Analog protein, preparation method thereof and in-vitro diagnostic reagent

Abstract

The invention discloses an analogue protein, a preparation method thereof and an in-vitro diagnostic reagent, and belongs to the field of in-vitro diagnosis. The target stable protein in the in-vitro diagnostic reagent is mutated to obtain the analogue protein, and the analogue protein is used as a protein stabilizer of the in-vitro diagnostic reagent, so that the stability of the target stable protein can be improved, and the detection accuracy of the in-vitro diagnostic reagent is improved.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and in particular to an analog protein and a preparation method thereof, and an in vitro diagnostic reagent. Background Art

[0002] In the field of clinical medical testing, the stability of reagents is an important parameter for the quality of in vitro diagnostic reagents. For example, the stability of calibrators or quality control reagents affects the accuracy of the measured values ​​after calibration. When the stability of the calibrators is poor, it will lead to large deviations in the measured values, affecting the objectivity of the measurement results.

[0003] There are many factors that lead to the poor stability of the above-mentioned in vitro diagnostic reagents. Among them, the antigens participating in the immune response in the in vitro diagnostic reagents have poor stability, and aggregation and degradation are common factors. Taking SAA (Serum amyloid A, SAA) as an example, SAA is an acute phase protein produced by liver cells and secreted into the serum. Clinically, blood routine tests are combined with CRP and SAA for detection. SAA increases in response to both viral and bacterial infections, and also responds sensitively to local inflammation; with the recovery of infection or the disappearance of inflammation, the SAA concentration will quickly drop to normal levels, and is usually used clinically to indicate inflammatory responses and identify infection types. However, as a relatively unstable protein, the SAA antigen has poor stability in vitro. The recombinantly expressed SAA antigen is prone to aggregation and degradation in vitro, which is the main reason for the poor stability of SAA, and directly affects the service life and product quality of SAA-related diagnostic reagent products.

[0004] In the prior art, stable diagnostic reagent products are obtained by using stable SAA mutants as immunogens; in addition, there are reports of using protein cross-linking agents to obtain SAA polymers as immunogens, thereby obtaining SAA immunogens with improved stability. However, both of the above methods directly change the antigen structure involved in the immune response, which may lead to differences in reactivity and real samples. Summary of the invention

[0005] The main purpose of the present invention is to provide an analog protein and a preparation method thereof, and an in vitro diagnostic reagent to solve the technical problem of poor stability of the in vitro diagnostic reagent.

[0006] To achieve the above object, the present invention provides an in vitro diagnostic reagent, which contains a protein stabilizer. The protein stabilizer includes an analog protein, and the analog protein is obtained by mutating a target stable protein.

[0007] In some embodiments of the present application, the amino acid sequence of the analog protein overlaps with the amino acid sequence of the target stable protein by more than 80%;

[0008] And / or, the target stable protein includes serum amyloid A, C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide or myoglobin.

[0009] In some embodiments of the present application, the serum amyloid protein A is used as a template, the phenylalanine at position 3 in the serum amyloid protein A is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.1;

[0010] and / or, using the serum amyloid protein A as a template, mutating the phenylalanine at position 4 to leucine, the methionine at position 17 to leucine, the glutamine at position 66 to proline, and the phenylalanine at position 68 to leucine in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2;

[0011] and / or, using the serum amyloid protein A as a template, mutating the methionine at position 17 to leucine, mutating the glutamine at position 66 to proline, mutating the phenylalanine at position 68 to leucine, and mutating the glycine at position 72 to aspartic acid in the serum amyloid protein A to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.3;

[0012] and / or, using the serum amyloid protein A as a template, mutating at least one of the amino acid at position 3 and the amino acid at position 4 in the serum amyloid protein A to any amino acid except phenylalanine to obtain the analog protein;

[0013] and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 17 in the serum amyloid protein A to any amino acid except methionine to obtain the analog protein;

[0014] and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 44 in the serum amyloid protein A to any amino acid except alanine to obtain the analog protein;

[0015] And / or, using the serum amyloid protein A as a template, the amino acids at positions 66 to 68 in the serum amyloid protein A are mutated to any amino acid other than the amino acids in the serum amyloid protein A sequence to obtain the analog protein.

[0016] In some embodiments of the present application, the mass ratio of the analog protein to the target stable protein is (1:10)-(10:1);

[0017] Alternatively, the target stable protein is in liquid form, and based on the total volume L of the liquid target stable protein, the added amount of the analog protein is (1.0-100) mg / L.

[0018] In some embodiments of the present application, the in vitro diagnostic reagent includes a quality control product, a calibrator, or a kit;

[0019] and / or, the in vitro diagnostic reagent is a diagnostic reagent used for turbidimetry, enzyme-linked immunosorbent assay, radioimmunoassay or chemiluminescence assay;

[0020] And / or, the test sample of the in vitro diagnostic reagent includes serum, plasma, whole blood or body fluid.

[0021] In some embodiments of the present application, the in vitro diagnostic reagent is a kit, which includes a serum amyloid A detection kit, a C-reactive protein detection kit, a procalcitonin detection kit, an interleukin 6 detection kit, a brain natriuretic peptide detection kit or a myoglobin detection kit.

[0022] To achieve the above object, the present invention also provides a method for preparing an analog protein, wherein the analog protein is obtained by mutating less than 20% of the amino acids in the amino acid sequence of the wild-type protein.

[0023] In some embodiments of the present application, the wild-type protein includes serum amyloid A, C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide or myoglobin;

[0024] And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, the phenylalanine at position 3 in the serum amyloid protein A is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1;

[0025] And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template, and the phenylalanine at position 4 in the serum amyloid protein A is mutated to leucine, the methionine at position 17 is mutated to leucine, the glutamine at position 66 is mutated to proline, and the phenylalanine at position 68 is mutated to leucine to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2;

[0026] And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template to mutate the methionine at position 17 to leucine, the glutamine at position 66 to proline, the phenylalanine at position 68 to leucine, and the glycine at position 72 to aspartic acid in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.3;

[0027] And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, at least one of the amino acid at position 3 and the amino acid at position 4 in the serum amyloid protein A is mutated to any amino acid except phenylalanine to obtain the analog protein;

[0028] And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template, and the amino acid at position 17 in the serum amyloid protein A is mutated to any amino acid except methionine to obtain the analog protein;

[0029] and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 44 in the serum amyloid protein A to any amino acid except alanine to obtain the analog protein;

[0030] And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, the amino acids at positions 66 to 68 in the serum amyloid protein A are mutated to any amino acid other than the amino acids in the serum amyloid protein A sequence to obtain the analog protein.

[0031] To achieve the above object, the present invention also provides an analog protein, which is obtained by mutation of a wild-type protein, and the amino acid sequence of the analog protein overlaps with that of the wild-type protein by more than 80%.

[0032] In some embodiments of the present application, the analog protein has an amino acid sequence as shown in SEQ ID NO.1;

[0033] And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.2;

[0034] And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.3;

[0035] And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.4;

[0036] And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.5;

[0037] And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.6;

[0038] And / or, the analog protein has the amino acid sequence shown in SEQ ID NO.7.

[0039] The beneficial effects that can be achieved by the present invention are:

[0040] The present invention uses an analog protein obtained by mutating a target stable protein as a protein stabilizer and adds it to an in vitro diagnostic reagent. The analog protein has lower reactivity than the target stable protein. If the target stable protein is used to participate in an antigen-antibody immune reaction, such as participating as an antigen or antibody in an immune reaction used as a diagnostic reagent, the activity of the analog protein in the corresponding antigen-antibody reaction can be reduced by more than 90% compared with the target stable protein, so that it has the effect of enhancing the stability of the target protein and preventing the target stable protein from aggregation and degradation; if the target stable protein is used for an enzyme catalytic reaction, the corresponding enzyme catalytic activity of the analog protein can be reduced by more than 90%, so that it has the effect of enhancing the stability of the target enzyme and preventing the target enzyme from aggregation and degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0042] Figure 1 Schematic diagram of a method for constructing an expression vector according to an embodiment of the present invention.

[0043] Figure 2 This is a test result diagram of the accelerated experiment of Example 1 of the present invention.

[0044] Figure 3 This is a test result diagram of the accelerated experiment of Example 2 of the present invention.

[0045] Figure 4 This is a test result diagram of the accelerated experiment of Example 3 of the present invention.

[0046] Figure 5 This is a test result diagram of the accelerated experiment of Example 4 of the present invention.

[0047] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0048] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] In the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their 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 at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0051] The present invention provides an in vitro diagnostic reagent, which contains a protein stabilizer. The protein stabilizer includes an analog protein, and the analog protein is obtained by mutation of a target stable protein.

[0052] It should be noted that the above-mentioned target stable proteins are enzymes, antibodies, antigens, etc., which need to improve the stability in in vitro diagnostic reagents.

[0053] The present invention uses an analog protein obtained by mutating a target stable protein as its protein stabilizer. When the target stable protein participates in the immune response of an in vitro diagnostic reagent, the activity of the analog protein can be reduced by more than 90% compared with the target stable protein. When the target stable protein is used in an enzyme catalytic reaction, the enzyme catalytic activity of the analog protein can also be reduced by more than 90%, which is beneficial to improving the stability of the target stable protein and preventing the target stable protein from aggregation and degradation.

[0054] In some embodiments, the amino acid sequence of the analog protein overlaps with the amino acid sequence of the target stable protein by more than 80% and less than 100%.

[0055] In some embodiments, the amino acid sequence of the analog protein overlaps with the amino acid sequence of the target stable protein at any one of the ranges of 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 97%, 98%, 99%, etc., which is more than 80% and less than 100%. Under the above overlap conditions, when the target stable protein participates in an in vitro diagnostic reagent or an enzyme-catalyzed reaction, the analog protein, as a protein stabilizer, is conducive to reducing the activity of the analog protein by more than 90% compared with the target stable protein, thereby achieving the effect of enhancing the stability of the target stable protein and preventing the aggregation and degradation of the target stable protein.

[0056] The present invention does not limit the type of target stable protein. In some embodiments, the target stable protein can be serum amyloid A (SAA), C-reactive protein (CRP), procalcitonin (PCT), interleukin 6 (IL-6), brain natriuretic peptide (BNP) or myoglobin (MYO). The above types of proteins are used as templates for mutation to obtain the corresponding analog proteins, and the analog proteins are added to the in vitro diagnostic reagents as protein stabilizers. The analog proteins have lower reactivity than the target stable protein. If the target stable protein is used to participate in the antigen-antibody immune reaction, such as participating in the immune reaction of the diagnostic reagent as an antigen or antibody, the activity of the analog protein in the corresponding antigen-antibody reaction can be reduced by more than 90% compared with the target stable protein, so that it has the effect of enhancing the stability of the target protein and preventing it from aggregation and degradation; if the target stable protein is used for enzyme catalysis, the corresponding enzyme catalytic activity of the analog protein can be reduced by more than 90%, so that it has the effect of enhancing the stability of the target enzyme.

[0057] It should be noted that the analog proteins obtained by mutating SAA, CRP, PCT, IL-6, BNP, and MYO as template proteins can not only serve as protein stabilizers for the corresponding template proteins, but in some embodiments, can also serve as protein stabilizers for proteins other than the template proteins. For example, when SAA is used as a template protein to mutate to obtain an SAA analog protein, the SAA analog protein can not only serve as a protein stabilizer for SAA, but also has good stability for CRP and can improve the stability of the SAA / CRP composite antigen.

[0058] The present invention is now described using SAA antigen as an example, but this is not to be construed as a limitation of the present invention.

[0059] As a relatively unstable protein, SAA antigen has poor in vitro stability, which directly affects the shelf life and product quality of SAA-related diagnostic reagent products. The aggregation and degradation of recombinantly expressed SAA antigen in vitro are the main reasons for the poor stability of SAA antigen. In the present invention, serum amyloid A is used as a template for mutation, and the analog protein of serum amyloid A is obtained and used as a protein stabilizer, which can solve the problem of poor stability of SAA antigen.

[0060] The present invention now lists some amino acid sequences of SAA analog proteins, but the following examples are only some examples of the present invention and do not constitute a limitation to the technical solution of the present invention.

[0061] In some embodiments, using serum amyloid A as a template, the phenylalanine at position 3 in serum amyloid A is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1.

[0062] In some embodiments, using serum amyloid protein A as a template, the phenylalanine at position 4 in serum amyloid protein A is mutated to leucine, the methionine at position 17 is mutated to leucine, the glutamine at position 66 is mutated to proline, and the phenylalanine at position 68 is mutated to leucine to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2.

[0063] In some embodiments, using serum amyloid A as a template, the methionine at position 17 in serum amyloid A is mutated to leucine, the glutamine at position 66 is mutated to proline, the phenylalanine at position 68 is mutated to leucine, and the glycine at position 72 is mutated to aspartic acid to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.3.

[0064] In some embodiments, using serum amyloid protein A as a template, at least one of the 3rd amino acid and the 4th amino acid in serum amyloid protein A is mutated to any amino acid other than phenylalanine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.4; illustratively, the 3rd amino acid and the 4th amino acid in serum amyloid protein A are mutated to glycine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.4.

[0065] In some embodiments, using serum amyloid A as a template, the amino acid at position 17 in serum amyloid A is mutated to any amino acid other than methionine to obtain an analog protein. For example, the amino acid at position 17 in serum amyloid A is mutated to aspartic acid to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.5;

[0066] In some embodiments, using serum amyloid A as a template, the amino acid at position 44 in serum amyloid A is mutated to any amino acid other than alanine to obtain an analog protein. For example, the amino acid at position 44 in serum amyloid A is mutated to arginine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.6;

[0067] In some embodiments, using serum amyloid protein A as a template, the amino acids at positions 66 to 68 in serum amyloid protein A are mutated to any amino acid other than the amino acids in the serum amyloid protein A sequence to obtain an analog protein. For example, the amino acids at positions 66 to 68 in serum amyloid protein A are mutated to phenylalanine, aspartic acid, and glutamine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.7.

[0068] The analog protein obtained in the above embodiment has an amino acid sequence that is more than 80% and less than 100% identical to the amino acid sequence of the template protein serum amyloid A. It is used as a protein stabilizer for serum amyloid A, which can improve the stability of serum amyloid A when used as an in vitro diagnostic reagent product, making serum amyloid A more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0069] The present invention can also use C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide, myoglobin, etc. as template proteins, mutate to obtain corresponding analog proteins, so that the amino acid sequence of the analog protein and the amino acid sequence of the template protein have an overlap rate of more than 80% and less than 100%. Using the analog protein as a protein stabilizer can improve the stability of the template protein or proteins other than the template protein as in vitro diagnostic reagent products, make the immune reaction or enzyme-catalyzed reaction more stable, and the obtained detection effect is more objective.

[0070] The present invention does not limit the mass ratio of the analog protein and the target stabilizing protein in the in vitro diagnostic reagent when the analog protein is used as a protein stabilizer. In some embodiments, the mass ratio of the analog protein and the target stabilizing protein is (1:10)-(10:1); for example, it can be any ratio in the range of (1:10)-(10:1), such as 1:10, 1:8, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. Under the above mass ratio conditions, the analog protein can exert a good stabilizing effect and improve the stability of the target stabilizing protein during the immune response or enzyme catalytic reaction in the in vitro diagnostic test.

[0071] In addition, the present invention also provides another method for determining the amount of analog protein added to an in vitro diagnostic reagent as a protein stabilizer. When the target stable protein is in liquid form, that is, the target stable protein stock solution, the amount of analog protein added can be (1.0-100) mg / L based on the total volume L of the target stable protein stock solution, for example, it can be 1.0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, 100 mg / L, etc. Any one of the addition amounts in the range of (1.0-100) mg / L. Under the above addition amount range conditions, the analog protein can exert a good stabilizing effect and improve the stability of the target stable protein during the immune response or enzyme catalytic reaction of in vitro diagnostic reagents.

[0072] The present invention does not limit the type of in vitro diagnostic reagents, which may be quality control products, calibrators, kits, etc. Exemplarily, the in vitro diagnostic reagent is a quality control product or a calibrator, and the analog protein of the present invention is used as a protein stabilizer for the target stable protein. The calibrator or quality control product has good stability, and the accuracy of the measured value after calibration is high, the measured value deviation is small, and it is not easy to affect the objectivity of the measurement result. Exemplarily, the in vitro diagnostic reagent is a kit, and the analog protein of the present invention is used as a protein stabilizer for the target stable protein. The target stable protein in the kit has good stability, the measured value deviation is small, and it is not easy to affect the objectivity of the measurement result.

[0073] In addition, the present invention does not limit the application of the in vitro diagnostic reagent, and the in vitro diagnostic reagent can be used as a diagnostic reagent for turbidimetry, enzyme-linked immunosorbent assay, radioimmunoassay or chemiluminescence assay.

[0074] In addition, the present invention does not limit the type of test sample of the in vitro diagnostic reagent, including serum, plasma, whole blood or body fluid.

[0075] In addition, the present invention does not limit the type of the kit. In some embodiments, the kit is a serum amyloid A detection kit, a C-reactive protein detection kit, a procalcitonin detection kit, an interleukin 6 detection kit, a brain natriuretic peptide detection kit or a myoglobin detection kit.

[0076] The present invention also provides a method for preparing an analog protein, wherein less than 20% of the amino acids in the amino acid sequence of a wild-type protein are mutated to obtain the corresponding analog protein. The present invention does not limit the mutation method, and the mutation can be performed using methods commonly used by those skilled in the art.

[0077] The present invention is not limited to the type of wild-type protein. In some embodiments, the wild-type protein is serum amyloid A, C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide or myoglobin.

[0078] The present invention now lists some embodiments of the method for preparing analog proteins, but the following embodiments are only some embodiments of the present invention and do not constitute a limitation to the technical solution of the present invention.

[0079] In some embodiments, using serum amyloid A as a template, the phenylalanine at position 3 in serum amyloid A is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1.

[0080] In some embodiments, using serum amyloid protein A as a template, the phenylalanine at position 4 in serum amyloid protein A is mutated to leucine, the methionine at position 17 is mutated to leucine, the glutamine at position 66 is mutated to proline, and the phenylalanine at position 68 is mutated to leucine to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2.

[0081] In some embodiments, using serum amyloid A as a template, the methionine at position 17 in serum amyloid A is mutated to leucine, the glutamine at position 66 is mutated to proline, the phenylalanine at position 68 is mutated to leucine, and the glycine at position 72 is mutated to aspartic acid to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.3.

[0082] In some embodiments, using serum amyloid protein A as a template, at least one of the amino acids at positions 3 and 4 in serum amyloid protein A is mutated to any amino acid other than phenylalanine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.4;

[0083] In some embodiments, using serum amyloid A as a template, the amino acid at position 17 in serum amyloid A is mutated to any amino acid other than methionine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.5;

[0084] In some embodiments, using serum amyloid A as a template, the amino acid at position 44 in serum amyloid A is mutated to any amino acid except alanine to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.6;

[0085] In some embodiments, using serum amyloid A as a template, the amino acids at positions 66 to 68 in serum amyloid A are mutated to any amino acid other than the amino acids in the serum amyloid A sequence to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.7.

[0086] The analog protein corresponding to serum amyloid A prepared by the above preparation method has an amino acid sequence that is more than 80% and less than 100% identical to the amino acid sequence of the template protein serum amyloid A. The analog protein serves as a protein stabilizer for serum amyloid A and can improve the stability of serum amyloid A, making serum amyloid A more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0087] In some embodiments, C-reactive protein is used as a mutation template to obtain an analog protein corresponding to the C-reactive protein, and the amino acid sequence of the analog protein has an overlap rate of more than 80% and less than 100% with the amino acids of the template protein C-reactive protein. The analog protein serves as a protein stabilizer for C-reactive protein, which can improve the stability of C-reactive protein, making the C-reactive protein more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0088] In some embodiments, procalcitonin is used as a mutation template to obtain an analog protein corresponding to procalcitonin, and the amino acid sequence of the analog protein has an overlap rate of more than 80% and less than 100% with the amino acid of the template protein procalcitonin. The analog protein serves as a protein stabilizer for procalcitonin, which can improve the stability of procalcitonin, making procalcitonin more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0089] In some embodiments, interleukin 6 is used as a mutation template to obtain an analog protein corresponding to interleukin 6, and the amino acid sequence of the analog protein has an overlap rate of more than 80% and less than 100% with the amino acid of the template protein interleukin 6. The analog protein serves as a protein stabilizer for interleukin 6, which can improve the stability of interleukin 6 and make interleukin 6 more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0090] In some embodiments, brain natriuretic peptide is used as a mutation template to obtain an analog protein corresponding to brain natriuretic peptide, and the amino acid sequence of the analog protein has an overlap rate of more than 80% and less than 100% with the amino acid of the template protein brain natriuretic peptide. The analog protein serves as a protein stabilizer for brain natriuretic peptide, which can improve the stability of brain natriuretic peptide, making brain natriuretic peptide more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0091] In some embodiments, myoglobin is used as a mutation template to obtain an analog protein corresponding to myoglobin, and the amino acid sequence of the analog protein has an overlap rate of more than 80% and less than 100% with the amino acid of the template myoglobin. The analog protein serves as a protein stabilizer for myoglobin, which can improve the stability of myoglobin, making myoglobin more stable when participating in immune reactions or enzyme-catalyzed reactions, and the resulting detection effect is more objective.

[0092] The present invention also provides an analog protein, which is obtained by mutation of a wild-type protein, and the overlap rate between the amino acid sequence of the analog protein and the amino acid sequence of the wild-type protein is more than 80% and less than 100%.

[0093] In some embodiments, the wild-type protein can be serum amyloid A, C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide or myoglobin. The above types of proteins are used as templates for mutation to obtain the corresponding analog proteins, and the analog proteins are added to the in vitro diagnostic reagents as protein stabilizers. The analog proteins have lower reactivity than the target stable protein. If the target stable protein is used to participate in the antigen-antibody immune reaction, such as as an antigen or antibody in the immune reaction used for diagnostic reagents, the activity of the analog protein in the corresponding antigen-antibody reaction can be reduced by more than 90% compared with the target stable protein, so that it has the effect of enhancing the stability of the target protein; if the target stable protein is used for enzyme catalysis, the corresponding enzyme catalytic activity of the analog protein can be reduced by more than 90%, so that it has the effect of enhancing the stability of the target enzyme.

[0094] The present invention does not limit the specific amino acid sequence of the analog protein. The analog proteins in some embodiments are now shown, but the following embodiments are only some embodiments of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0095] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.1.

[0096] In some embodiments, the analog protein has the amino acid sequence shown in SEQ ID NO.2.

[0097] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.3.

[0098] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.4.

[0099] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.5.

[0100] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.6.

[0101] In some embodiments, the analog protein has an amino acid sequence as shown in SEQ ID NO.7.

[0102] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0103] Determine the reactivity of analog proteins and target stable proteins

[0104] 1. Preparation of analog proteins

[0105] Using serum amyloid A (SAA) as a template, the phenylalanine at position 3 in SAA is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1, and the analog protein is marked as mutant A.

[0106] Using serum amyloid A (SAA) as a template, the phenylalanine at position 4 in SAA is mutated to leucine, the methionine at position 17 is mutated to leucine, the glutamine at position 66 is mutated to proline, and the phenylalanine at position 68 is mutated to leucine to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2, and the analog protein is marked as mutant B.

[0107] Using serum amyloid A (SAA) as a template, the methionine at position 17 in SAA is mutated to leucine, the glutamine at position 66 is mutated to proline, the phenylalanine at position 68 is mutated to leucine, and the glycine at position 72 is mutated to aspartic acid to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.3, and the analog protein is marked as mutant C.

[0108] Reference Figure 1 According to the amino acid sequences of the original and mutant SAA, and in accordance with the codon optimization principle of E. coli, the corresponding nucleotide sequences were designed, synthesized, and constructed into the modified E. coli expression vector pET28a-His-TEV. The constructed expression vectors are:

[0109] Wild-type SAA: pET28a-SAA-V1;

[0110] SAA mutant A: pET28a-SAA-V2;

[0111] SAA mutant B: pET28a-SAA-V3;

[0112] SAA mutant C: pET28a-SAA-V4;

[0113] The nucleotide sequences corresponding to SAA mutants A, B, and C are SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The above plasmids were transformed into E. coli host BL21 (DE3), and the transformants were inoculated into LB medium and cultured at 37°C overnight. They were transferred to fresh LB medium at 1 / 100 and continued to be cultured at 37°C until OD600 = 0.6-0.8, and IPTG was added at a final concentration of 0.5 mM to induce expression. After 5 hours of induction, the cells were collected, broken with a homogenizer, and affinity chromatography was performed. The purified fusion protein was removed from the Trx tag with TEV enzyme, and the tag protein was removed again by affinity chromatography to obtain wild-type SAA and SAA mutants without tags for subsequent evaluation.

[0114] The above-mentioned SAA mutants A, B, and C were used as protein stabilizers of SAA antigens, respectively. The reactivity of the above-mentioned SAA mutants in SAA reagents at different concentrations was measured using latex turbidimetric SAA reagents produced by different manufacturers. The test results of the reactivity are shown in Table 1.

[0115] Among them, manufacturer A source: brand Zhongyuan Huiji, reagent batch number H220901, serum amyloid A detection kit (latex enhanced immunoturbidimetry), biochemical line;

[0116] Manufacturer B source: Brand Jiuqiang Biological, reagent batch number 22-0418;

[0117] Manufacturer C source: Brand Ipronocon, reagent batch number 2022061102;

[0118] Manufacturer D Source: Brand Ningbo Prebo, reagent batch number SS3265.

[0119] Table 1

[0120]

[0121]

[0122] As shown in the results of Table 1, the SAA of mutant C and mutant B are almost unreactive under the test of reagents from different manufacturers, and will not react with SAA, so they are suitable for being added alone as a protective agent; the SAA of mutant A has a low reactivity under the test of reagents from most manufacturers, but has reactivity under the test of reagents from manufacturer B. When added as a protective agent, sufficient concentration adjustment and calibration efficacy evaluation can be performed.

[0123] Example 1

[0124] SAA mutant A, SAA mutant B, and SAA mutant C were added to the SAA antigen at a concentration of 25 mg / L as protein stabilizers, and then compared with the SAA antigen solution without the above protein stabilizer to compare the stability of the 25 mg / L SAA antigen under the action of different SAA mutants. The SAA solution without the addition of different SAA mutants was subjected to a 37°C thermal acceleration test and compared with the SAA solution stored at 4°C. The SAA antigen solution was tested using the Zhongyuan Huiji Biochemical SAA (latex enhanced immunoturbidimetry) reagent. The test results are as follows: Figure 2 shown.

[0125] from Figure 2 It can be seen that compared with the SAA antigen solution without adding the protein stabilizer SAA mutant, the SAA antigen solution with the above-mentioned SAA mutant as a protein stabilizer can be observed to have better stability in the observation group accelerated for more than 7 days. Mutants A, B, and C have a good protective effect on the target stable protein SAA, and the effect of mutant C is better. It can be seen that the analog protein of the present invention can be added as a protein stabilizer to in vitro diagnostic reagents such as quality control products and calibrators to improve the stability of the target stable protein and improve the accuracy of the test results.

[0126] Example 2

[0127] The mutant C with better protective effect in Example 1 was added as a protein stabilizer at a concentration of 25 mg / L to SAA antigen solutions with concentrations of 10 mg / L, 120 mg / L, and 240 mg / L, respectively, and the stability was compared with that of the SAA antigen solution without adding a protective agent. The stability experiment specifically involved: a 37°C thermal acceleration test was performed on the SAA solutions with different concentrations of mutant C added, and then compared with the SAA solutions stored at 4°C. The experimental results are shown in Figure 3 shown.

[0128] Depend on Figure 3 It can be seen that under the 14-day 37°C heat acceleration experiment, the addition of mutant C as a protein stabilizer can significantly improve the stability of the SAA antigen, especially for the SAA antigen solution with a target concentration of 10 mg / L.

[0129] Example 3

[0130] Taking the total volume L of the SAA antigen solution with a concentration of 10 mg / L as the benchmark, mutant C was added to the SAA antigen solution as a protein stabilizer at 1.0 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, and 100 mg / L, and then the stability was compared with the SAA antigen solution without adding a protective agent. The stability experiment is as follows: the SAA solution with added mutant C was subjected to a 37°C thermal acceleration test and the measurement values ​​were tracked for 14 days. The experimental results are as follows Figure 4 As shown. Figure 4 It can be seen that adding the mutant as a protein stabilizer has different degrees of protein stabilization effect in the range of 1 to 100 mg / L.

[0131] Example 4

[0132] The CRP / SAA antigen composite calibrator was prepared using a buffer formula with mutant A added as a protein stabilizer (25 mg / L). The mass concentration of CRP antigen was 20 mg / L, the mass concentration of SAA antigen was 10 mg / L, and the mass concentration of protein stabilizer was 25 mg / L. Long-term tracking of CRP / SAA antigen stability, the stability results are as follows Figure 5 shown.

[0133] Depend on Figure 5 It can be seen that the CRP / SAA composite antigen composite calibrator with mutant A as a protein stabilizer can simultaneously meet the stability requirements of SAA and CRP antigens. It can be seen that the analog protein obtained by SAA mutation in the present invention can be used as a protein stabilizer for SAA antigen and a stabilizer for CRP antigen, and can be applied to the project of joint detection of CRP and SAA.

[0134] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An in vitro diagnostic reagent comprising a protein stabilizer, characterized in that: The protein stabilizer includes an analog protein, and the analog protein is obtained by mutation of the target stabilized protein.

2. The in vitro diagnostic reagent according to claim 1, characterized in that: The amino acid sequence of the analog protein overlaps with the amino acid sequence of the target stable protein by more than 80%; And / or, the target stable protein includes serum amyloid A, C-reactive protein, procalcitonin, interleukin 6, brain natriuretic peptide or myoglobin.

3. The in vitro diagnostic reagent according to claim 2, characterized in that: Using the serum amyloid protein A as a template, mutating the phenylalanine at position 3 to alanine, the alanine at position 44 to valine, and the glutamine at position 66 to proline in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1; and / or, using the serum amyloid protein A as a template, mutating the phenylalanine at position 4 to leucine, the methionine at position 17 to leucine, the glutamine at position 66 to proline, and the phenylalanine at position 68 to leucine in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2; and / or, using the serum amyloid protein A as a template, mutating the methionine at position 17 to leucine, mutating the glutamine at position 66 to proline, mutating the phenylalanine at position 68 to leucine, and mutating the glycine at position 72 to aspartic acid in the serum amyloid protein A to obtain an analog protein having an amino acid sequence as shown in SEQ ID NO.3; and / or, using the serum amyloid protein A as a template, mutating at least one of the amino acid at position 3 and the amino acid at position 4 in the serum amyloid protein A to any amino acid except phenylalanine to obtain the analog protein; and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 17 in the serum amyloid protein A to any amino acid except methionine to obtain the analog protein; and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 44 in the serum amyloid protein A to any amino acid except alanine to obtain the analog protein; And / or, using the serum amyloid protein A as a template, the amino acids at positions 66 to 68 in the serum amyloid protein A are mutated to any amino acid other than the amino acids in the serum amyloid protein A sequence to obtain the analog protein.

4. The in vitro diagnostic reagent according to any one of claims 1 to 3, characterized in that: The mass ratio of the analog protein to the target stable protein is (1:10)-(10:1); Alternatively, the target stable protein is a target stable protein stock solution, and based on the total volume L of the target stable protein stock solution, the added amount of the analog protein is (1.0-100) mg / L.

5. The in vitro diagnostic reagent according to any one of claims 1 to 3, wherein the in vitro diagnostic reagent comprises a quality control product, a calibrator or a kit; and / or, the in vitro diagnostic reagent is a diagnostic reagent used for turbidimetry, enzyme-linked immunosorbent assay, radioimmunoassay or chemiluminescence assay; And / or, the test sample of the in vitro diagnostic reagent includes serum, plasma, whole blood or body fluid.

6. The in vitro diagnostic reagent according to claim 5, characterized in that: The in vitro diagnostic reagent is a kit, which includes a serum amyloid A detection kit, a C-reactive protein detection kit, a procalcitonin detection kit, an interleukin 6 detection kit, a brain natriuretic peptide detection kit or a myoglobin detection kit.

7. A method for preparing an analog protein, characterized in that: The analog protein is obtained by mutating less than 20% of the amino acids in the amino acid sequence of the wild-type protein.

8. The method for preparing the analog protein according to claim 7, characterized in that: The wild-type protein includes serum amyloid A, C-reactive protein, procalcitonin, interleukin-6, brain natriuretic peptide or myoglobin; And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, the phenylalanine at position 3 in the serum amyloid protein A is mutated to alanine, the alanine at position 44 is mutated to valine, and the glutamine at position 66 is mutated to proline to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.1; And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template, and the phenylalanine at position 4 is mutated to leucine, the methionine at position 17 is mutated to leucine, the glutamine at position 66 is mutated to proline, and the phenylalanine at position 68 is mutated to leucine in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.2; And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template to mutate the methionine at position 17 to leucine, the glutamine at position 66 to proline, the phenylalanine at position 68 to leucine, and the glycine at position 72 to aspartic acid in the serum amyloid protein A to obtain an analog protein with an amino acid sequence as shown in SEQ ID NO.3; And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, at least one of the amino acid at position 3 and the amino acid at position 4 in the serum amyloid protein A is mutated to any amino acid except phenylalanine to obtain the analog protein; And / or, the wild-type protein is serum amyloid protein A, and the serum amyloid protein A is used as a template, and the amino acid at position 17 in the serum amyloid protein A is mutated to any amino acid except methionine to obtain the analog protein; and / or, using the serum amyloid protein A as a template, mutating the amino acid at position 44 in the serum amyloid protein A to any amino acid except alanine to obtain the analog protein; And / or, the wild-type protein is serum amyloid protein A, and using the serum amyloid protein A as a template, the amino acids at positions 66 to 68 in the serum amyloid protein A are mutated to any amino acid other than the amino acids in the serum amyloid protein A sequence to obtain the analog protein.

9. An analog protein, characterized in that The analog protein is obtained by mutation of the wild-type protein, and the coincidence rate between the amino acid sequence of the analog protein and the amino acid sequence of the wild-type protein is above 80%.

10. The analog protein according to claim 9, characterized in that The analog protein has an amino acid sequence as shown in SEQ ID NO.1; And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.2; And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.3; And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.4; And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.5; And / or, the analog protein has an amino acid sequence as shown in SEQ ID NO.6; And / or, the analog protein has the amino acid sequence shown in SEQ ID NO.7.

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