Fc fragment, recombinant vector, recombinant cell, fusion antigen and preparation method thereof

Abstract

The invention discloses an Fc fragment, a recombinant vector, a recombinant cell, a fusion antigen and a preparation method thereof, and relates to the field of antigen preparation. The Fc fragment comprises a first Fc fragment and a second Fc fragment capable of forming a pestle mortar structure with the first Fc fragment, the first Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 1, and the second Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 2. The Fc fragment structure provided by the invention has no influence on the performance of the antigen, the Fc fragment can be specifically combined with the antigen, the stability of the antigen is improved, and the expression quantity of the antigen in mammalian cells is improved, so that the fusion antigen with high expression quantity, high stability and high purity can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of antigen preparation, and in particular to an Fc fragment, a recombinant vector, a recombinant cell and a fusion antigen and a preparation method thereof. Background Art

[0002] Antigen refers to a substance that can induce an immune response in the matrix, produce antibodies, and specifically bind to antibodies to produce an immune response. It can be used to prepare diagnostic materials such as calibrators, quality control products, and standards, and is the core raw material for immunodiagnosis. Therefore, it is particularly important to obtain high-quality, suitable, and stable antigens.

[0003] Currently, antigens can be divided into natural antigens and recombinant antigens according to different preparation methods. Among them, natural antigens have complex structures, are difficult to purify, have non-specific binding and are easily inactivated, and have certain limitations. Recombinant antigens are usually expressed through five major expression systems, such as Escherichia coli, mammals, insect cells, Bacillus subtilis, and yeast. Although recombinant antigens have obvious advantages in long-acting technology, higher purity, and antigenicity that is closest to natural antigens compared to natural antigens, recombinant antigens prepared by existing methods have the disadvantages of low expression and poor stability, which leads to higher production costs.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] Based on the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide an Fc fragment, a recombinant vector, a recombinant cell and a fusion antigen and a method for preparing the same, aiming to solve the problems of low expression level and poor stability of the recombinant antigens prepared by the existing methods.

[0006] The technical solution of the present invention is as follows:

[0007] According to a first aspect of the present invention, an Fc fragment is provided, wherein the Fc fragment comprises a first Fc fragment and a second Fc fragment capable of forming a knob-and-hole structure with the first Fc fragment, the first Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 1, and the second Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 2.

[0008] Optionally, the first Fc fragment further comprises a protein purification tag sequence connected to one end of the amino acid sequence shown in SEQ ID NO:1.

[0009] Optionally, the second Fc fragment further comprises a protein purification tag sequence connected to one end of the amino acid sequence shown in SEQ ID NO:2.

[0010] The second aspect of the present invention provides a fusion antigen, wherein the fusion antigen comprises an antigen and the Fc fragment of the present invention as described above connected to the antigen via a linker.

[0011] The third aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains a nucleotide sequence encoding the fusion antigen of the present invention as described above.

[0012] A fourth aspect of the present invention provides a recombinant cell, wherein the recombinant cell contains the recombinant vector of the present invention as described above.

[0013] A fifth aspect of the present invention provides a method for preparing a fusion antigen, which comprises the following steps:

[0014] Inserting DNA fragments encoding a first Fc fragment, a linker and an antigen respectively into a first empty plasmid to obtain a first recombinant plasmid; the first Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 1;

[0015] Inserting a DNA fragment encoding a second Fc fragment into a second empty plasmid to obtain a second recombinant plasmid; the second Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 2;

[0016] The first recombinant plasmid and the second recombinant plasmid are mixed and then transfected into cells for eukaryotic expression. After purification, the fusion protein is obtained.

[0017] Optionally, the nucleotide sequence of the DNA fragment encoding the first Fc fragment is shown in SEQ ID NO: 3;

[0018] The nucleotide sequence of the DNA fragment encoding the second Fc fragment is shown in SEQ ID NO:4.

[0019] Optionally, the nucleotide sequences of the first empty vector plasmid and the second empty vector plasmid are both as shown in SEQ ID NO: 5;

[0020] The nucleotide sequence of the first recombinant plasmid is shown in SEQ ID NO:6; the nucleotide sequence of the second recombinant plasmid is shown in SEQ ID NO:7.

[0021] Optionally, the cell is a mammalian cell.

[0022] Beneficial effects: The present invention performs mutation design based on the Fc fragment of IgG1, introduces complementary mutation sites, mutates the small amino acid in the CH3 region on one chain into a large amino acid, and the corresponding large amino acid on the other chain into a small amino acid, respectively forming a knob and hole structure (i.e., a convex and concave structure), and obtains an Fc fragment with a special structure, i.e., the Fc fragment contains a first Fc fragment and a second Fc fragment that can form a knob and hole structure with the first Fc fragment. The Fc fragment structure has no effect on the performance of the antigen, and the Fc fragment can specifically bind to the antigen, improve the stability of the antigen, and increase the expression of the antigen in mammalian cells, thereby obtaining a fusion antigen with high expression, high stability and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structure of the fusion antigen.

[0024] Figure 2 This is the SDS-PAGE electrophoresis diagram of the pM06-kiHk-Apo plasmid and the pM06-Hd plasmid after transfection at a mass ratio of 1:1 and purification by Ni-Ted in Example 1.

[0025] Figure 3 This is the SDS-PAGE electrophoresis diagram after pM06-Apo plasmid transfection and Ni-Ted purification. DETAILED DESCRIPTION

[0026] The present invention provides an Fc fragment, a recombinant vector, a recombinant cell, and a fusion antigen and a method for preparing the same. To make the purpose, technical solution, and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0028] An embodiment of the present invention provides an Fc fragment, wherein the Fc fragment includes a first Fc fragment and a second Fc fragment capable of forming a knob-in-hole structure with the first Fc fragment, the first Fc fragment includes the amino acid sequence shown in SEQ ID NO: 1, and the second Fc fragment includes the amino acid sequence shown in SEQ ID NO: 2.

[0029] In the embodiment of the present invention, mutation design is performed based on the Fc fragment of IgG1 (whose amino acid sequence is shown in SEQ ID NO: 8, i.e., the crystallizable fragment of IgG1), complementary mutation sites are introduced, and the small amino acid in the CH3 region on one chain is mutated into a large amino acid, and the corresponding large amino acid on the other chain is mutated into a small amino acid, thereby forming a knob and hole structure (i.e., a convex and concave structure), respectively, to obtain an Fc fragment with a special structure, i.e., the Fc fragment contains a first Fc fragment and a second Fc fragment that can form a knob and hole structure with the first Fc fragment. Specifically, the Fc fragment of IgG1 (whose amino acid sequence is shown in SEQ ID NO: 8) was subjected to S134C (cysteine ​​at position 134 serves as an element for forming the disulfide bond of the first Fc fragment), E136D, M138L and T146W site mutations to obtain the amino acid sequence shown in SEQ ID NO: 1, and the Fc fragment of IgG1 (whose amino acid sequence is shown in SEQ ID NO: 8) was subjected to E136D, E137K, M138L, T146S, L148A and Y187V site mutations to obtain the amino acid sequence shown in SEQ ID NO: 2.

[0030] Among them, E136D means that E (glutamic acid) at position 136 is mutated to D (aspartic acid), and the meanings of other similar expressions are explained here.

[0031] The Fc fragment structure provided in the embodiment of the present invention has no effect on the performance of the antigen. The Fc fragment can specifically bind to the antigen (to form a fusion antigen) and change the inherent structure of the antigen. The Fc fragment binds to the antigen to form a structure similar to a "Y" shape, which stabilizes the antigen morphology, reduces antigen degradation (i.e., improves antigen stability), and increases the expression level of the antigen in mammalian cells, thereby obtaining a fusion antigen with high expression level, high stability, high activity and high purity.

[0032] In some embodiments, the first Fc fragment further comprises a protein purification tag sequence connected to one end (N-terminus or C-terminus) of the amino acid sequence shown in SEQ ID NO: 1. In this way, the Fc fragment can specifically bind to the Ni-Ted column, simplifying the purification step of the fusion protein.

[0033] In some embodiments, the second Fc fragment further comprises a protein purification tag (e.g., His) sequence connected to one end (N-terminus or C-terminus) of the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence of the second Fc fragment can be shown in SEQ ID NO: 9. In this way, the Fc fragment can specifically bind to the Ni-Ted column, simplifying the purification step of the fusion protein. In other words, the amino acid sequence of the second Fc fragment can be shown in SEQ ID NO: 2 or in SEQ ID NO: 9.

[0034] The present invention also provides a DNA fragment, wherein the DNA fragment contains a nucleotide sequence encoding the Fc fragment described in the present invention. The DNA fragment in the present invention contains two DNA fragments, wherein one DNA fragment encodes a first Fc fragment, and its nucleotide sequence is shown in SEQ ID NO: 3; and the other DNA fragment encodes a second Fc fragment, and its nucleotide sequence can be shown in SEQ ID NO: 4.

[0035] The present invention also provides a fusion antigen, wherein: Figure 1 As shown, the fusion antigen includes an antigen and the Fc fragment of the embodiment of the present invention as described above, which is connected to the antigen through a linker. The Fc fragment is composed of a first Fc fragment and a second Fc fragment that forms a knob-in-hole structure (i.e., a Knob-in-hole structure) with the first Fc fragment. Specifically, the fusion antigen includes an antigen and an Fc fragment connected to the antigen through a peptide chain linker. The peptide chain linker may be (G4S)5, wherein the polyglycine-serine (G4S) linker is a flexible unstructured synthetic peptide linker sequence, and G4S is composed of a core pentapeptide sequence Gly-Gly-Gly-Gly-Ser. The Fc fragment is located at the N-terminus of the fusion antigen, and the antigen is located at the C segment of the fusion antigen.

[0036] The Fc fragment specifically binds to the antigen (forming a fusion antigen with a non-traditional antigen protein structure), that is, the Fc fragment binds to the antigen to form a structure similar to the "Y" shape, which changes the inherent structure of the antigen, stabilizes the antigen form, reduces the degradation of the antigen, enhances the stability of the antigen, and increases the expression of the antigen in mammalian cells. The Fc fragment structure has no effect on the performance of the antigen. Therefore, the fusion antigen provided in this embodiment has high stability and high expression. The fusion antigen can be applied to the production of standard products, quality control products, and calibration products, and can also be used to label conjugates, coated solid phases, etc.

[0037] In the embodiment of the present invention, the type of the antigen is not limited. As an example, the antigen bound to the Fc fragment is apolipoprotein Apo.

[0038] The embodiment of the present invention further provides a recombinant vector, wherein the recombinant vector contains a nucleotide sequence encoding the fusion antigen of the present invention as described above. The recombinant vector may be a recombinant plasmid.

[0039] An embodiment of the present invention further provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described above in the embodiment of the present invention.

[0040] The present invention also provides a method for preparing a fusion antigen, which comprises the following steps:

[0041] S1, inserting DNA fragments encoding a first Fc fragment, a linker (such as (G4S)5, etc.) and an antigen into a first empty plasmid to obtain a first recombinant plasmid; the first Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 1;

[0042] S2, inserting a DNA fragment encoding a second Fc fragment into a second empty plasmid to obtain a second recombinant plasmid; the second Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 2;

[0043] S3. The first recombinant plasmid and the second recombinant plasmid are mixed, and then transfected into cells for eukaryotic expression. After purification, the fusion protein is obtained.

[0044] In the embodiment of the present invention, the Fc fragment of IgG1 is mutated and designed to obtain the first Fc fragment and the second Fc fragment that can form a knob-and-hole structure. The first Fc fragment and the second Fc fragment are combined with the antigen to form a structure similar to a "Y" shape, thereby changing the inherent structure of the antigen, stabilizing the antigen morphology, reducing antigen degradation, enhancing the stability of the antigen, and increasing the expression level of the antigen in mammals.

[0045] Therefore, the present invention also provides an application of the Fc fragment or the DNA fragment described above in the present invention in preparing a fusion antigen. The present invention also provides a method for increasing the expression of a fusion protein based on the Fc fragment.

[0046] In step S1, in some embodiments, the nucleotide sequence of the DNA fragment encoding the first Fc fragment is shown in SEQ ID NO: 3; the nucleotide sequence of the first empty vector plasmid is shown in SEQ ID NO: 5; and the nucleotide sequence of the first recombinant plasmid is shown in SEQ ID NO: 6.

[0047] In step S1, a linker can also be connected to the C-terminus of the first Fc fragment to obtain protein fragment a (i.e., KiHk fragment hereinafter), and then the sequence encoding protein fragment a is inserted into the first empty plasmid, and then the DNA fragment encoding the antigen is inserted into the first empty plasmid.

[0048] In step S2, in some embodiments, the nucleotide sequence of the DNA fragment encoding the second Fc fragment is shown in SEQ ID NO: 4; the nucleotide sequence of the second empty vector plasmid is shown in SEQ ID NO: 5; and the nucleotide sequence of the second recombinant plasmid is shown in SEQ ID NO: 7.

[0049] In step S3, in some embodiments, the cells are mammalian cells, specifically HEK-293 cells, but not limited thereto.

[0050] In some embodiments, the first recombinant plasmid and the second recombinant plasmid are mixed at a mass ratio of 1:1.

[0051] In some embodiments, the purification uses Ni-Ted column purification.

[0052] The present invention will be further described below by means of specific examples.

[0053] Example 1

[0054] This embodiment provides a method for preparing a fusion antigen, comprising the following steps:

[0055] (1) The Fc fragment of IgG1 was searched through NCBI and it has 227 amino acids in total. Its amino acid sequence is shown in SEQ ID NO: 8.

[0056] (2) Using the amino acid sequence of the Fc fragment of IgG1 in step (1) as a template, S134C, E136D, M138L and T146W site mutations were performed, and then a (G4S)5 fragment was connected to the back of the entire fragment to obtain a KiHk fragment (247aa), whose amino acid sequence is shown in SEQ ID NO: 10. After optimization, the nucleotide sequence of the DNA fragment encoding the KiHk fragment is shown in SEQ ID NO: 11.

[0057] The DNA fragment encoding the KiHk fragment was inserted into the empty plasmid pM06 to construct the pM06-KiHk-cMCS plasmid, wherein the nucleotide sequence of the empty plasmid pM06 is shown in SEQ ID NO: 5, and the nucleotide sequence of the pM06-KiHk-cMCS plasmid is shown in SEQ ID NO: 12.

[0058] According to the pM06-KiHk-cMCS plasmid constructed above, the cMCS region is replaced with the target antigen sequence, that is, the target antigen sequence is the nucleotide sequence encoding apolipoprotein Apo, to obtain the pM06-KiHk-Apo plasmid. The nucleotide sequence of the pM06-KiHk-Apo plasmid is shown in SEQ ID No: 6.

[0059] (3) Using the amino acid sequence of the Fc fragment of IgG1 in step (1) as a template, mutations were made to the E136D, E137K, M138L, T146S, L148A and Y187V sites, and a His tag was added to the C-terminus to obtain an Hd fragment, the amino acid sequence of which is shown in SEQ ID NO: 9 (240aa). After optimization, the nucleotide sequence of the DNA fragment encoding the Hd fragment is shown in SEQ ID NO: 4.

[0060] The DNA fragment encoding the Hd fragment was inserted into the empty vector plasmid pM06 (the nucleotide sequence of which is shown in SEQ ID NO: 5) to construct the pM06-Hd plasmid. The nucleotide sequence of the pM06-Hd plasmid is shown in SEQ ID NO: 7.

[0061] (4) The pM06-KiHk-Apo plasmid in step (2) and the pM06-Hd plasmid in step (3) were mixed at a mass ratio of 1:1 and transfected into HEK293 cells for expression. For specific operations, refer to the instructions for transient transfection of HEK-293 cells.

[0062] (5) Prepare Ni-Ted binding buffer and Ni-Ted elution buffer according to Table 1 and Table 2; then mix Ni-Ted binding buffer and Ni-Ted elution buffer in a volume ratio of 19:1 to prepare Ni-Ted elution buffer.

[0063] Table 1. Preparation of Ni-Ted binding buffer

[0064]

[0065] Table 2. Preparation of Ni-Ted elution buffer

[0066]

[0067] In Table 1 and Table 2, MR represents relative molecular mass; taking the preparation of Ni-Ted elution buffer as an example, a total of 1000 mL of Ni-Ted binding buffer was prepared, wherein the concentration of NaH2PO4·2H2O was 20 mM, the concentration of Na2HPO4·12H2O was 20 mM, the concentration of NaCl was 500 mM, and the concentration of imidazole was 500 mM. The preparation of Ni-Ted binding buffer is similar.

[0068] (6) The expression product was collected according to the process shown in Table 3 and purified using a Ni-Ted column to obtain a fusion antigen (denoted as Apo-1).

[0069] Table 3. Ni-Ted purification process

[0070] step Chromatography solution CIP (Cleaning in Place) 1M NaOH rinse Purified water balance Ni-Ted binding buffer (pH 7.4) Loading Filter the fermentation supernatant Complex balance Ni-Ted binding buffer (pH 7.4) Rinse 1 Ni-Ted washing buffer (pH 7.4) Elution Ni-Ted elution buffer (pH 7.4) Rinse 1 1M NaOH Rinse 2 Purified water Rinse 3 Ni-Ted binding buffer (pH 7.4) save 20% ethanol

[0071] Comparative Example 1

[0072] The nucleotide sequence encoding the antigen Apo with a His tag added to the C-terminus was directly inserted into the pM06 plasmid (the nucleotide sequence of which is described above) to construct the pM06-Apo plasmid, and the nucleotide sequence of the pM06-Apo plasmid is shown in SEQ ID NO: 13. The same conditions as in Comparative Example 1 were used to transfect into HEK-293 cells for expression as a control experiment, and the obtained antigen was recorded as Apo-2.

[0073] test:

[0074] (1) The products of Example 1 and Comparative Example 1 were subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis detection and concentration detection. The SDS-PAGE electrophoresis diagram of the product in Example 1 is as follows: Figure 2 As shown, the meanings of each lane are as follows:

[0075] Lane 1: protein marker (molecular weight standard protein); Lane 2: antigen supernatant; Lane 3: antigen flow-through; Lane 4: elution non-reduction; Lane 5: elution reduction.

[0076] The SDS-PAGE electrophoresis diagram of the product in Comparative Example 1 is as follows: Figure 3 As shown, the meanings of each lane are as follows:

[0077] Lane 1: antigen supernatant; Lane 2: antigen flow-through; Lane 3: elution non-reduced; Lane 4: eluate reduced; Lane 5: secondary purification eluate non-reduced; Lane 6: secondary purification eluate reduced; Lane M: molecular weight standard protein.

[0078] It can be seen that in Example 1, the fusion antigen designed based on the IgG1-Fc fragment has two reduced electrophoresis bands, one of which has a molecular weight of 35kD, which is different from the traditional antigen electrophoresis diagram, and can more quickly and accurately determine whether the obtained product is correct. At the same time, under the same conditions, the yield and stability of the fusion antigen obtained in Example 1 are significantly higher than the antigen yield obtained by the conventional method pM06-Apo plasmid transfection (Comparative Example 1). Among them, the expression level of the fusion antigen obtained in Example 1, i.e., Apo-1, is 25 mg / L, which is 67.33% higher than that in Comparative Example 1, so the present invention can prepare antigens with fewer resources.

[0079] (2) Apo-1 in Example 1 and Apo-2 in Example 2 were subjected to performance verification, including appearance, blank limit, linear range and repeatability.

[0080] a. In terms of appearance, Apo-1 in Example 1 and Apo-2 in Example 2 are both colorless or light yellow clear liquids.

[0081] b. Blank limit verification: Measure the luminescence value of the sample 20 times, and calculate the minimum detection limit concentration of the blank limit through the established standard curve. If it is less than 2.0 mg / dL, it meets the requirements. The results are shown in Table 4 and Table 5.

[0082] Table 4. Apo-1 blank limit detection data

[0083]

[0084] Table 5. Apo-2 blank limit detection data

[0085]

[0086] Among them, when the concentration of the reference standard is 0 mg / dL, the corresponding average light intensity value is 11.74, and when the concentration of the reference standard is 11.71 mg / dL, the corresponding average light intensity value is 19623.

[0087] According to the above results, the minimum detection limit concentration of Apo-1 is 0.04, and the minimum detection limit concentration of Apo-2 is 0.1, both of which meet the verification requirements.

[0088] c. Linear range: The sample detection concentration is between 2.0 and 20.0 mg / dL. The concentration gradient is set to measure the light intensity values ​​of different concentration gradients (three samples for each concentration gradient, i.e., three repeated measurements). The curve is fitted according to the measured values. The linear correlation coefficient |R|≥0.990 meets the requirements; at the same time, within the absolute deviation interval measured in the range of [2.0, 6.0] mg / dL, the absolute linear deviation does not exceed ±0.6 mg / dL; within the range of [6.0, 20.0] mg / dL, the relative deviation of the measurement is within the range of ±10%. The results are shown in Tables 6 and 7.

[0089] Table 6. Apo-1 linear range detection data

[0090]

[0091] Table 7. Apo-2 linear range detection data

[0092]

[0093] From the above results, it can be seen that both Apo-1 and Apo-2 meet the linear range verification requirements.

[0094] d. Repeatability: Test the light intensity values ​​corresponding to low concentration and high concentration of the sample, repeat five groups of experiments, and calculate the repeatability coefficient of variation (CV) ≤ 6%, which meets the requirements. The results are shown in Tables 8 and 9.

[0095] Table 8. Apo-1 repeatability experimental data

[0096]

[0097] Table 9. Apo-2 repeatability experimental data

[0098]

[0099] After analysis, the repeatability verification of Apo-1 and Apo-2 were both qualified.

[0100] In summary, the performance of Apo-1 and Apo-2 both meet the requirements.

[0101] (3) The antigens obtained in Example 1 and Comparative Example 1 were tested for high temperature and shelf life stability

[0102] a. High temperature stability test: Place the sample at 37°C for 7 days and measure the concentration change. 0.05,n-2 S(b1) calculates the statistics of concentration change. The results are shown in Tables 10 and 11.

[0103] Table 10. Apo-1 high temperature stability test results

[0104]

[0105]

[0106] Table 11. Apo-2 high temperature stability test results

[0107]

[0108] According to the above analysis, the regression coefficient of Apo-1 |b1|<t 0.05,n-2 ·S(b1), indicating that the change trend of the assignment results during the stable period is not significant, and the high temperature stability is good; while the regression coefficient |b1| of Apo-2 is much larger than t 0.05,n-2 ·S(b1), indicating that the change trend of the assignment results during the stable period is significant, there is degradation phenomenon, and the high temperature stability is poor. 0.05,n-2 represents the statistic calculated using t distribution and n-2 degrees of freedom at a significance level of 0.05, where n represents the degrees of freedom and S(b1) represents the sample standard deviation.

[0109] b. Shelf life stability test: Take two bottles of samples, under the condition of 2-8°C, with a shelf life of 12 months, set a time gradient to conduct concentration detection regularly, and take 3 samples from each sample bottle for concentration detection in each time gradient. Perform regression analysis based on the obtained data. The specific test data of Apo-1 and Apo-2 are shown in Tables 12 and 13 below.

[0110] Table 12. Apo-1 shelf life stability test

[0111]

[0112]

[0113] Table 13. Apo-2 shelf life stability test results

[0114]

[0115] Among them, t 0.05,n-2 , n and S(b1) have the same meanings as above. From the above results, it can be seen that Apo-1 has good shelf life stability and is suitable for long-term storage; while the concentration of Apo-2 has decreased to 34.13% of the initial concentration after 12 months, and it is turbid and has poor shelf life stability. Therefore, while ensuring performance, Apo-1 in Example 1 has better stability.

[0116] In summary, while ensuring performance, the antigen obtained by the present invention has better stability.

[0117] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Fc fragment, characterized in that The Fc fragment includes a first Fc fragment and a second Fc fragment capable of forming a knob-and-hole structure with the first Fc fragment, the first Fc fragment includes the amino acid sequence shown in SEQ ID NO: 1, and the second Fc fragment includes the amino acid sequence shown in SEQ ID NO:

2.

2. The Fc fragment according to claim 1, characterized in that The first Fc fragment further includes a protein purification tag sequence connected to one end of the amino acid sequence shown in SEQ ID NO:

1.

3. The Fc fragment according to claim 1, characterized in that The second Fc fragment further includes a protein purification tag sequence connected to one end of the amino acid sequence shown in SEQ ID NO:

2.

4. A fusion antigen, characterized in that The fusion antigen comprises an antigen and the Fc fragment according to any one of claims 1 to 3 connected to the antigen via a linker.

5. A recombinant vector, characterized in that: The recombinant vector contains a nucleotide sequence encoding the fusion antigen according to claim 4.

6. A recombinant cell, characterized in that The recombinant cell contains the recombinant vector according to claim 5.

7. A method for preparing a fusion antigen, characterized in that: The steps include: Inserting DNA fragments encoding a first Fc fragment, a linker and an antigen respectively into a first empty plasmid to obtain a first recombinant plasmid; the first Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 1; Inserting a DNA fragment encoding a second Fc fragment into a second empty plasmid to obtain a second recombinant plasmid; the second Fc fragment comprises an amino acid sequence as shown in SEQ ID NO: 2; The first recombinant plasmid and the second recombinant plasmid are mixed and then transfected into cells for eukaryotic expression. After purification, the fusion protein is obtained.

8. The preparation method according to claim 7, characterized in that: The nucleotide sequence of the DNA fragment encoding the first Fc fragment is shown in SEQ ID NO: 3; The nucleotide sequence of the DNA fragment encoding the second Fc fragment is shown in SEQ ID NO:

4.

9. The preparation method according to claim 7, characterized in that: The nucleotide sequences of the first empty vector plasmid and the second empty vector plasmid are both shown in SEQ ID NO: 5; The nucleotide sequence of the first recombinant plasmid is shown in SEQ ID NO:6; the nucleotide sequence of the second recombinant plasmid is shown in SEQ ID NO:

7.

10. The preparation method according to claim 7, characterized in that: The cells are mammalian cells.

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