody library, the method comprising the steps of: screening the antibody library of the third aspect to obtain an antigen-specific antibody library.

In some embodiments, the method of constructing an antigen-specific antibody library comprises the steps of:

(i) culturing the antibody library to release viruses;

(ii) incubating the virus with an antigen, removing the virus non-specifically bound to the antigen, and retaining the virus specifically bound to the antigen;

(iii) infecting bacteria with the virus specifically bound to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

In some embodiments, the bacterium of step (iii) is escherichia coli.

In a fifth aspect, the present invention provides an antigen-specific antibody library obtained by the above-described method for constructing an antigen-specific antibody library, or a polyclonal antibody specifically binding to an antigen produced by expression of the antigen-specific antibody library.

In a sixth aspect, the present invention provides a method of preparing an antigen binding protein, antibody or antibody active fragment, said method comprising the steps of: screening the antibody library of the third aspect to obtain an antigen-binding protein, an antibody or an antibody active fragment specifically binding to an antigen.

In some embodiments, the method of making an antigen binding protein, antibody or antibody active fragment comprises the steps of:

(a) culturing the antibody library to release viruses;

(b) incubating the virus with an antigen, removing the virus non-specifically bound to the antigen, and retaining the virus specifically bound to the antigen;

(c) infecting bacteria with the virus specifically bound to the antigen, smearing the infected bacteria on a plate culture medium for culture, and selecting a single colony.

In some embodiments, the bacterium of step (c) is escherichia coli.

In some embodiments, the single colony may be expanded for culture prior to antigen-specific binding identification.

In some embodiments, the single colony may be expanded and then subjected to step (d): the DNA is extracted, transformed into host cells and expressed to obtain monoclonal antibodies.

In a seventh aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by the above-described method for producing an antigen-binding protein, an antibody or an antibody active fragment.

In an eighth aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds pepsinogen I; the antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5;

CDR2 shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9; and

CDR3 shown in SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 6 and CDR3 shown in SEQ ID NO. 10.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 7 and CDR3 shown in SEQ ID NO. 11.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 12.

In some embodiments, the heavy chain variable region has: CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 9 and CDR3 shown in SEQ ID NO. 13.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 14 or conservative variants obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 14.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 15 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 15.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 16 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 16.

In some embodiments, the heavy chain variable region has: the amino acid sequence shown as SEQ ID NO. 17 or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 17.

In some embodiments, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

In a ninth aspect, the present invention provides a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO 2 to SEQ ID NO 17 or an antigen binding protein, antibody or antibody active fragment as described above.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 18.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 19.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 20.

In some embodiments, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is set forth in SEQ ID NO 21.

In a tenth aspect, the present invention provides an expression vector comprising the nucleotide sequence described above.

In some embodiments, the expression vector is a phage expression vector, preferably a phage surface display screening vector.

In some embodiments, the expression vector further comprises a nucleotide sequence encoding the phage envelope protein pIII.

In an eleventh aspect, the present invention provides a virus exogenously transferred with the expression vector described above.

In some embodiments, the virus is a bacteriophage.

In a twelfth aspect, the present invention provides a host cell exogenously transformed with the expression vector described above, or infected with the virus described above.

In some embodiments, the host cell is e.

In a thirteenth aspect, the invention provides a method of expressing an antigen binding protein, antibody or antibody active fragment using a host cell as described above.

In a fourteenth aspect, the present invention provides an antigen binding protein, antibody or antibody active fragment obtained by expression using a host cell as described above.

In a fifteenth aspect, the present invention provides a humanized antigen binding protein, antibody or antibody active fragment obtained by humanizing said antigen binding protein, antibody or antibody active fragment described above.

In a sixteenth aspect, the present invention provides a protein conjugate comprising an antigen binding protein, antibody or antibody active fragment as described above or a humanized antigen binding protein, antibody or antibody active fragment as described above and a ligand.

In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorophore, and a delivery vehicle.

In a seventeenth aspect, the present invention provides a kit for detecting the amount of pepsinogen I in a sample comprising an antigen binding protein, antibody or antibody active fragment as described above or a humanized antigen binding protein, antibody or antibody active fragment as described above.

In some embodiments, the antigen binding protein, antibody or antibody active fragment is labeled with a label. Preferably, the label is selected from the group consisting of an enzyme, a chemiluminescent group and an isotopic group.

In some embodiments, the sample is animal serum, preferably human serum.

In an eighteenth aspect, the present invention provides the use of an antigen binding protein, antibody or antibody active fragment as described above, a humanized antigen binding protein, antibody or antibody active fragment as described above, a protein conjugate as described above or a kit as described above for detecting the pepsinogen I content in a sample.

In some embodiments, the sample is animal serum, preferably human serum.

In a nineteenth aspect, the present invention provides a method for detecting pepsinogen I content in a sample using an antigen binding protein, an antibody or an antibody active fragment as described above, a humanized antigen binding protein, an antibody or an antibody active fragment as described above, a protein conjugate as described above, or a kit as described above.

In some embodiments, the sample is animal serum, preferably human serum.

Compared with the prior art, the technical scheme provided by the invention has the following remarkable advantages: the antibody provided by the invention can be used for detecting the content of PGI in serum, is further used for judging stomach pathological changes, and has potential clinical diagnosis and treatment values; the antibody provided by the invention has a simple structure, is easy to carry out genetic engineering transformation, has a mature optimization strategy for enhancing the affinity of the nano antibody, prolonging the half-life period in vivo and coupling with other molecules for drug development, such as connecting radioactive isotopes, coupling transfer drugs, CART, fluorescence labeling high-resolution imaging and the like; the antibody sequence provided by the invention has high homology with the VH region sequence of human IgG, and the humanization of the single-domain antibody can be realized through a few amino acid mutations; the antibody provided by the invention has high stability, acid and pH resistance and high temperature resistance, can avoid the requirement that the conventional antibody needs low-temperature storage and transportation, is beneficial to large-scale popularization and application, has low volume production cost, and is easy for large-scale recombinant preparation. The monoclonal nano antibody designed by the invention can be well recombined and expressed in an escherichia coli expression system with low cost, the mass production cost is low, and the yield can reach dozens of milligrams per liter of escherichia coli. The escherichia coli recombinant expression system is mature in technology and simple in quality control, and is beneficial to reducing the production cost and realizing large-scale production.

Drawings

FIG. 1 is a schematic diagram showing the result of detecting the affinity between monoclonal antibody PGI _1G1 and antigen;

FIG. 2 is a schematic diagram showing the result of detecting the affinity between monoclonal antibody PGI _1G9 and antigen;

FIG. 3 is a schematic diagram showing the result of detecting the affinity between monoclonal antibody PGI _1H10 and antigen;

FIG. 4 is a schematic diagram of the result of detecting the affinity between monoclonal antibody PGI _2F1 and antigen.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Definition of

Pepsinogen I: pepsinogen I (PGI) is one of the two pepsin precursors secreted by the stomach and involved in digestion, synthesized by the cells of the pepsin and mucous neck cells of the fundal glands of the stomach and/or synthesized by the cells of the mucous neck of the cardia and antral pyloric glands of the stomach and the upper part of the duodenum and secreted into the stomach or intestine. Human PG I is a 388-amino acid polypeptide chain which is inactive per se and is cleaved by gastric acid or activated Pepsin in the gastric cavity to form mature Pepsin (Pepsin). Pepsin is capable of hydrolyzing proteins in food, and it acts mainly on peptide bonds containing phenylalanine or tyrosine in protein and polypeptide molecules. Typically, about 1% of the pepsinogen permeates the capillaries of the gastric mucosa and enters the blood circulation, and the pepsinogen entering the blood circulation is very stable in the blood. When the gastric mucosa is pathologically changed, the content of pepsinogen in serum is changed.

kd value: dissociationThe constant (kd) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from another smaller component, and is the reciprocal of the association constant in mol/L (M) or nmol/L (nM). A smaller kd value indicates a stronger binding ability of the two substances.

Nanobodies: naturally light chain-deficient antibodies found in the peripheral blood of camelids, which antibodies comprise only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but do not adhere to each other, or even aggregate, as readily as artificially engineered single chain antibody fragments; the VHH structure which is cloned and expressed independently has the structural stability which is equivalent to that of the original heavy chain antibody and the binding activity with antigen, and is the minimum unit which is known to be combined with target antigen; the VHH crystal is 2.5nm, 4nm long and has a molecular weight of only 15KDa, so the VHH crystal is also called a Nanobody (Nb). Compared with the traditional animals such as mice, rabbits and the like which can only recognize the polypeptide with flat antigen surface, the immune system in the camelid animal body can recognize the complex spatial structure of the antigen surface and can generate the nano antibody with high specificity and high affinity.

According to the technical scheme of the invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see the following table 1:

TABLE 1

Furthermore, because of the degeneracy of bases, substitutions can be made to bases of a polynucleotide sequence without altering the activity or function of the polynucleotide sequence, see table 2 below:

TABLE 2

Example 1: preparation of antigens

Constructing a DNA sequence coding the pepsinogen I into a pFASTBac-HTA expression vector, carrying out recombinant expression on PGI with a histidine tag (6 His-tag) in SF9 insect cells, extracting and purifying to obtain the antigen.

The antigen has an amino acid sequence shown as SEQ ID NO. 1, and specifically comprises the following components: MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNPARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGSSNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEGSCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

Example 2: alpaca immune injection

This example immunizes alpaca with the antigen prepared in example 1. The method comprises the following specific steps:

(1) the antigen prepared in example 1 was divided into 4 portions on average, each of about 0.25 mg;

(2) 4 times of immunization is carried out on the alpaca in an accumulated way, the antigen is injected into the animal body subcutaneously, the first immunization is recorded as the first day, and the subsequent immunizations are respectively carried out on the 10 th day, the 19 th day and the 28 th day; on day 28, about 200mL of alpaca venous peripheral blood was collected before the fourth immunization injection, and on day 42, 14 days after the fourth immunization, about 300mL of alpaca venous peripheral blood was collected.

Compared with the traditional immunization technical scheme of animal antibodies such as mice and rabbits, the method provided by the embodiment collects a large amount of alpaca vein peripheral blood, and is beneficial to obtaining highly diversified nano antibodies through subsequent screening.

Example 3: construction of antibody libraries

Two batches of alpaca venous peripheral blood collected in example 2 were used as raw materials to construct a highly diverse nanobody library. The method for treating the peripheral blood of the alpaca veins of two batches is the same, and specifically comprises the following steps:

(1) separating lymphocytes from the peripheral blood of the alpaca veins by using a density gradient centrifugation method;

(2) extracting total mRNA of the lymphocyte and performing reverse transcription to obtain cDNA;

(3) using proper DNA primer, using the cDNA as template, obtaining VHH fragment of alpaca immunoglobulin IgG2 and IgG3 by Polymerase Chain Reaction (PCR) amplification, namely DNA fragment of nano antibody;

(4) connecting the DNA of the VHH to a phage surface display screening vector to form a VHH-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on a bacteriophage surface flagellum;

(5) transforming the DNA connecting product to TG1 competent bacteria by an electric transformation method, and collecting all colonies after proper culture, namely the nano antibody library of the alpaca.

Compared with the traditional method for separating the antibody from the serum or the lymphocyte of the animal such as the mouse, the rabbit and the like, the method can obtain and store all nano antibody fragments (namely the library) of the alpaca for a long time, and can continuously support the follow-up continuous screening and development of the nano antibody.

Example 4: phage surface display screening specific nano antibody

In this embodiment, the nanobody library obtained in example 3 is used as a source, and the phage surface display screening is performed to obtain the antigen-specific nanobody. The method comprises the following specific steps:

(1) taking a proper amount of the cryopreserved nano antibody library obtained in the embodiment 3, inoculating the cryopreserved nano antibody library to a bacterial culture medium, adding a proper amount of helper phage after proper culture, and continuously culturing under a proper amount of conditions;

(2) extracting the amplified phage in the bacterial culture supernatant by a PEG-NaC method;

(3) fixing the antigen obtained in example 1 in an immune test tube, and taking phage and incubating the antigen;

(4) elutriation: discarding the phage, rinsing the antigen for a proper number of times by using PBS buffer solution, elutriating and removing the phage non-specifically combined with the antigen, and reserving the phage specifically combined with the antigen;

(5) and (3) elution: and treating the bacteriophage specifically bound with the antigen by using an acidic glycine solution to dissociate and retain the bacteriophage from the antigen.

Thus, the phage expressing the specific nano antibody is obtained.

Example 5: construction of an antigen-specific antibody library

This example uses the phage obtained in example 4 to construct an antigen-specific nanobody library. The method comprises the following specific steps:

(1) infecting the phage expressing the specific nano antibody with escherichia coli cultured to a proper state, but not adding auxiliary phage;

(2) after the phage is completely infected, the specific nano antibody exists in Escherichia coli in the form of DNA plasmid, and all Escherichia coli are collected to obtain antigen-specific nano antibody library.

The library obtained in this example can be returned as a raw material to example 4 for phage surface display screening.

Example 6: obtaining monoclonal antibody colonies

This example uses the phage obtained in example 4 to obtain monoclonal nanobody colonies. The method comprises the following specific steps:

(1) infecting and culturing the phage with the specific nano antibody to Escherichia coli in a proper state, but not adding auxiliary phage;

(2) after the bacteriophage is completely infected, the escherichia coli is evenly smeared on a bacterial culture dish for culture, and then the monoclonal colony containing the DNA plasmid of the nano antibody can be obtained.

Example 7: identification of Positive monoclonal antibodies

This example identifies the monoclonal colonies obtained in example 6. The method comprises the following specific steps:

(1) selecting 4 groups of the monoclonal colonies to be cultured in a micropore plate respectively;

(2) adding IPTG to induce expression of VHH-pIII (namely the fusion protein containing the nano antibody);

(3) collecting bacterial culture supernatant containing the nano antibody, and incubating with the antigen;

(4) and detecting whether the monoclonal nano antibody is combined with the pepsinogen I antigen or not by adopting an enzyme-linked immunosorbent assay (ELISA) method, and detecting the affinity of the antibody and the antigen according to the combination strength.

The results of the detection of the affinity between the antibody corresponding to the 4 groups of monoclonal colonies and the pepsinogen I antigen are shown in FIGS. 1 to 4, and the results of the affinity values kd are shown in Table 3 below.

Table 3: results of affinity assay

	kd(nM)
		PGI_1G1	152.9
PGI_1G9	1034.0
		PGI_1H10	123.3
PGI_2F1	404.0

From the above results, it is understood that the antibody obtained by selecting the monoclonal colonies of the present example has a kd value of 1200nM or less, preferably 500nM or less, more preferably 200nM or less, and still more preferably 150nM or less, for binding to the pepsinogen I.

Respectively carrying out amplification culture on the 4 groups of monoclonal colonies, extracting DNA plasmids, carrying out DNA sequencing to obtain a nucleotide sequence of the antibody, and translating to obtain a complete amino acid sequence. Specifically, the amino acid sequences and nucleotide sequences corresponding to 4 groups of monoclonal antibody microbial colonies are specifically shown in tables 4 to 7 below:

table 4: amino acid sequence and nucleotide sequence of monoclonal antibody PGI _1G1

Table 5: amino acid sequence and nucleotide sequence of monoclonal antibody PGI _1G9

Table 6: amino acid sequence and nucleotide sequence of monoclonal antibody PGI _1H10

Table 7: amino acid sequence and nucleotide sequence of monoclonal antibody PGI _2F1

Example 8: small batch antibody production and preparation

The DNA plasmid of the nanobody obtained in example 7 was transformed into BL21(DE3) competent cells, and the monoclonal nanobody was expressed and purified in small batches with the aid of an E.coli expression system, with a batch yield of about several milligrams.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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<400> 11

Tyr Val His Arg Ser Ser Ile Phe Thr Asn Arg Glu

1 5 10

<210> 12

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody PGI _1H10

<400> 12

Thr Phe Thr Arg Trp Leu Val Arg Gly Asp

1 5 10

<210> 13

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 of monoclonal antibody PGI _2F1

<400> 13

Ala Ala Asn Gly Pro Pro Phe Gly Ala Tyr Trp Trp Pro Thr Ala Arg

1 5 10 15

Glu Tyr Asp

<210> 14

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody PGI _1G1

<400> 14

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Asn Trp Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Val Val Arg Gly Thr Val Val Glu Gly Val Phe Ser Asn Arg Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 15

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody PGI _1G9

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Ser Ala His Leu Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val His Arg Ser Ser Ile Phe Thr Asn Arg Glu Tyr Trp Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 16

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody PGI _1H10

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Asn Phe Ser Ser Phe

20 25 30

Asp Arg Tyr Thr Met Ser Trp Val Arg Gln Ile Pro Gly Lys Gly Leu

35 40 45

Glu Trp Val Ser Ala Ile Asn Gly Gly Gly Ile Lys Thr Ala Tyr Ala

50 55 60

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

65 70 75 80

Thr Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Ala Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Thr Phe Thr Arg Trp Leu Val Arg Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 17

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of monoclonal antibody PGI _2F1

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Asp

1 5 10 15

Ser Leu Lys Leu Ser Cys Leu Ala Ser Gly Arg Thr Phe Ser Ser Arg

20 25 30

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

35 40 45

Ala Ala Ile Ser Thr Ser Gly Val Leu Lys Tyr Tyr Ala Pro Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Asn Gly Pro Pro Phe Gly Ala Tyr Trp Trp Pro Thr Ala Arg

100 105 110

Glu Tyr Asp Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 18

<211> 369

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody PGI _1G1

<400> 18

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcgg cctctggacg caccttcagt aggtatgcca tgggctggtt ccgccaggct 120

ccaggaaagg agcgtgagtt tgtcgcggct attaattgga gtggtcgtag cacatactat 180

gcagactccg tgaagggccg attcatcatt tccagagaca atgccaagaa cacggtgcat 240

ctgcagatga acaacctgaa acctgaggac acagccgtct atatttgtgt agtccgtggg 300

acggtagtag aaggtgtctt ctcgaatcgt gattattggg gtcaggggac ccaggtcacc 360

gtctcctca 369

<210> 19

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody PGI _1G9

<400> 19

caggtgcagc tggtgcagtc tgggggaggc tcggcgcacc ttggggggtc tctgaggctc 60

tcctgtgcag cctccggatt catcttcagt gactactaca tggcctgggt ccgccaggct 120

cctgggaagg agcgcgaatg ggtctcagaa attaatacta gtggtcgcac atactatgca 180

gattccgtga agggccgatt caccatctcc agagacaacg ccaagaacac gctgtatctg 240

caaatggaca gcttgaaacc tgaggacacg gccgtctatt actgttatgt ccacaggtcg 300

tccatattta ctaataggga gtactggggc caggggaccc aggtcaccgt ctcctca 357

<210> 20

<211> 363

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody PGI _1H10

<400> 20

caggtgcagc tggtgcagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgtag tctctggatt caactttagc agcttcgata ggtatactat gagttgggtc 120

cgccagattc cagggaaggg gctagagtgg gtctccgcta ttaatggtgg tggtattaag 180

actgcatatg cagactccgt gaagggccga ttcaccatct ccagagacaa cgccaagaac 240

acggtttatc tacaaatgaa cgacctcaaa cctgcggaca cggccgtgta ctactgtact 300

tttactcggt ggctggttcg gggtgactac tggggccagg ggacccaggt cactgtctcc 360

tca 363

<210> 21

<211> 381

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of monoclonal antibody PGI _2F1

<400> 21

caggtgcagc tggtggagtc tgggggagga ttggtgcaga ctggggactc tctgaaactc 60

tcctgtctag cctctggacg caccttcagt tcccgtgcca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct atcagcacta gtggtgttct aaaatactat 180

gcaccctccg tgaagggccg attcactatc tccagagaca acgccaagaa caccatgtat 240

ctagaaatga acagcccgga acctgaggac acggccgttt attactgtgc agcaaatggt 300

cccccgttcg gtgcttactg gtggccgacc gctcgtgagt atgacgcctg gggtcagggg 360

acccaggtca ccgtctcctc a 381

Claims

1. An antigen binding protein, antibody or antibody active fragment obtained by immunizing a camelid with pepsinogen I.

2. The antigen binding protein, antibody or antibody active fragment according to claim 1, wherein the camelid is selected from the group consisting of dromedary, bactrian camels, llamas, alpacas and llamas.

3. The antigen binding protein, antibody or antibody active fragment of claim 1, wherein the antibody is a nanobody and the antibody active fragment is a nanobody active fragment;

and/or, the antibody is a monoclonal antibody or a polyclonal antibody.

4. The antigen-binding protein, antibody or antibody active fragment according to any one of claims 1 to 3, wherein said antigen-binding protein, antibody or antibody active fragment binds to pepsinogen I with a kd value of 1200nM or less, preferably 500nM or less, more preferably 200nM or less, more preferably 150nM or less.

5. The antigen-binding protein, antibody or antibody active fragment according to claim 1 or 4, wherein the pepsinogen I has an amino acid sequence shown in SEQ ID NO 1;

preferably, the pepsinogen I is prepared by a method comprising the following steps: constructing a nucleotide sequence encoding the pepsinogen I into a vector plasmid; and (3) transfecting the vector plasmid into a eukaryotic cell line for expression and purification.

6. A method of constructing an antibody library comprising the steps of:

(3) inserting the amplified DNA into a virus expression vector, preferably a phage expression vector, transforming into bacteria, preferably escherichia coli, and collecting colonies to obtain an antibody library;

preferably, in the step (1), the camelid is alpaca, and/or the immunization adopts a subcutaneous injection mode, and/or the immunization times are 3-5 times, and/or the venous peripheral blood of the immunized animal is collected before and after the last immunization respectively.

7. An antibody library obtained by the method of claim 6, or polyclonal antibodies produced by expression of said antibody library.

8. A method of constructing an antigen-specific antibody library comprising the steps of: screening the antibody library of claim 7 to obtain an antigen-specific antibody library;

preferably, the method comprises the following steps:

(i) culturing the antibody library to release viruses;

(iii) infecting bacteria, preferably Escherichia coli, with the virus specifically binding to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

9. An antigen-specific antibody library obtained by the method of claim 8, or polyclonal antibodies specifically binding to an antigen produced by expression of the antigen-specific antibody library.

10. A method of producing an antigen binding protein, antibody or active fragment of an antibody comprising the steps of: screening the antibody library of claim 7 to obtain an antigen binding protein, antibody or antibody active fragment that specifically binds to an antigen;

preferably, the method comprises the following steps:

(a) culturing the antibody library to release viruses;

(c) infecting bacteria, preferably escherichia coli, with the virus specifically bound to the antigen, smearing the infected bacteria to a plate culture medium for culture, selecting a single colony, and optionally performing antigen-specific binding identification on the single colony;

more preferably, the method further comprises the following steps: (d) DNA of the single colony is extracted, transformed into a host cell and expressed.

11. An antigen binding protein, antibody or antibody active fragment obtained by the method of claim 10.

12. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds pepsinogen I, characterized in that said antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5;

CDR2 shown in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9; and

CDR3 shown in SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13;

preferably, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

13. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds pepsinogen I, characterized in that said antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 6 and CDR3 shown in SEQ ID NO. 10;

or, CDR1 shown in SEQ ID NO. 3, CDR2 shown in SEQ ID NO. 7 and CDR3 shown in SEQ ID NO. 11;

or, CDR1 shown in SEQ ID NO. 4, CDR2 shown in SEQ ID NO. 8 and CDR3 shown in SEQ ID NO. 12;

or, CDR1 shown in SEQ ID NO. 5, CDR2 shown in SEQ ID NO. 9, and CDR3 shown in SEQ ID NO. 13;

14. An antigen binding protein, antibody or antibody active fragment that specifically recognizes and/or binds pepsinogen I, characterized in that said antigen binding protein, antibody or antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

an amino acid sequence shown as SEQ ID NO. 14, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 14;

or the amino acid sequence shown as SEQ ID NO. 15, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 15;

or the amino acid sequence shown as SEQ ID NO. 16, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown as SEQ ID NO. 16;

or the amino acid sequence shown in SEQ ID NO. 17, or conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 17;

15. A nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO 2 to SEQ ID NO 17 or an antigen binding protein, antibody or antibody active fragment as set forth in any one of claims 1 to 5 or 11 to 14;

preferably, the nucleotide sequence encoding the antigen binding protein, antibody or antibody active fragment is selected from the group consisting of: 18, 19, 20 and 21.

16. An expression vector comprising the nucleotide sequence of claim 15;

preferably, the expression vector is a phage expression vector, preferably a phage surface display screening vector;

more preferably, the expression vector also contains a nucleotide sequence for coding the phage envelope protein pIII.

17. A virus exogenously introduced with the expression vector of claim 16; the virus is preferably a bacteriophage.

18. A host cell, preferably E.coli, which is exogenously transfected with an expression vector according to claim 8 or infected with a virus according to claim 9.

19. A method of expressing an antigen binding protein, antibody or antibody active fragment using the host cell of claim 18.

20. Expressing the obtained antigen binding protein, antibody or antibody active fragment using the host cell of claim 18.

21. A humanized antigen-binding protein, antibody or antibody active fragment obtained by humanizing the antigen-binding protein, antibody or antibody active fragment according to claim 1 to 5, 11 to 14 or 20.

22. A protein conjugate comprising the antigen binding protein, antibody or antibody active fragment of claim 1 to 5, 11 to 14 or 20 or the humanized antigen binding protein, antibody or antibody active fragment of claim 21 and a ligand;

preferably, the ligand is selected from the group consisting of radioisotopes, fluorophores, and delivery vehicles.

23. A kit for detecting the amount of pepsinogen I in a sample, comprising the antigen binding protein, antibody or antibody active fragment of claims 1 to 5, 11 to 14 or 20 or the humanized antigen binding protein, antibody or antibody active fragment of claim 21;

preferably, the antigen binding protein, antibody or antibody active fragment is labeled with a label; the label is preferably an enzyme, a chemiluminescent group or an isotopic group;

more preferably, the sample is animal serum, preferably human serum.

24. Use of the antigen binding protein, antibody or antibody active fragment of claim 1-5, 11-14 or 20, the humanized antigen binding protein, antibody or antibody active fragment of claim 21, the protein conjugate of claim 22 or the kit of claim 23 for detecting pepsinogen I content in a sample;

preferably, the sample is animal serum, preferably human serum.

25. A method for detecting the pepsinogen I content in a sample using the antigen binding protein, antibody or antibody active fragment of claims 1 to 5, 11 to 14 or 20, the humanized antigen binding protein, antibody or antibody active fragment of claim 21, the protein conjugate of claim 22 or the kit of claim 23;

preferably, the sample is animal serum, preferably human serum.