Recombinant PIC protein, preparation method and application

Abstract

The invention discloses a recombinant PIC protein as well as a preparation method and application thereof. The preparation method comprises the following steps: obtaining a gene segment of the recombinant PIC protein; connecting the gene segment to a starting vector to form a recombinant vector; and transfecting the recombinant vector to a cell host for expression to obtain the recombinant PIC protein. The PIC is recombinant truncated protein and is obtained through heterologous expression of mammalian cells (H293F), the molecular weight of natural PIC is about 140-150 kDa, the molecular weight of the recombinant truncated PIC protein is about 100 kDa, the pressure of heterologous expression of the mammalian cells (H293F) is relieved due to reduction of the molecular weight, and the PIC expression quantity is increased; the recombinant high-purity truncated PIC antigen and antibody prepared by the invention have strong immunoreactivity, and completely meet the requirements of thrombus detection on the PIC antigen.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical detection, in particular to a recombinant PIC protein, a preparation method and application thereof. BACKGROUND

[0002] Plasminogen (Plg) is a precursor of plasmin (P), which is a serine protease essential for fibrinolysis. Native Plg contains 791 residues (Glu1-Asn791) and is a single-chain protein with a molecular weight of about 85 kDa. Plg contains seven domains, including an N-terminal peptide domain and a C-terminal protease domain, which can be activated by various activators, including five Kringle domains (K1-K5) composed of Lys78-Arg561. Plg is cleaved between Arg561-Val562 to form plasmin, which consists of two peptide chains, with the A chain as the binding center and the B chain as the enzyme active center.

[0003] Alpha2-plasmin inhibitor (α2-PI) is a single-chain glycoprotein with a molecular weight of 60 to 70 kDa. Mature α2-PI loses the amino-terminal 12 residues during blood circulation and is converted to the Asn form at the NH2 terminus, with the Asn form accounting for 60-70% of the total α2-PI in plasma. The reaction site of α2-PI with the active center of plasmin is composed of Arg376-Met377, and α2-PI has strong affinity for Plg and non-covalently binds to the lysine binding site of Plg.

[0004] Thrombosis is a disease with occult onset, sudden onset, and high mortality and disability rate, which can be seen in various clinical departments. Imbalance between coagulation and fibrinolysis systems will directly lead to the occurrence of thrombotic diseases. Plasmin-α2-plasmin inhibitor complex (PIC) is formed by the combination of α2-plasmin inhibitor (α2-PI) and plasmin (P) in a 1:1 ratio when the body's fibrinolytic system is activated. Plasmin is a direct evidence of fibrinolytic system activation, and due to its short half-life and non-specific antigenicity, it is difficult to measure. Therefore, the complex PIC formed by plasmin and its inhibitor can be a good marker for plasmin generation and inhibitor consumption. PIC has a molecular weight of about 150 kDa, and the production of this complex indicates the formation of Plg and changes in the fibrinolytic system in the human body, which is an important marker for thrombosis.

[0005] PIC formation mechanism: first, the C-terminal of alpha2-PI is combined with the lysine binding site of plasmin by non-covalent binding, and the site exists in the K1 loop domain of A chain. The crystal structure of natural Plg shows that in Glu-Plg (closed conformation), the initial binding of alpha2-PI is actually mainly the independent action of the lysine binding site, and in Lys-Plg (open conformation), K2 of the lysine binding site is mainly involved in the binding. Earlier studies have shown that K4 of the lysine binding site is important in the interaction of plasmin and alpha2-PI, but K1, 2, 3 and 5 are also involved. The Arg376 of alpha2-PI in the reactive center loop acts on the Ser741 active site of the B chain of plasmin by covalent bond, which leads to the formation of PIC complex, accompanied by complete loss of plasmin activity and cleavage of the scissile peptide bond of alpha2-PI. In the formation of PIC, P is connected to alpha2-PI by acyl covalent bond, so its structure is very stable.

[0006] Currently, there are mainly two methods for preparing PIC at home and abroad: the first method is to directly add urokinase to fresh plasma to activate PIC, and then purify PIC by affinity chromatography step by step; the second method is to first purify alpha2-fibrinolysis inhibitor from plasma, then purify plasminogen, and then form plasmin by the action of urokinase, and then react to form PIC, and then obtain PIC by affinity chromatography. However, both of these two methods are obtained from natural plasma, and the extraction process is complicated, the cost is high, and it is not suitable for large-scale production. SUMMARY

[0007] The application discloses a recombinant PIC protein, a preparation method and application, to solve the problems of complicated extraction process, high cost and unsuitable for large-scale production in the prior art.

[0008] In order to solve the above technical problems, the application adopts the following technical solutions:

[0009] A recombinant PIC protein, wherein the amino acid sequence of the recombinant PIC protein is shown as SEQ ID NO: 1.

[0010] A preparation method of a recombinant PIC protein, comprising the following steps:

[0011] obtaining a gene fragment of the recombinant PIC protein;

[0012] connecting the gene fragment to a starting vector to form a recombinant vector;

[0013] transfecting the recombinant vector into a cell host for expression to obtain the recombinant PIC protein.

[0014] Further, obtaining a gene fragment of the recombinant PIC protein comprises:

[0015] According to the 40-491 amino acid sequence of alpha2-PI and the K1-K5 amino acid sequence of Plg, the corresponding alpha2-pi nucleic acid sequence and Plg-K1-K5 nucleic acid sequence are synthesized, respectively.

[0016] The alpha2-pi nucleic acid sequence and Plg-K1-K5 nucleic acid sequence are connected through a flexible linker to obtain a gene fragment of the recombinant PIC protein.

[0017] Further, the K1-K5 amino acid sequence of Plg includes the Kringle1 domain of the 103-181th amino acid sequence of Plg, the Kringle2 domain of the 184-262th amino acid sequence of Plg, the Kringle3 domain of the 275-352th amino acid sequence of Plg, the Kringle4 domain of the 377-454th amino acid sequence of Plg, and the Kringle5 domain of the 481-560th amino acid sequence of Plg.

[0018] Further, the Plg amino acid sequence is shown in SEQ ID No: 3.

[0019] Further, the K1-K5 amino acid sequence of Plg is shown in SEQ ID No: 4.

[0020] Further, the amino acid sequence of alpha2-PI is shown in SEQ ID No: 5.

[0021] Further, the nucleic acid sequence of the gene fragment is shown in SEQ ID NO: 2.

[0022] Further, the starting vector is pcDNA3.4G, and the cell host is Expi293F.

[0023] The recombinant PIC protein is applied in a thrombus detection kit.

[0024] The present application has the following advantages by adopting the above technical solutions.

[0025] The PIC of the present application is a recombinant truncated protein, which is obtained by heterologous expression of mammalian cells (Expi293F). The natural PIC has a molecular weight of about 140-150kDa, while the recombinant truncated PIC protein of the present application has a molecular weight of about 100kDa. The reduction of molecular weight reduces the pressure of heterologous expression of mammalian cells (Expi293F), which is beneficial to improve the expression amount of PIC. The recombinant high-purity truncated PIC antigen and antibody prepared by the present application have strong immunoreactivity, which fully meets the requirements of thrombus detection for PIC antigen. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure is a construction map of the pcDNA3.4G-His-PIC recombinant vector.

[0027] Figure 2 The electrophoresis comparison chart before and after the purification of the recombinant PIC protein, wherein M represents the band value, 1 is the electrophoresis chart of the recombinant PIC protein before purification, 2 is the electrophoresis chart of the recombinant PIC protein obtained by flow-through, and 3 is the electrophoresis chart of the recombinant PIC protein after nickel column purification;

[0028] Figure 3 The electrophoresis chart of the recombinant PIC protein after purification. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0030] The main test materials and reagents involved in the present application are as follows: 1, cells and vectors: the expression host is Expi293F, and the expression plasmid vector is pcDNA3.4G; 2, enzymes and other biochemical reagents: endonuclease / high-fidelity DNA polymerase and recombinase; 3, Expi293F cell transfection reagent: PEI-MAX (2 mg / mL, polyscience); 4, Expi293F cell culture medium: Opti-MEM medium (Gibco), OPM-293CD05 Medium (Aopmai), OPM-293 Profeed (F081918).

[0031] The present application discloses a recombinant PIC protein, the amino acid sequence of which is shown as SEQ ID NO: 1, which can be applied to the preparation of a thrombus detection kit.

[0032] The nucleic acid sequence of the gene fragment for preparing the above-mentioned recombinant PIC protein is shown as SEQ ID NO: 2.

[0033] The preparation method of the recombinant PIC protein of the present application is as follows:

[0034] The gene fragment of the recombinant PIC protein is obtained;

[0035] The gene fragment is connected to the starting vector to form a recombinant vector;

[0036] The recombinant vector is transfected into a cell host for expression to obtain the recombinant PIC protein.

[0037] Further, the gene fragment of the recombinant PIC protein is obtained, which comprises:

[0038] According to the amino acid sequence 40-491 of α2-PI and the amino acid sequence K1-K5 of Plg, the corresponding α2-pi nucleic acid sequence and Plg-K1-K5 nucleic acid sequence were synthesized respectively;

[0039] The α2-pi nucleic acid sequence and the Plg-K1-K5 nucleic acid sequence are connected through a flexible linker to obtain a gene fragment of a recombinant PIC protein.

[0040] Among them, the K1-K5 amino acid sequence of Plg includes the Kringle1 domain at positions 103-181, the Kringle2 domain at positions 184-262, the Kringle3 domain at positions 275-352, the Kringle4 domain at positions 377-454, and the Kringle5 domain at positions 481-560 of the Plg amino acid sequence.

[0041] The amino acid sequence of Plg is shown in SEQ ID No: 3, and its domain structure is as follows:

[0042] The amino acid sequence 1-19 is the signal peptide sequence, the amino acid sequence 103-181 is the Kringle1 domain, the amino acid sequence 184-262 is the Kringle2 domain, the amino acid sequence 275-352 is the Kringle3 domain, the amino acid sequence 377-454 is the Kringle4 domain, the amino acid sequence 481-560 is the Kringle5 domain, the amino acid sequence 20-580 is the heavy chain, and the amino acid sequence 581-810 is the light chain, that is, the plasmin domain.

[0043] Furthermore, the amino acid sequence of K1-K5 of Plg is shown in SEQ ID No:4.

[0044] Furthermore, the amino acid sequence of α2-PI is shown in SEQ ID No: 5, and its domain structure is as follows:

[0045] The amino acid sequence from 1 to 27 is a signal peptide sequence, the amino acid sequence from 28 to 39 is a leader peptide sequence, and the amino acid sequence from 40 to 491 is an α2-PI domain.

[0046] Furthermore, the starting vector is pcDNA3.4G, the recombinant vector is pcDNA3.4G-His-PIC; and the cell host is Expi293.

[0047] The preparation method of the present invention is characterized in that the K1-K5 amino acid sequence of Plg and the 40-491 amino acid sequence of α2-PI can be efficiently expressed in the Expi293F expression system after being fused, and a highly immunogenic and high-purity recombinant PIC protein can be obtained, which can be used in the preparation of a thrombosis detection kit.

[0048] The method for preparing the PIC at home and abroad at present is mainly obtained from natural plasma, and the PIC prepared in the application is a recombinant truncated protein, which is obtained by heterologous expression of mammalian cells (Expi293F). The molecular weight of the natural PIC is about 140-150 kDa, and the molecular weight of the recombinant truncated PIC protein in the application is about 100 kDa. The reduction of the molecular weight reduces the pressure of heterologous expression of mammalian cells (Expi293F), which is beneficial to improve the expression amount of the PIC. The recombinant high-purity truncated PIC antigen and antibody prepared in the application have strong immunoreactivity, which fully meets the requirements of thrombus detection for PIC antigen.

[0049] In order to further explain the application, the preparation process and application of the recombinant PIC protein are described in detail through Example 1 and Example 2 shown as follows, respectively.

[0050] Example 1

[0051] (1) Construction of recombinant vector pcDNA3.4G-His-PIC

[0052] 1) Amplifying the nucleic acid sequence of 40-491 amino acid sequence of alpha2-PI

[0053] The alpha2-pi gene fragment is amplified by the method of PCR, and a nucleic acid fragment of part of flexible linker (GGGGS)*3 is introduced as a homologous region with Plg-K1-K5.

[0054] The primers used in PCR are as follows: alpha2-PI-F (SEQ ID No: 6, 51 bp); alpha2-PI-R (SEQ ID No: 7, 39 bp), wherein alpha2-PI-F and alpha2-PI-R are used to amplify the gene coding sequence of alpha2-PI.

[0055] 2) Amplifying the nucleic acid sequence of K1-K5 amino acid sequence of Plg

[0056] The Plg-K1-K5 gene fragment is amplified by the method of PCR, and a nucleic acid fragment of part of flexible linker (GGGGS)*3 is introduced as a homologous region with alpha2-PI.

[0057] The primers used in PCR are as follows: Plg-K1-K5-F (SEQ ID No: 8, 48 bp); Plg-K1-K5-R (SEQ ID No: 9, 61 bp): wherein Plg-K1-K5-F and Plg-K1-K5-R are used to amplify the gene coding sequence of Plg-K1-K5.

[0058] 3) Amplifying the nucleic acid sequence of truncated PIC amino acid sequence pic

[0059] The gene fragment of recombinant PIC protein was obtained by connecting the nucleic acid sequences of α2-pi and Plg-K1-K5 using the method of Overlap PCR.

[0060] The primers used in PCR were as follows: α2-PI-F (SEQ ID No: 6, 51 bp); Plg-K1-K5-R (SEQ ID No: 9, 61 bp); wherein, α2-PI-F and Plg-K1-K5-R were used to amplify the gene coding sequence of truncated PIC.

[0061] 4) Construction of recombinant vector pcDNA3.4G-His-PIC

[0062] The nucleic acid fragment of vector pcDNA3.4G was obtained by double enzyme digestion method, and the gene fragment of PIC and the starting vector were connected by T4 DNA ligase to obtain the recombinant vector pcDNA3.4G-His-PIC as shown in Figure 1 , and the recombinant vector pcDNA3.4G-His-PIC was transformed into E. coli DH5α.

[0063] (2) Expression and purification of high-purity recombinant PIC protein

[0064] 1. After the recombinant vector pcDNA3.4G-His-PIC was transformed into E. coli DH5α, the plasmid containing the recombinant vector pcDNA3.4G-His-PIC was extracted from E. coli DH5α.

[0065] 2. Cell transfection and culture

[0066] 1) Day-1 seed cell (Expi293F) culture: p18 generation Expi293F cells were inoculated at a density of 2.0-3*10^6 cells / mL with OPM-293CD05 Medium expression medium, and continued to be cultured at 37°C in a carbon dioxide shaking incubator for 24h.

[0067] 2) Day-0 cell culture: the seed cells were diluted to 3.0*10^6 cells / mL;

[0068] Day-0 transfection: prepare the transfection complex according to the following ratio for transfection; filter the plasmid using a 0.22μm sterile filter and a disposable syringe;

[0069] A: 10mL Opti-MEM + 100μg plasmid, mix well;

[0070] B: 10mL Opti-MEM + 1000μg transfection reagent, mix well;

[0071] Add A liquid into B liquid drop by drop, mix well by blowing, incubate at room temperature for 15 min, and add the transfection system while shaking the cell bottle containing the seed cells (Expi293F).

[0072] 3) Feed addition: add feed medium OPM-293Profeed 20 mL at 20 h after transfection;

[0073] 4) Cell culture and expression: continue to culture until the 6th day after transfection.

[0074] 3, Isolation and purification of recombinant PIC protein

[0075] After fermentation expression, centrifuge the cell liquid at 6000 rpm for 5 min, collect the supernatant, concentrate and change the liquid to 20 mM PB, 0.15 M NaCl, 20 mM imidazole, pH 7.4 using a 10 kDa membrane bag. Then purify the supernatant of the concentrated and changed liquid of the fermentation liquid using a nickel column, and the purification uses A liquid 20 mM PB + 0.15 M NaCl, 20 mM imidazole, pH 7.4, and B liquid 20 mM PB + 0.15 M NaCl + 500 mM imidazole, pH 7.4. The purified protein is collected and eluted, which is the target protein as shown in Figure 2 The eluted target protein is changed to a dialysis buffer: 20 mM PB, 0.15 M NaCl, pH 7.4, and finally a high-purity recombinant truncated PIC protein is obtained, as shown in Figure 3 The molecular weight of the recombinant truncated PIC is 100 KDa, the purity is higher than 80%, and the protein expression amount is about 50 mg / L.

[0076] Example 3

[0077] High-purity recombinant PIC protein immunogenicity detection: The double-antibody sandwich method is used for immunogenicity detection and antigen titer analysis of the recombinant truncated PIC. First, the PIC antigen is gradiently diluted, and then the luminescence value when the PIC antigen and the corresponding antibody are combined at each dilution is detected. The detection result is shown in Table 1. The luminescence value of the recombinant truncated PIC antigen diluted by 10,000 times is about 270,000, the luminescence value of the recombinant truncated PIC antigen diluted by 100,000 times is about 25,000, and the luminescence value of the recombinant truncated PIC antigen diluted by 1,000,000 times is about 3,000. It shows that the recombinant truncated PIC antigen and the two antibodies have strong affinity, which completely meets the application in thrombus detection.

[0078]

[0079] The recombinant truncated PIC protein obtained by the application has many advantages compared with the PIC protein obtained from natural plasma:

[0080] 1) The production process of recombinant proteins can be carried out in a strictly controlled laboratory environment, avoiding possible contamination by pathogens. In contrast, natural proteins can be contaminated by viruses, bacteria or other pathogens; 2) The production of recombinant proteins can be precisely controlled to ensure consistency in each batch of product. In contrast, the purity and consistency of natural proteins can vary due to the variability of biological sources; 3) The production of recombinant proteins does not depend on specific biological sources, thus having an advantage in terms of resource sustainability; 4) Although the initial cost of research and development and production of recombinant proteins can be high, once the production process is established, the cost of production will be lower than that of natural proteins; 5) Using cell culture technology, recombinant proteins can be produced on a large scale, while the availability of natural proteins is usually limited by the availability of biological sources.

[0081] The Expi293F expression system used in the present application is a special human embryonic kidney 293 (HEK293) cell line that has been optimized to improve its transfection efficiency and protein expression level; Expi293F expression system can be used to produce various types of proteins, including single-chain and double-chain antibodies, membrane proteins, enzymes and vaccines, etc. Expi293F expression system can easily be scaled from small-scale laboratory scale to large-scale production scale, making it an ideal choice for industrial-grade protein production. Expi293F expression system is compatible with various protein purification and analysis techniques, such as affinity chromatography, gel electrophoresis and immunoblotting, etc. In general, Expi293F expression system is a powerful protein expression tool that combines efficient transfection, optimized cell proliferation and enhanced protein expression, providing an efficient and scalable solution for protein production.

[0082] The above is only an embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application is included in the scope of the claims of the present application.

Claims

1. A recombinant PIC protein, characterized in that, The amino acid sequence of the recombinant PIC protein is shown as SEQ ID NO:

1.

2. A method of producing a recombinant PIC protein, characterized by, The method comprises the following steps: obtaining a gene fragment of the recombinant PIC protein; connecting the gene fragment to a starting vector to form a recombinant vector; transfecting the recombinant vector into a cell host for expression to obtain the recombinant PIC protein.

3. The method for preparing a recombinant PIC protein according to claim 2, characterized in that: The method for obtaining the gene fragment of the recombinant PIC protein comprises: According to the 40-491 amino acid sequence of α2-PI and the K1-K5 amino acid sequence of Plg, their corresponding α2-pi nucleic acid sequences and Plg-K1-K5 nucleic acid sequences were synthesized respectively. by a flexible linker α2-pi The nucleic acid sequence and the Plg-K1-K5 nucleic acid sequence are connected to obtain a gene fragment of the recombinant PIC protein.

4. The method of claim 3, wherein the recombinant PIC protein is prepared by the steps of: The K1-K5 amino acid sequence of the Plg comprises a Kringle1 domain at positions 103-181, a Kringle2 domain at positions 184-262, a Kringle3 domain at positions 275-352, a Kringle4 domain at positions 377-454, and a Kringle5 domain at positions 481-560 of the Plg amino acid sequence.

5. The method for preparing a recombinant PIC protein according to claim 4, characterized in that: The Plg amino acid sequence is shown as SEQ ID No:

3.

6. The method of claim 3, wherein the recombinant PIC protein is prepared by the steps of: The K1-K5 amino acid sequence of the Plg is shown as SEQ ID No:

4.

7. The method for preparing a recombinant PIC protein according to claim 3, characterized in that: The amino acid sequence of the α2-PI is shown as SEQ ID No:

5.

8. The method for preparing a recombinant PIC protein according to claim 2, characterized in that: The nucleic acid sequence of the gene fragment is shown as SEQ ID NO:

2.

9. The method for preparing a recombinant PIC protein according to claim 2, characterized in that: The starting vector is pcDNA3.4G, and the cell host is Expi293F.

10. The recombinant PIC protein of claim 1 is used in a thrombus detection kit.

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