Human β2-microglobulin mutant

Abstract

The present invention discloses a human β2-microglobulin mutant. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant of the present invention has a mutation in at least one of the following sites: I1C, K19C, K41C, K48C, K58C, K91C, K94C; the wild-type human β2-microglobulin has an amino acid sequence as shown in SEQ ID NO: 1. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has a free thiol group and can be coupled with a small molecule polyethylene glycol derivative (e.g., methoxypolyethylene glycol maleimide; mPEG-Maleimide). The conjugate formed can be used for immunization to obtain antibodies with higher titers against natural β2-microglobulin.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a human β2-microglobulin mutant. More specifically, the present invention relates to a protein conjugate, a method for preparing a protein conjugate and a method for improving the immunogenicity of human β2-microglobulin. Background Art

[0002] Human β2-microglobulin (Beta-2-microglobulin) is a small molecule globulin with a molecular weight of 11.8Kd. The increase of β2-microglobulin in serum can reflect the damage of glomerular filtration function or the increase of filtration load; and the increase of β2-microglobulin excreted in urine indicates renal tubular damage or increased filtration load; this indicator is used clinically to monitor the function of proximal renal tubules and is widely used in renal function-related tests such as acute tubular injury, chronic interstitial nephritis, and chronic renal failure.

[0003] Since there are N-glycosylation sites on the molecule, each site will have a certain covering effect on the nearby amino acids after glycosylation. This causes the recombinantly expressed protein to differ from the natural protein in structure and immune target. When using recombinant human β2-microglobulin for immunization, there will be more "invalid" sites of the natural protein that are shielded by glycosylation, thereby reducing the content of effective antibodies. In addition, the molecular weight of the protein itself is only 11.8kd, and its immunogenicity is relatively weak. The most direct manifestation is that the antibody titer against the recombinant antigen used for immunization is acceptable, but the titer against the natural protein is very low.

[0004] Therefore, there is an urgent need to prepare recombinant antigens with structures and immune targets similar to natural antigens in order to prepare polyclonal antibodies that can efficiently recognize natural antigens. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] The present invention is accomplished based on the following findings of the inventors:

[0007] The inventors found that the molecular weight of β2-MG is 11.8kd, which is a typical small molecule antigen. Its own immunogenicity is not high. A considerable part of the antibodies obtained by recombinant protein immunization are invalid antibodies that cannot recognize natural proteins. Specifically, the antibody has a good titer for the immunogen and recombinant antigen, but a very low titer for the naturally extracted protein, which is prone to missed detection and insufficient detection sensitivity. In order to overcome this problem, the inventors chose to mutate the glycosylation sites on human β2-microglobulin to cysteine. The free thiol groups of cysteine ​​can be used for coupling reaction with small molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL) to form protein conjugates. Due to the presence of the polyethylene glycol structure, a "shielding effect" similar to glycosylation is formed at the modified site, and the surrounding amino acids are "shielded" by the hydration radius of the polyethylene glycol structure and do not participate in the immune response, while the amino acids outside the hydration radius participate in the immune response normally, thus simulating the structure and immune response of the natural protein, thereby preparing polyclonal antibodies that can efficiently recognize natural antigens.

[0008] Based on this, in the first aspect of the present invention, a human β2-microglobulin mutant is proposed. According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations at the following sites: I1C, K19C, K41C, K48C, K58C, K91C, K94C; the wild-type human β2-microglobulin has an amino acid sequence as shown in SEQ ID NO: 1. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein to form a protein conjugate with improved immunogenicity.

[0009] In the second aspect of the present invention, the present invention proposes a protein conjugate. According to an embodiment of the present invention, the protein conjugate comprises: the human β2-microglobulin mutant described in the first aspect of the present invention; a conjugate, wherein the human β2-microglobulin mutant is connected to the conjugate. The protein conjugate according to an embodiment of the present invention has significantly improved immunogenicity compared to wild-type recombinant human β2-microglobulin.

[0010] In the third aspect of the present invention, the present invention provides a method for preparing a protein conjugate. According to an embodiment of the present invention, the method comprises: contacting the human β2-microglobulin mutant described in the first aspect of the present invention with a conjugate to obtain the protein conjugate. According to the method of the embodiment of the present invention, a protein conjugate with significantly improved immunogenicity compared with wild-type recombinant human β2-microglobulin can be prepared.

[0011] In a fourth aspect of the present invention, the present invention proposes a method for improving the immunogenicity of human β2-microglobulin. According to an embodiment of the present invention, the method comprises: contacting the human β2-microglobulin with methoxy PEG maleimide; wherein the human β2-microglobulin is selected from the β2-microglobulin mutant described in the first aspect of the present invention, and the methoxy PEG maleimide is consistent with the conjugate defined in the protein conjugate described in the second aspect of the present invention. The method according to the embodiment of the present invention can improve the immunogenicity of human β2-microglobulin.

[0012] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0014] Figure 1 The present invention is a technical implementation flow chart.

[0015] Figure 2 This is the molecular structure of β2-MG.

[0016] Figure 3 It is the molecular structure of mPEG-maleimide.

[0017] Figure 4 Schematic diagram of β2-microglobulin after mPGE-maleimide modification mutation.

[0018] Figure 5 This is a comparison chart of the immune effects of different types of β2-microglobulin molecules in Example 1. DETAILED DESCRIPTION

[0019] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0020] In the present invention, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0021] In the present invention, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or situation can but does not necessarily occur, and the description includes cases where the event or situation occurs and cases where it does not occur.

[0022] In the present invention, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited as long as they show the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N terminal) of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl terminal (C terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and the H chain are called VL and VH, respectively.

[0023] In the present invention, the term "antigen" refers to a substance that can stimulate the immune system to produce antibodies or induce immune cell responses. In some optional embodiments of the present invention, "antigen" refers to BMG, BMG mutants, protein conjugates, etc., which can stimulate the immune system to produce antibodies or induce immune cell responses.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0025] The present invention provides a human β2-microglobulin mutant, a protein conjugate, a method for preparing the protein conjugate and a method for improving the immunogenicity of human β2-microglobulin, which will be described in detail below.

[0026] Human β2-microglobulin mutant

[0027] In the first aspect of the present invention, a human β2-microglobulin mutant is provided. According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations at the following sites: I1C, K19C, K41C, K48C, K58C, K91C, K94C; the wild-type human β2-microglobulin has an amino acid sequence as shown in SEQ ID NO: 1. Compared with the wild-type β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein to form a protein conjugate with improved immunogenicity.

[0028] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSK DWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:1)

[0029] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has at least one of the following mutation combinations (1) to (5): (1) K58C; (2) K41C, K58C and K91C; (3) K41C and K91C; (4) I1C, K19C and K58C; (5) I1C, K41C, K58C and K91C. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0030] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations of K41C, K58C and K91C. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0031] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has a K58C mutation. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0032] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has K41C and K91C mutations. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0033] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations of I1C, K19C and K58C. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0034] According to an embodiment of the present invention, compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations of I1C, K41C, K58C and K91C. Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant according to an embodiment of the present invention has a free thiol group, which can be coupled with a small molecule PEG derivative (such as methoxy PEG maleimide mPEG-MAL) to simulate the structure and immune response of the natural β2-microglobulin protein, and further form a protein conjugate with improved immunogenicity.

[0035] Protein conjugates

[0036] In the second aspect of the present invention, the present invention proposes a protein conjugate. According to an embodiment of the present invention, the protein conjugate comprises: the human β2-microglobulin mutant described in the first aspect of the present invention; a conjugate, wherein the human β2-microglobulin mutant is connected to the conjugate. The protein conjugate according to an embodiment of the present invention has significantly improved immunogenicity compared to wild-type human β2-microglobulin.

[0037] According to an embodiment of the present invention, the conjugate is selected from small molecule polyethylene glycol derivatives. Among them, it needs to be explained that "small molecule polyethylene glycol derivatives" refer to small molecule polyethylene glycol derivatives with a single branch chain, and an active group is connected to the end of the polyethylene glycol. The end of the polyethylene glycol of the "small molecule polyethylene glycol derivative" in the embodiment of the present invention is connected to a maleimide and its analogs. It needs to be further explained that the "maleimide and its analogs" in the present invention are a class of compounds with high reactivity to sulfhydryl groups, and are therefore often used as labeling reagents or cross-linking agents for sulfhydryl compounds. By reacting with sulfhydryl groups, maleimide and its analogs can be chemically combined with proteins, polypeptides or other sulfhydryl-containing biomolecules to form a stable covalent connection. The "maleimide analogs" in the present invention are a class of compounds with similar functions or structures to maleimide, especially maleimide analogs can be obtained by replacing the H of maleimide with a substituent group. In an optional embodiment of the present invention, the substituent group has the following structure: The range of n is 1 to 3.

[0038] In an alternative embodiment of the present invention, the substituent group has the following structure:

[0039] According to an embodiment of the present invention, the small molecule polyethylene glycol derivative is selected from a polyethylene glycol derivative having an activating group, and the human β2-microglobulin mutant and the small molecule polyethylene glycol derivative are connected through the activating group. Among them, it needs to be explained that the "activating group" in the present invention refers to a chemical group that can react with the amino acid residues of the protein. In the protein structure, the amino acid residue usually has a reactive amino group (-NH2) group that can be covalently bound to other compounds. In the present invention, the small molecule polyethylene glycol derivative can form a covalent connection with the human β2-microglobulin mutant through maleimide, and the polyethylene glycol is combined with the protein. This connection can form a shielding structure at the site and its surrounding amino acids, which is similar to the effect of natural antigen glycosylation. Thereby making the immune target of the recombinant β2-microglobulin closer to the natural protein, so as to obtain more effective antibodies.

[0040] According to an embodiment of the present invention, the activation group is selected from at least one of a hydroxyl group, an amino group, a thiol group, a carboxyl group, an ester group, an aldehyde group, an acrylic group and a maleimide group.

[0041] According to an embodiment of the present invention, the activating group is selected from maleimide groups.

[0042] According to an embodiment of the present invention, the ester group is selected from a succinimidyl acetate group, a succinimidyl propionate group or a succinimidyl carbonate group.

[0043] According to an embodiment of the present invention, the small molecule polyethylene glycol derivative is selected from methoxy polyethylene glycol maleimide and its analogs, the human β2-microglobulin mutant and the small molecule polyethylene glycol maleimide and its analogs are connected via maleimide, and the thiol group of the human β2-microglobulin mutant and the maleimide of the small molecule polyethylene glycol maleimide and its analogs are connected.

[0044] According to an embodiment of the present invention, the small molecule polyethylene glycol derivative is a methoxy polyethylene glycol derivative.

[0045] According to an embodiment of the present invention, the small molecule polyethylene glycol derivative is a methoxy polyethylene glycol maleimide analog, which is a commercially available product (PS1-M-350). The methoxy polyethylene glycol derivative has the following structure:

[0046]

[0047] According to an optional embodiment of the present invention, the molecular weight of the methoxy polyethylene glycol derivative is 350.

[0048] Methods for preparing protein conjugates

[0049] In the third aspect of the present invention, the present invention provides a method for preparing a protein conjugate. According to an embodiment of the present invention, the method comprises: contacting the human β2-microglobulin mutant described in the first aspect of the present invention with a conjugate to obtain the protein conjugate. According to the method of the embodiment of the present invention, a protein conjugate with significantly improved immunogenicity compared with wild-type human β2-microglobulin can be prepared.

[0050] According to an embodiment of the present invention, the conjugate is selected from small molecule polyethylene glycol derivatives, and the small molecule polyethylene glycol derivatives are consistent with the small molecule polyethylene glycol derivatives defined in the protein conjugate described in the second aspect of the present invention.

[0051] According to an embodiment of the present invention, the contact treatment is carried out under the condition that the molar ratio of the human β2-microglobulin mutant to the conjugate is 1:1, and the final concentration of the conjugate in the solution containing the conjugate and the human β2-microglobulin mutant is 0.5mM to 2mM; the final concentration of the human β2-microglobulin mutant in the solution containing the conjugate and the human β2-microglobulin mutant is 0.5mM to 2mM. According to the method of the embodiment of the present invention, a protein conjugate with significantly further improved immunogenicity compared with wild-type human β2-microglobulin can be prepared.

[0052] Method for improving the immunogenicity of human β2-microglobulin

[0053] In a fourth aspect of the present invention, the present invention proposes a method for improving the immunogenicity of human β2-microglobulin. According to an embodiment of the present invention, the method comprises: contacting the human β2-microglobulin with a small molecule polyethylene glycol derivative; wherein the human β2-microglobulin is selected from the human β2-microglobulin mutant described in the first aspect of the present invention, and the small molecule polyethylene glycol derivative is consistent with the conjugate defined in the protein conjugate described in the second aspect of the present invention. The method according to the embodiment of the present invention can improve the immunogenicity of human β2-microglobulin.

[0054] According to an embodiment of the present invention, the contact treatment is carried out under the condition that the molar ratio of the human β2-microglobulin mutant to the conjugate is 1:1, the final concentration of the small molecule polyethylene glycol derivative in the solution containing the small molecule polyethylene glycol derivative and the human β2-microglobulin mutant is 0.5mM to 2mM; the final concentration of the human β2-microglobulin mutant in the solution containing the small molecule polyethylene glycol derivative and the human β2-microglobulin mutant is 0.5mM to 2mM. The method according to an embodiment of the present invention can further improve the immunogenicity of human β2-microglobulin.

[0055] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0056] Example 1

[0057] (1) Preparation of β2-microglobulin mutants:

[0058] First, in this example, the human β2-microglobulin mutants were prepared using the molecular cloning method (refer to the NEB site-directed mutagenesis kit instructions; E0552). The specific experimental process is as follows:

[0059] The isoleucine (I) at position 1 of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 1#;

[0060] The 19th lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 2#;

[0061] The 41st lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 3#;

[0062] The 48th lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 4#;

[0063] The 58th lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 5#;

[0064] The 91st lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 6#;

[0065] The 94th lysine (K) of human β2-microglobulin was mutated to cysteine ​​(C), recorded as 7#;

[0066] The lysine (K) at positions 41, 58, and 91 of human β2-microglobulin were mutated to cysteine ​​(C), recorded as 8#;

[0067] The 41st and 91st lysine (K) of human β2-microglobulin were mutated to cysteine ​​(C), recorded as 9#;

[0068] The isoleucine (I) at position 1 of human β2-microglobulin was mutated to cysteine ​​(C), and the lysine (K) at positions 19 and 58 were mutated to cysteine ​​(C), recorded as 10#;

[0069] The isoleucine (I) at position 1 of human β2-microglobulin was mutated to cysteine ​​(C), and the lysine (K) at positions 41, 58, and 91 were mutated to cysteine ​​(C), recorded as 11#;

[0070] (2) Chemical modification of mutant β2-microglobulin:

[0071] Secondly, 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#, and 11# were induced to express in Escherichia coli to obtain the target protein, and then coupled with maleimide according to the method of Yin, Ying et al. "Self-conjugated protective antigen elicits strong and durable protective antibody response against anthrax." International journal of biological macromolecules vol. 137 (2019): 790-800., and then purified to obtain 1# protein conjugate, 2# protein conjugate, 3# protein conjugate, 4# protein conjugate, 5# protein conjugate, 6# protein conjugate, 7# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, and 11# protein conjugate respectively. Subsequently, the method of Sun, Xiaowei et al. "Conjugation Reaction with 8-Arm PEG Markedly Improves the Immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 Fusion Protein." Bioconjugate chemistry vol. 28, 6 (2017): 1658-1668. was used to remove the unpolymerized methoxymaleamide in 1# protein conjugate, 2# protein conjugate, 3# protein conjugate, 4# protein conjugate, 5# protein conjugate, 6# protein conjugate, 7# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, and 11# protein conjugate for use.

[0072] (3) Animal immunization and titer detection: The protein conjugates 1# to 11# obtained in step (2) were used to immunize New Zealand white mackerel according to the standard immunization process described by Wang Chuanwu et al. (2002) to prepare polyclonal antibodies. A total of five immunizations were performed, and after each immunization, ear blood was collected and the ELISA method was used in combination with natural antigen coating to detect the immune effect.

[0073] The experimental results of ELISA during the immunization process of 1# protein conjugate, 2# protein conjugate, 3# protein conjugate, 4# protein conjugate, 5# protein conjugate, 6# protein conjugate, 7# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, and 11# protein conjugate showed that 5# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, and 11# protein conjugate were better than the wild-type recombinant antigen immunization control group, and the antiserum titer was improved. This example exemplifies the experimental results of ELISA during the immunization process of 5# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, and 11# protein conjugate.

[0074] The experimental results are as follows Figure 5 As shown, with the increase of immunization times, the titer of antiserum against natural β2 microglobulin of 5#, 8#, 9#, 10#, and 11# protein conjugates was significantly higher than that of wild-type recombinant protein and other single-site mutant antigen control groups. The titer of antiserum against 9# protein conjugate increased by about 5 times to 5500 relative to the wild-type recombinant antigen control group; the titer of antiserum against 8# protein conjugate increased by about 7 times to 7300 relative to the wild-type recombinant antigen control group; and the titer of antiserum against 5# protein conjugate increased significantly higher than that of wild-type recombinant antigen control group with the increase of immunization times. The titer of antiserum against 5# protein conjugate increased by about 3 times to 1:3400 relative to the wild-type recombinant antigen control group. The titer of antiserum against 10# protein conjugate increased by about 4 times to 1:4200 relative to the wild-type recombinant antigen control group. The titer of antiserum against 11# protein conjugate increased by about 3 times to 1:3000 relative to the wild-type recombinant antigen control group.

[0075] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0076] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A human β2-microglobulin mutant, characterized in that: Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has one of the following mutation combinations (1) to (4): (1) K41C, K58C and K91C; (2) K41C and K91C; (3) I1C, K19C and K58C; (4) I1C, K41C, K58C and K91C; The wild-type human β2-microglobulin has an amino acid sequence as shown in SEQ ID NO:

1.

2. The human β2-microglobulin mutant according to claim 1, characterized in that Compared with the wild-type human β2-microglobulin, the human β2-microglobulin mutant has mutations of K41C, K58C and K91C.

3. A protein conjugate, characterized in that include: The human β2-microglobulin mutant according to claim 1 or 2; The conjugate is connected to the human β2-microglobulin mutant.

4. The protein conjugate according to claim 3, characterized in that The conjugate is selected from small molecule polyethylene glycol derivatives.

5. The protein conjugate according to claim 4, characterized in that The small molecule polyethylene glycol derivative is selected from a small molecule polyethylene glycol derivative having an activation group, and the human β2-microglobulin mutant and the small molecule polyethylene glycol derivative are connected via the activation group.

6. The protein conjugate according to claim 5, characterized in that The activating group is selected from at least one of a hydroxyl group, an amino group, a thiol group, a carboxyl group, an ester group, an aldehyde group, an acrylic group and a maleimide group.

7. The protein conjugate according to claim 6, characterized in that The ester group is selected from succinimidyl acetate, succinimidyl propionate or succinimidyl carbonate.

8. The protein conjugate according to claim 6, characterized in that The aldehyde group is selected from propionaldehyde, butyraldehyde, acetaldehyde or valeraldehyde.

9. The protein conjugate according to claim 4, characterized in that The small molecule polyethylene glycol derivative is selected from methoxy polyethylene glycol maleimide, the human β2-microglobulin mutant and the methoxy polyethylene glycol maleimide are connected through maleimide, and the thiol group of the human β2-microglobulin mutant is connected to the maleimide of the methoxy polyethylene glycol maleimide.

10. A method for preparing a protein conjugate, characterized in that: include: The human β2-microglobulin mutant according to claim 1 or 2 and a conjugate are contacted to obtain the protein conjugate.

11. The method according to claim 10, characterized in that The conjugate is selected from small molecule polyethylene glycol derivatives, and the small molecule polyethylene glycol derivatives are consistent with the small molecule polyethylene glycol derivatives defined in the protein conjugate according to any one of claims 3 to 9.

12. The method according to claim 10, characterized in that The contact treatment is carried out under the condition that the molar ratio of the human β2-microglobulin mutant to the conjugate is 1:1, and the final concentration of the conjugate in the solution containing the conjugate and the human β2-microglobulin mutant is 0.5 mM to 2 mM; The final concentration of the human β2-microglobulin mutant in the solution containing the conjugate and the human β2-microglobulin mutant is 0.5 mM to 2 mM.

13. A method for improving the immunogenicity of human β2-microglobulin, characterized in that: include: contacting the human β2-microglobulin with a small molecule polyethylene glycol derivative; Wherein, the human β2-microglobulin is selected from the human β2-microglobulin mutant described in claim 1 or 2, and the small molecule polyethylene glycol derivative is consistent with the conjugate defined in the protein conjugate described in any one of claims 3 to 9.

14. The method according to claim 13, characterized in that The contact treatment is carried out under the condition that the molar ratio of the human β2-microglobulin mutant to the conjugate is 1:1, and the final concentration of the small molecule polyethylene glycol derivative in the solution containing the small molecule polyethylene glycol derivative and the human β2-microglobulin mutant is 0.5 mM to 2 mM; The final concentration of the human β2-microglobulin mutant in the solution containing the small molecule polyethylene glycol derivative and the human β2-microglobulin mutant is 0.5 mM to 2 mM.

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