CN118027176B - Human beta 2-microglobulin mutant - Google Patents
Human beta 2-microglobulin mutant Download PDFInfo
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Abstract
本发明公开了一种人β2‑微球蛋白突变体。其中，本发明的人β2‑微球蛋白突变体与人β2‑微球蛋白野生型相比，所述人β2‑微球蛋白突变体具有如下如下至少之一位点的突变：I1C、K19C、K41C、K48C、K58C、K91C、K94C；所述人β2‑微球蛋白野生型具有如SEQ ID NO:1所示的氨基酸序列。该人β2‑微球蛋白突变体，相比于野生型人β2‑微球蛋白，具有游离的巯基，可与小分子聚乙二醇衍生物(例如甲氧基聚乙二醇马来酰亚胺；mPEG‑Maleimide)进行偶联反应，形成的缀合物用于免疫可以得到针对天然β2微球蛋白效价更高的抗体。
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
The invention belongs to the technical field of biology, in particular to a human beta 2-microglobulin mutant, and more particularly relates to a protein conjugate, a method for preparing the protein conjugate and a method for improving immunogenicity of the human beta 2-microglobulin.
Background
Human Beta 2-microglobulin (Beta-2-microglobulin) is a small molecular globulin with a molecular mass of 11.8Kd. The elevation of beta 2-microglobulin in serum can reflect the condition of damaged glomerular filtration function or increased filtration load, while the elevation of beta 2-microglobulin discharged in urine prompts the damage of tubular or increased filtration load, and the index is clinically used for monitoring the function of proximal tubular and is widely applied to the relevant detection of renal functions such as acute tubular injury, chronic interstitial nephritis, chronic renal failure and the like.
Because the molecule has N-glycosylation sites, each site can play a certain role in covering nearby amino acids after glycosylation. This results in a recombinant expressed protein that differs from the native protein in structure and immune target. Immunization with recombinant human β2-microglobulin also increases the number of "null" sites where some native proteins are masked by glycosylation, thereby reducing the amount of effective antibodies. In addition, the molecular weight of the protein is only 11.8kd, and the immunogenicity is relatively weak. The most direct expression is that antibodies have a very good potency against recombinant antigens for immunization and a very low potency against natural proteins.
Thus, there is a need to prepare recombinant antigens that are similar to the structure and immune targets of natural antigens in order to prepare polyclonal antibodies that can recognize natural antigens with high efficiency.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent.
The present invention has been completed based on the following findings by the inventors:
The inventor finds that the beta 2-MG molecular weight is 11.8kd, is a typical small molecular antigen, has low immunogenicity, and a considerable part of antibodies obtained by immunization of recombinant proteins are ineffective antibodies which cannot recognize natural proteins, and the antibodies are particularly effective against immunogens and recombinant antigens, but have low titers against the naturally extracted proteins, so that the problems of missed detection and insufficient detection sensitivity are easy to occur. To overcome this problem, the inventors have chosen to mutate the glycosylation sites on human β2-microglobulin to cysteines, respectively, which carry free sulfhydryl groups for coupling reactions with PEG derivatives of small molecules (e.g. methoxy PEG maleimide mPEG-MAL) to form protein conjugates. Because of the existence of polyethylene glycol structure, the modified site forms a 'shielding effect' similar to glycosylation, peripheral amino acids are not involved in immune response under the 'shielding' of the hydration radius of the polyethylene glycol structure, and amino acids outside the hydration radius are normally involved in immune response, so that the structure and immune response of the natural protein are simulated, and the polyclonal antibody capable of efficiently recognizing the natural antigen is prepared.
Based on this, the present invention proposes, in a first aspect, a human β2-microglobulin mutant. According to an embodiment of the invention, the human beta 2-microglobulin mutant has a mutation of the following site compared with the human beta 2-microglobulin wild type, I1C, K19C, K41C, K48C, K58C, K3891C, K C, and the human beta 2-microglobulin wild type has an amino acid sequence as shown in SEQ ID NO. 1. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with a small-molecule PEG derivative (methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and forms a protein conjugate with improved immunogenicity.
In a second aspect of the invention, the invention provides a protein conjugate. According to an embodiment of the invention, the protein conjugate comprises the human beta 2-microglobulin mutant according to the first aspect of the invention, and the conjugate, wherein the human beta 2-microglobulin mutant is connected with the conjugate. The protein conjugate according to the embodiment of the invention has significantly improved immunogenicity compared to wild-type recombinant human beta 2-microglobulin.
In a third aspect of the invention, the invention provides a method of preparing a protein conjugate. According to an embodiment of the invention, the method comprises contacting the human β2-microglobulin mutant according to the first aspect of the invention with a conjugate to obtain the protein conjugate. According to the method of the embodiment of the invention, protein conjugates with significantly improved immunogenicity compared to wild-type recombinant human beta 2-microglobulin can be prepared.
In a fourth aspect of the invention, the invention provides a method of increasing the immunogenicity of human β2-microglobulin. According to an embodiment of the invention, the method comprises contacting the human β2-microglobulin with a methoxy PEG maleimide, wherein the human β2-microglobulin is selected from the group consisting of the β2-microglobulin mutants according to the first aspect of the invention, and wherein the methoxy PEG maleimide is identical to the conjugate defined in the protein conjugate according to the second aspect of the invention. According to the method of the embodiment of the invention, the immunogenicity of the human beta 2-microglobulin can be improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a flow chart of an embodiment of the present invention.
FIG. 2 shows the beta.2-MG molecular structure.
FIG. 3 is a molecular structure of mPEG-maleimide.
FIG. 4 is a schematic representation of a mPGE-maleimide modified mutated beta 2-microglobulin.
FIG. 5 is a graph showing the comparison of the immunological effects of different types of beta 2-microglobulin molecules in example 1.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
In the present invention, the terms "comprising" or "including" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In the present invention, the terms "optionally," "optional," or "optionally" mean generally that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited so long as they exhibit the desired biological activity. It generally comprises a light chain of relatively light molecular weight and a heavy chain of relatively heavy molecular weight, the heavy chain (H chain) and the light chain (L chain) being linked by disulfide bonds to form an antibody molecule. Wherein the amino terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxy terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively.
In the present invention, the term "antigen" refers to a substance that is capable of eliciting antibodies or inducing an immune cell response from the immune system. In some alternative embodiments of the invention, "antigen" refers to a substance, such as BMG, BMG mutants, protein conjugates, or the like, that is capable of eliciting antibodies or inducing an immune cell response by the immune system.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The present invention provides a human beta 2-microglobulin mutant, a protein conjugate, a method for preparing the protein conjugate and a method for improving the immunogenicity of human beta 2-microglobulin, which will be described in detail below, respectively.
Human beta 2-microglobulin mutant
In a first aspect of the invention, the invention provides a human β2-microglobulin mutant. According to an embodiment of the invention, the human beta 2-microglobulin mutant has a mutation of the following site compared with the human beta 2-microglobulin wild type, I1C, K19C, K41C, K48C, K58C, K3891C, K C, and the human beta 2-microglobulin wild type has an amino acid sequence as shown in SEQ ID NO. 1. Compared with wild type beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulate the structure and immune reaction of beta 2-microglobulin, and form protein conjugate with improved immunogenicity.
IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSK DWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:1)
According to an embodiment of the invention, the human β2-microglobulin mutant has a mutation combination of at least one of (1) K58C, (2) K41C, K C and K91C, (3) K41C and K91C, (4) I1C, K C and K58C, (5) I1C, K41C, K C and K91C, and (1) compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
According to an embodiment of the invention, the human β2-microglobulin mutant has mutations of K41C, K C and K91C compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
According to an embodiment of the invention, the human β2-microglobulin mutant has a mutation of K58C compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
According to an embodiment of the invention, the human β2-microglobulin mutant has mutations of K41C and K91C compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
According to an embodiment of the invention, the human β2-microglobulin mutant has mutations of I1C, K C and K58C compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
According to an embodiment of the invention, the human β2-microglobulin mutant has mutations of I1C, K41C, K C and K91C compared to the human β2-microglobulin wild type. Compared with wild type human beta 2-microglobulin, the human beta 2-microglobulin mutant provided by the embodiment of the invention has free sulfhydryl groups, can perform coupling reaction with small-molecule PEG derivatives (such as methoxy PEG maleimide mPEG-MAL), simulates the structure and immune reaction of beta 2-microglobulin, and further forms protein conjugate with improved immunogenicity.
Protein conjugates
In a second aspect of the invention, the invention provides a protein conjugate. According to an embodiment of the invention, the protein conjugate comprises the human beta 2-microglobulin mutant according to the first aspect of the invention, and the conjugate, wherein the human beta 2-microglobulin mutant is connected with the conjugate. The protein conjugate according to the embodiment of the invention has significantly improved immunogenicity compared to wild-type human beta 2-microglobulin.
According to an embodiment of the invention, the conjugate is selected from small molecule polyethylene glycol derivatives. The small molecular polyethylene glycol derivative is a small molecular polyethylene glycol derivative with a single branched chain, and the tail end of the polyethylene glycol is connected with an active group. The tail end of the polyethylene glycol of the small molecular polyethylene glycol derivative is connected with maleimide and analogues thereof. It should be further explained that "maleimide and the like" in the present invention is a type of a compound having a high reactivity to mercapto groups and is therefore often used as a labeling agent or crosslinking agent for mercapto compounds. Maleimide and its analogues can chemically bind to biomolecules contained in proteins, polypeptides or other thiols by reaction with thiols, forming stable covalent linkages. "maleimide analogs" in the present invention are a class of compounds having a function or structure similar to that of maleimide, and in particular maleimide analogs can be obtained by substitution of the H of maleimide with a substituent. In an alternative embodiment of the invention, the substituent has the structure: Wherein n ranges from 1 to 3.
In an alternative embodiment of the invention, the substituent has the structure:
According to an embodiment of the invention, the small molecule polyethylene glycol derivative is selected from polyethylene glycol derivatives having an activating group through which the human β2-microglobulin mutant and the small molecule polyethylene glycol derivative are linked. In the present invention, the "activating group" refers to a chemical group that can react with an amino acid residue of a protein. In protein structures, amino acid residues typically have a reactive amino (-NH 2) group that can be covalently bound to other compounds. In the invention, the small molecule polyethylene glycol derivative can form covalent connection with human beta 2-microglobulin mutant through maleimide, so as to combine polyethylene glycol with protein. Such a linkage may form a shielding structure at the site and its surrounding amino acids, similar to the effect of natural antigen glycosylation. So that the immune target of the recombinant beta 2-microglobulin is more similar to the natural protein, and more effective antibodies are obtained.
According to an embodiment of the present invention, the activating group is selected from at least one of a hydroxyl group, an amino group, a mercapto group, a carboxyl group, an ester group, an aldehyde group, an acrylic group, and a maleimide group.
According to an embodiment of the invention, the activating group is selected from maleimide groups.
According to an embodiment of the invention, the ester group is selected from succinimidoacetate group, succinimidopropionate group or succinimidocarbonate group.
According to an embodiment of the invention, the small-molecule polyethylene glycol derivative is selected from methoxy polyethylene glycol maleimide and analogues thereof, the human beta 2-microglobulin mutant and the small-molecule polyethylene glycol maleimide and analogues thereof are connected through maleimide, and the sulfhydryl group of the human beta 2-microglobulin mutant and the maleimide of the small-molecule polyethylene glycol maleimide and analogues thereof are connected.
According to an embodiment of the invention, the small molecule polyethylene glycol derivative is a methoxypolyethylene glycol derivative.
According to the embodiment of the invention, the small-molecule polyethylene glycol derivative is a methoxy polyethylene glycol maleimide analogue, and is a commercially available product (PS 1-M-350), and the methoxy polyethylene glycol derivative has the following structure:
according to an alternative embodiment of the invention, the methoxypolyethylene glycol derivative has a molecular weight of 350.
Method for preparing protein conjugates
In a third aspect of the invention, the invention provides a method of preparing a protein conjugate. According to an embodiment of the invention, the method comprises contacting the human β2-microglobulin mutant according to the first aspect of the invention with a conjugate to obtain the protein conjugate. According to the method of the embodiment of the invention, protein conjugates with significantly improved immunogenicity compared to wild-type human beta 2-microglobulin can be prepared.
According to an embodiment of the invention, the conjugate is selected from small molecule polyethylene glycol derivatives, which are identical to the small molecule polyethylene glycol derivatives defined in the protein conjugate according to the second aspect of the invention.
According to an embodiment of the invention, the contacting treatment is performed under the condition that the molar ratio of the human beta 2-microglobulin mutant to the conjugate is 1:1, the final concentration of the conjugate in a solution containing the conjugate and the human beta 2-microglobulin mutant is 0.5 mM-2 mM, and the final concentration of the human beta 2-microglobulin mutant in a solution containing the conjugate and the human beta 2-microglobulin mutant is 0.5 mM-2 mM. According to the method of the embodiment of the invention, protein conjugates with significantly further improved immunogenicity compared to wild-type human beta 2-microglobulin can be prepared.
Method for improving immunogenicity of human beta 2-microglobulin
In a fourth aspect of the invention, the invention provides a method of increasing the immunogenicity of human β2-microglobulin. According to an embodiment of the invention, the method comprises contacting the human beta 2-microglobulin with a small molecule polyethylene glycol derivative, wherein the human beta 2-microglobulin is selected from the human beta 2-microglobulin mutant according to the first aspect of the invention, and the small molecule polyethylene glycol derivative is identical to the conjugate defined in the protein conjugate according to the second aspect of the invention. According to the method of the embodiment of the invention, the immunogenicity of the human beta 2-microglobulin can be improved.
According to the embodiment of the invention, the contact treatment is carried out under the condition that the molar ratio of the human beta 2-microglobulin mutant to the conjugate is 1:1, the final concentration of the small molecular polyethylene glycol derivative in a solution containing the small molecular polyethylene glycol derivative and the human beta 2-microglobulin mutant is 0.5 mM-2 mM, and the final concentration of the human beta 2-microglobulin mutant in a solution containing the small molecular polyethylene glycol derivative and the human beta 2-microglobulin mutant is 0.5 mM-2 mM. According to the method of the embodiment of the invention, the immunogenicity of the human beta 2-microglobulin can be further improved.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
(1) Preparation of beta 2-microglobulin mutant:
Firstly, the method of molecular cloning (refer to NEB site-directed mutagenesis kit instruction; E0552) is used for preparing human beta 2-microglobulin mutant respectively in the embodiment, and the specific experimental process is as follows:
the isoleucine (I) at position 1 of human beta 2-microglobulin was mutated to cysteine (C), recorded as # 1;
mutation of lysine (K) at position 19 of human β2-microglobulin to cysteine (C), recorded as # 2;
mutation of lysine (K) at position 41 of human beta 2-microglobulin to cysteine (C), recorded as 3#;
Mutation of lysine (K) at position 48 of human beta 2-microglobulin to cysteine (C), recorded as # 4;
mutation of lysine (K) at position 58 of human beta 2-microglobulin to cysteine (C), recorded as 5#;
Mutation of lysine (K) at position 91 of human beta 2-microglobulin to cysteine (C), recorded as 6#;
mutation of lysine (K) at position 94 of human beta 2-microglobulin to cysteine (C), recorded as 7#;
mutation of lysine (K) at positions 41, 58, 91 of human beta 2-microglobulin to cysteine (C), recorded as 8#;
mutation of lysine (K) at positions 41 and 91 of human beta 2-microglobulin to cysteine (C), recorded as 9#;
mutation of isoleucine (I) at position 1 of human beta 2-microglobulin to cysteine (C), mutation of lysine (K) at positions 19 and 58 to cysteine (C), and recording as 10#;
mutation of isoleucine (I) at position 1 of human beta 2-microglobulin to cysteine (C), mutation of lysine (K) at positions 41, 58, 91 to cysteine (C), recording as 11#;
(2) Chemical modification of mutant β2-microglobulin:
Next, the target proteins were obtained by inducing expression of 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#, 11#, and then coupling with maleimide according to the method described in 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., followed by purification 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, 11# protein conjugate, respectively. Subsequently, the non-polymerized methoxy maleamide in the 1# protein conjugate, the 2# protein conjugate, the 3# protein conjugate, the 4# protein conjugate, the 5# protein conjugate, the 6# protein conjugate, the 7# protein conjugate, the 8# protein conjugate, the 9# protein conjugate, the 10# protein conjugate, and the 11# protein conjugate was removed using a molecular sieve using a 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. method for later use.
(3) Animal immunization and potency detection, namely, selecting New Zealand white fungus to immunize according to the standard immunization procedure of Wang Chuanwu et al (2002) to prepare the polyclonal antibody by using the 1# to 11# protein conjugate obtained in the step (2). Five times of immunization are carried out, and the ELISA method for taking the ear blood after each immunization is matched with the natural antigen coating to detect the immune effect.
Experimental results of ELISA during immunization of the 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, 11# protein conjugate showed that the 5# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, 11# protein conjugate were better than the wild-type recombinant antigen immune control group, and the antiserum titers were all increased, and the present example demonstrated experimental results of ELISA during immunization of the 5# protein conjugate, 8# protein conjugate, 9# protein conjugate, 10# protein conjugate, 11# protein conjugate.
As shown in the experimental results in FIG. 5, the titers of the 5#, 8#, 9#, 10#, 11# protein conjugates against the natural beta 2 microglobulin are obviously higher than those of the wild-type recombinant protein and other single-site mutant antigen control groups along with the increase of the immunization times, the titers of the 9# protein conjugates are obviously higher than those of the wild-type recombinant antigen control groups along with the increase of the immunization times, the titers of the 9# protein conjugates are improved by about 5 times to 5500 relative to the wild-type recombinant antigen control groups, the titers of the 8# protein conjugates are improved by about 7 times to 7300 relative to the wild-type recombinant antigen control groups along with the increase of the immunization times, and the titers of the 5# protein conjugates are obviously higher than those of the wild-type recombinant antigen control groups along with the increase of the immunization times. The antisera titer was increased about 3-fold to 1:3400 for the 5# protein conjugate relative to the wild-type recombinant antigen control. The antisera titer was increased about 4-fold to 1:4200 relative to the wild-type recombinant antigen control. The antisera titer was increased about 3-fold to 1:3000 relative to the wild-type recombinant antigen control.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (14)
1.A human β2-microglobulin mutant, characterized in that the human β2-microglobulin mutant has a combination of mutations of one of the following (1) - (4) compared with the human β2-microglobulin wild type:
(1) K41C, K C and K91C;
(2) k41C and K91C;
(3) I1C, K C and K58C;
(4) i1C, K41C, K C and K91C;
The wild type human beta 2-microglobulin has an amino acid sequence shown as SEQ ID NO. 1.
2. The human β2-microglobulin mutant of claim 1, wherein the human β2-microglobulin mutant has mutations in K41C, K C and K91C as compared to the human β2-microglobulin wild type.
3. A protein conjugate of the present invention, which comprises a protein, characterized by comprising the following steps:
the human β2-microglobulin mutant of claim 1 or 2;
a conjugate to which the human β2-microglobulin mutant is linked.
4. A protein conjugate according to claim 3, wherein the conjugate is selected from small molecule polyethylene glycol derivatives.
5. The protein conjugate of claim 4, wherein said small molecule polyethylene glycol derivative is selected from the group consisting of small molecule polyethylene glycol derivatives having an activating group through which said human β2-microglobulin mutant and said small molecule polyethylene glycol derivative are linked.
6. The protein conjugate of claim 5, wherein said 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 of claim 6, wherein said ester group is selected from the group consisting of a succinimidoacetate group, a succinimidopropionate group, and a succinimidocarbonate group.
8. The protein conjugate of claim 6, wherein the aldehyde group is selected from the group consisting of propionaldehyde group, butyraldehyde group, acetaldehyde group, and valeraldehyde group.
9. The protein conjugate of claim 4, wherein said small molecule polyethylene glycol derivative is selected from the group consisting of methoxy polyethylene glycol maleimide, said human β2-microglobulin mutant and said methoxy polyethylene glycol maleimide are linked by maleimide, and said thiol group of said human β2-microglobulin mutant and said methoxy polyethylene glycol maleimide are linked by maleimide.
10. A method of preparing a protein conjugate comprising:
Contacting the human β2-microglobulin mutant of claim 1 or 2 with a conjugate to obtain the protein conjugate.
11. The method according to claim 10, wherein the conjugate is selected from small molecule polyethylene glycol derivatives, which are identical to the small molecule polyethylene glycol derivatives defined in any one of claims 3 to 9.
12. The method of claim 10, wherein the contacting is performed at a molar ratio of the human β2-microglobulin mutant to the conjugate of 1:1, the final concentration of the conjugate in a solution comprising the conjugate and human β2-microglobulin mutant being between 0.5mm and 2mm;
the final concentration of the human beta 2-microglobulin mutant in a solution containing the conjugate and the human beta 2-microglobulin mutant is 0.5 mM-2 mM.
13. A method of increasing the immunogenicity of a human β2-microglobulin comprising:
Contacting the human beta 2-microglobulin with a small molecule polyethylene glycol derivative;
wherein the human β2-microglobulin is selected from the human β2-microglobulin mutant of claim 1 or 2, and the small molecule polyethylene glycol derivative is identical to the conjugate defined in any one of claims 3 to 9.
14. The method of claim 13, wherein the contacting is performed at a molar ratio of the human β2-microglobulin mutant to the conjugate of 1:1, and wherein the final concentration of the small polyethylene glycol derivative in the solution comprising the small polyethylene glycol derivative and the human β2-microglobulin mutant is 0.5mm to 2mm;
The final concentration of the human beta 2-microglobulin mutant in the solution containing the small molecular polyethylene glycol derivative and the human beta 2-microglobulin mutant is 0.5 mM-2 mM.
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