Preparation method and application of PSA-ACT compound recombinant protein

Abstract

The invention discloses a preparation method and application of a PSA-ACT compound recombinant protein. The preparation method comprises the following steps: obtaining gene segments of recombinant PSA and ACT proteins; respectively connecting the gene segments to starting vectors to form corresponding connectors; carrying out conversion and screening operation on the linker to obtain a corresponding recombinant plasmid; carrying out transformation, transfection, induction and fermentation broth separation and purification operation on the recombinant plasmid to obtain recombinant PSA and ACT proteins; then activating PSA recombinant protein by using metalloproteinase, and then mixing ACT with the activated PSA to obtain a mixture containing the PSA, ACT and a PSA-ACT compound; and finally, purifying by using an ion exchange chromatography technology and a hydrophobic chromatography technology in sequence to obtain the high-purity and high-activity PSA-ACT compound. According to the method, the spontaneous reaction of ACT and activated PSA is utilized, other chemical cross-linking agents are not needed, the cost can be reduced, and the recombinant protein can be produced on a large scale; the PSA-ACT recombinant protein prepared by the method disclosed by the invention has very strong immunoreactivity.

Description

Technical Field

[0001] This invention relates to the field of medical testing, specifically to a method for preparing and applying a recombinant PSA-ACT complex protein. Background Technology

[0002] Prostate cancer (PCa) is a malignant tumor that seriously threatens the health of middle-aged and elderly men, and its incidence varies significantly by race and region. Although the incidence of prostate cancer in my country is lower than in Europe and the United States, it has been on the rise in recent years, and its growth is even more rapid. Early diagnosis and early treatment of prostate cancer are of positive significance for prognosis. Currently, the main clinical methods for diagnosing prostate cancer include digital rectal examination, prostate-specific antigen (PSA) testing, and imaging examinations. Among these, PSA testing is widely used in clinical practice as a screening method for prostate cancer. The percentage of free prostate-specific antigen (f / tPSA), PSA density (PSAD), and PSA rate (PSAV) are parameters proposed in clinical practice to improve the ability of PSA to diagnose prostate cancer.

[0003] PSA is a single-chain glycoprotein with a molecular weight of approximately 28 kDa and containing 261 amino acid residues. It is secreted by prostate epithelial cells and possesses serine protease activity. Under normal circumstances, almost all PSA is secreted into the semen. However, in cases of prostate cancer (PCa), PSA leaks into the bloodstream. Therefore, serum PSA serves as a blood biomarker and is valuable for the diagnosis and early treatment of PCa. A small amount of PSA diffuses into the serum in various molecular forms. 70%–90% of PSA covalently binds to the endogenous protease inhibitor α1-Antichymotrypsin (ACT) to form the PSA-ACT complex. PSA also binds to another protease inhibitor similar to ACT, α2-macroglobulin (α2-M), but this binding is undetectable by current immunoassays because the antigenic determinant of PSA is blocked by α2-M. In addition, a very small amount of PSA binds to some members of the serine protease inhibitor family, but these forms are not clinically significant. The remaining 10% to 30% of serum PSA exists in an unbound form, called free PSA (fPSA).

[0004] α1-Antichymotrypsin (α1-ACT) is a major protease inhibitor and a member of the serine protease inhibitor family. The α1-ACT protein contains 423 amino acids, has multiple glycosylation active sites, and a molecular weight of 55-66 kDa. The N-terminus of the α1-ACT protein is a signal peptide and a SERPIN domain containing a serine protease inhibitor active group. The C-terminus contains a reactive center loop (RCL). The RCL extends outward from the protein body and directs binding to the target protease. The protease cleaves the serum protease inhibitor at the reactive site within the RCL, establishing a covalent link between the carboxyl group of the serum protease inhibitor's reactive site and the serine hydroxyl group of the protease. The resulting inactive serum protease inhibitor-protease complex is very stable.

[0005] Currently, there is relatively little research on the preparation of PSA-ACT. The mainstream preparation method for PSA-ACT products on the market is as follows: first, ACT monomers are obtained from fresh human plasma using multiple chromatography techniques; then, PSA monomers are obtained from fresh human semen using multiple chromatography techniques; finally, the two are reacted in vitro to synthesize the PSA-ACT complex. However, this method requires fresh human plasma and fresh human semen, which are limited in source and easily contaminated by pathogens. The protein extraction process is also cumbersome and costly, making it unsuitable for large-scale production. Summary of the Invention

[0006] This invention discloses a method for preparing recombinant PSA-ACT complex protein and its application, in order to solve the problems of cumbersome extraction process, high cost, easy influence of PSA monomers on detection results, and unsuitability for large-scale production in the prior art.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A first aspect of the present invention provides a method for preparing a recombinant PSA-ACT complex protein, comprising the following steps:

[0009] Obtain the PSA gene fragment and the ACT gene fragment respectively;

[0010] Gene fragments are ligated into the starting vector to form corresponding linkers;

[0011] The linkers are transformed and screened to obtain the corresponding recombinant plasmids;

[0012] The recombinant plasmids were transformed / transfected and induced to express to obtain ACT fermentation broth and PSA fermentation broth;

[0013] The fermentation broth was subjected to separation and affinity chromatography purification to obtain PSA recombinant protein and ACT recombinant protein;

[0014] The PSA recombinant protein was activated by metalloproteinases to obtain the active PSA recombinant protein;

[0015] The active PSA recombinant protein and ACT recombinant protein were mixed and reacted to obtain the crude PSA-ACT complex.

[0016] The crude PSA-ACT complex was separated and purified by ion exchange chromatography and hydrophobic chromatography to obtain high-purity and high-activity recombinant PSA-ACT complex protein.

[0017] Furthermore, the method for obtaining the PSA gene fragment is as follows:

[0018] The PSA amino acid sequence was retrieved from the UniProt database. The first 17 amino acids were the signal peptide. The PSA amino acid sequence was obtained by removing the first 17 amino acids.

[0019] The PSA gene fragment was synthesized based on the PSA amino acid sequence.

[0020] Furthermore, the PSA amino acid sequence is shown in SEQ ID NO:1; the nucleotide sequence of the PSA gene fragment is shown in SEQ ID NO:2.

[0021] Furthermore, the method for obtaining the ACT gene fragment is as follows:

[0022] The ACT amino acid sequence was retrieved from the UniProt database. The first 23 amino acid sequences were the signal peptide. The first 23 amino acids were removed to obtain the ACT amino acid sequence.

[0023] The ACT gene fragment was synthesized based on the ACT amino acid sequence.

[0024] Furthermore, the amino acid sequence of the ACT is shown in SEQ ID NO:3; the nucleotide sequence of the ACT gene fragment is shown in SEQ ID NO:4.

[0025] Furthermore, the starting vector for the PSA gene fragment is pcDNA 3.4G, and the starting vector for the ACT gene fragment is pET34.

[0026] Furthermore, the recombinant plasmids are pcDNA3.4G-PSA and pET34-ACT, respectively.

[0027] Further, the recombinant plasmid is transformed / transfected and expression is induced, including:

[0028] The pET34-ACT recombinant plasmid was transformed into BL21(DE3) for induced expression;

[0029] The pcDNA3.4G-PSA recombinant plasmid was transfected into Expi293F to induce expression.

[0030] A second aspect of the present invention relates to the use of the recombinant PSA-ACT complex protein prepared by any implementation of the first aspect in a prostate cancer diagnostic kit.

[0031] The present invention adopts the above technical solution and has the following advantages:

[0032] This invention utilizes the spontaneous reaction between ACT and activated PSA without the need for other chemical cross-linking agents, thus reducing costs and promoting green production. Cell culture technology enables large-scale production of recombinant proteins, avoiding the limitations of natural plasma and semen extraction. Multiple chromatographic techniques are used to purify the protein, significantly improving its purity and completely eliminating the influence of PSA monomers on detection results. The recombinant PSA-ACT antigen prepared by this invention exhibits strong immunoreactivity, fully meeting the requirements of prostate cancer diagnostic reagents for PSA-ACT antigens. Attached Figure Description

[0033] Figure 1 This is an electrophoresis image of recombinant ACT protein purified by Ni-NTA affinity chromatography, where M represents the band value;

[0034] Figure 2 This is an electrophoresis image of recombinant PSA protein purified by Ni-NTA affinity chromatography, where M represents the band value;

[0035] Figure 3 Electrophoresis images of the recombinant PSA-ACT complex after purification using ion exchange chromatography and hydrophobic chromatography.

[0036] Figure 4 For the recombinant PSA-ACT complex correlation. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This invention discloses a method for preparing recombinant PSA-ACT complex protein and its application.

[0039] The method for preparing the PSA-ACT complex recombinant protein involved in this invention includes the following steps:

[0040] Obtain the PSA gene fragment and the ACT gene fragment respectively;

[0041] Gene fragments are ligated into the starting vector to form corresponding linkers;

[0042] The linkers are transformed and screened to obtain the corresponding recombinant plasmids;

[0043] The recombinant plasmids were transformed / transfected and induced to express to obtain ACT fermentation broth and PSA fermentation broth;

[0044] The fermentation broth was subjected to separation and affinity chromatography purification to obtain PSA recombinant protein and ACT recombinant protein;

[0045] The PSA recombinant protein was activated by metalloproteinases to obtain the active PSA recombinant protein;

[0046] The active PSA recombinant protein and ACT recombinant protein were mixed and reacted to obtain the crude PSA-ACT complex.

[0047] The crude PSA-ACT complex was separated and purified by ion exchange chromatography and hydrophobic chromatography to obtain high-purity and high-activity recombinant PSA-ACT complex protein.

[0048] Furthermore, the method for obtaining the PSA gene fragment is as follows:

[0049] The PSA amino acid sequence was obtained by searching the UniProt database for P07288. The first 17 amino acids were the signal peptide. After removing the first 17 amino acids, the PSA amino acid sequence was obtained.

[0050] The PSA gene fragment was synthesized by codon optimization based on the PSA amino acid sequence.

[0051] Furthermore, the amino acid sequence of the PSA is shown in SEQ ID NO:1; the nucleotide sequence of the PSA gene fragment is shown in SEQ ID NO:2.

[0052] Furthermore, the method for obtaining the ACT gene fragment is as follows:

[0053] The ACT amino acid sequence was obtained by searching P01011 in the UniProt database. The first 23 amino acids were the signal peptide. After removing the first 23 amino acids, the ACT amino acid sequence was obtained.

[0054] The ACT gene fragment was obtained by codon optimization based on the ACT amino acid sequence.

[0055] Furthermore, the amino acid sequence of the ACT is shown in SEQ ID NO:3; the nucleotide sequence of the ACT gene fragment is shown in SEQ ID NO:4.

[0056] Furthermore, the starting vector for the PSA gene fragment is pcDNA 3.4G, and the starting vector for the ACT gene fragment is pET34.

[0057] Furthermore, the recombinant plasmids are pcDNA3.4G-PSA and pET34-ACT, respectively.

[0058] Further steps include transformation / transfection and induced expression of the recombinant plasmid, including:

[0059] The pcDNA3.4G-PSA recombinant plasmid was transfected into Expi293F cells, and the pET34-ACT recombinant plasmid was transformed into BL21(DE3) for induced expression, thus obtaining ACT fermentation broth and PSA fermentation broth.

[0060] Furthermore, the ACT and PSA fermentation broths were treated as follows:

[0061] The ACT and PSA fermentation broths were purified using a Ni-NTA affinity chromatography column to obtain PSA and ACT recombinant proteins.

[0062] Furthermore, the PSA recombinant protein and ACT recombinant protein were processed using the following steps to obtain the PSA-ACT complex recombinant protein:

[0063] The recombinant PSA protein was activated with metalloproteinase at a mass ratio of 1:20 to obtain active PSA;

[0064] Active PSA recombinant protein and ACT recombinant protein were mixed at a molar ratio of 1:2 and reacted at 37°C for 2 hours to obtain crude PSA-ACT complex containing PSA and ACT monomers.

[0065] Unreacted PSA monomers were removed using a DEAE ion-exchange chromatography column, and unreacted ACT monomers were removed using a hydrophobic chromatography column to obtain a highly immunogenic PSA-ACT complex recombinant protein, which can be used in the preparation of prostate cancer diagnostic kits.

[0066] The highly immunogenic PSA-ACT recombinant protein obtained by the method of this invention can provide an antigen for obtaining corresponding monoclonal antibodies and establishing detection methods for complexes, and is a key raw material required for the development of diagnostic reagents for prostate cancer.

[0067] Currently, the mainstream PSA-ACT products on the market are mainly prepared by obtaining ACT from natural plasma and PSA from natural semen, followed by in vitro synthesis of the PSA-ACT complex, but the purity of PSA-ACT is not particularly high. The PSA-ACT complex prepared in this invention is a recombinant protein. PSA and ACT are obtained through heterologous expression of Expi293F and BL21(DE3), and then the PSA-ACT complex is synthesized in vitro. High-purity recombinant PSA-ACT protein can be obtained using only three chromatography techniques, overcoming the bottleneck of the cumbersome and costly extraction process of the natural PSA-ACT complex, which is unsuitable for large-scale production. The recombinant high-purity PSA-ACT antigen prepared by this invention has strong immunoreactivity and fully meets the requirements of prostate cancer diagnostic reagents for PSA-ACT antigen.

[0068] The main experimental materials and reagents involved in this invention are as follows: 1. Cells and vectors: the expression hosts are Expi293F and BL21(DE3), and the expression plasmid vectors are pcDNA3.4G and pET34; 2. Enzymes and other biochemical reagents: endonucleases, T4 DNA ligases, metalloproteinases, etc.; 3. Expi293F cell transfection reagent: PEI-MAX (2mg / mL, Polyscience); 4. Expi293F cell culture medium: Opti-MEM medium (Gibco), OPM-293CD05 Medium (Opmai), OPM-293Profeed (F081918); 5. BL21(DE3) culture medium: 1% peptone, 0.5% yeast extract, 1% NaCl, natural pH; 6. Protein purification packing materials: Ni-NTA affinity chromatography medium (Genscript), DEAE ion exchange chromatography medium (Qianchun Biotechnology), phenyl hydrophobic agarose gel medium (Qianchun Biotechnology).

[0069] To further explain the preparation method of the present invention, the synthesis and purification process of PSA recombinant protein, ACT recombinant protein and PSA-ACT complex are described in detail through Examples 1, 2 and 3 shown below.

[0070] Example 1

[0071] (1) Preparation of recombinant strain BL21(DE3)(pET34-ACT)

[0072] The pET34 and ACT nucleic acid fragments were obtained using a double enzyme digestion method. The gene and the starting vector were ligated using T4 DNA ligase. The ligated compound was then transformed into *E. coli* DH5α competent cells and plated on LB agar plates (containing 100 μg / mL Amp) for selection. After successful sequencing, the recombinant plasmid pET34-ACT was extracted and transformed into BL21(DE3) to obtain the recombinant *E. coli* expression strain BL21(DE3)(pET34-ACT).

[0073] (2) Preparation of recombinant ACT protein

[0074] 2.1 Induced Expression of Recombinant ACT Protein

[0075] Single clones of the recombinant BL21(DE3)(pET34-ACT) expression strain obtained above were inoculated into 100 mL LB medium for seed culture. After culturing at 220 rpm and 37 °C for 12 h, they were transferred to 500 mL LB medium at a 1% inoculation rate and cultured at 220 rpm and 37 °C until OD600≈0.6-0.8. Then, IPTG was added to a final concentration of 0.1 mM, and the culture was placed in a shaker at 220 rpm and 37 °C for 4 h.

[0076] 2.2 Isolation and purification of recombinant ACT protein

[0077] The fermented bacterial culture was centrifuged at 6000 rpm for 5 min, and the precipitate was collected. The precipitate was then resuspended in buffer A (20 mM PB, 0.5 M NaCl, 20 mM imidazole, pH 7.4). Subsequently, the precipitate was sonicated at 50% power for 30 min under ice-water bath conditions, with sonication intervals of 2 s and 8 s. The supernatant and precipitate were then separated by centrifugation at 10000 rpm for 20 min. The supernatant was then purified using a Ni-NTA affinity chromatography column. Purification buffer A consisted of 20 mM PB, 0.5 M NaCl, 20 mM imidazole, pH 7.4, and buffer B consisted of 20 mM PB, 0.5 M NaCl, 500 mM imidazole, pH 7.4. The eluent was collected as the target protein. The eluted target protein was dialyzed with dialysis buffer (20 mM Hepes, 0.15 M NaCl, pH 7.8) to obtain the recombinant ACT protein, as shown below. Figure 1 As shown, the recombinant ACT has a molecular weight of 47.5 kDa, a purity of over 80%, and a protein expression level of approximately 30 mg / L.

[0078] Example 2

[0079] (1) Construction of recombinant vector pcDNA3.4G-PSA

[0080] The vector pcDNA3.4G and PSA gene fragments were obtained using a double enzyme digestion method. The gene fragments and the starting vector were ligated using T4 DNA ligase. The ligated compound was then transformed into *E. coli* DH5α competent cells and plated on LB agar plates (containing 100 μg / mL ampicillin) for selection. After successful sequencing, the recombinant plasmid pcDNA3.4G-PSA was extracted and used for transfection of Expi293F cells.

[0081] (2) Expression and purification of recombinant PSA protein

[0082] 2.1 Cell transfection and protein expression

[0083] 1) Seed cell (Expi293F) culture: p18 generation Expi293F cells were seeded in OPM-293CD05 Medium expression medium at a seeding density of 2.0-3*10^6 cells / mL and cultured in a 37℃ CO2 shaking incubator for 24 h.

[0084] 2) Cell transfection: Dilute seed cells to 3.0*10^6 cells / mL and prepare the transfection complex according to the following ratio for transfection:

[0085] A: 10 mL Opti-MEM + 100 μg plasmid, mix well;

[0086] B: 10 mL Opti-MEM + 1000 μg transfection reagent, mix well;

[0087] Add solution A dropwise to solution B, mix thoroughly by pipetting, incubate at room temperature for 15 minutes, and add the transfection system while shaking the cell flask containing seed cells (Expi293F).

[0088] 3) Feeding: 20 h after transfection, add 20 mL of OPM-293Profeed feeding medium;

[0089] 4) Cell culture and expression: Continue culturing until the 6th day after transfection, then stop culturing.

[0090] 2.2 Isolation and purification of recombinant PSA protein

[0091] The cell slurry after fermentation and expression was centrifuged at 6000 rpm for 5 min. The supernatant was collected and concentrated using a 10 kDa membrane buffer, and the buffer was changed to 20 mM PB, 0.15 M NaCl, 20 mM imidazole, pH 7.4. Purification was then performed using Ni-NTA affinity chromatography. Purification buffer A was 20 mM PB + 0.15 M NaCl, 20 mM imidazole, pH 7.4, and buffer B was 20 mM PB + 0.15 M NaCl + 500 mM imidazole, pH 7.4. The eluent was collected as the target protein. The eluted target protein was dialyzed with dialysis buffer: 20 mM Tris, 0.15 M NaCl, pH 8.0, and the buffer was changed to obtain high-purity recombinant PAS protein. Figure 2 As shown, the recombinant PSA has a molecular weight of 26.8 kDa (under reduction conditions), a purity of over 80%, and a protein expression level of approximately 20 mg / L.

[0092] Example 3

[0093] (1) Synthesis of PSA-ACT complex

[0094] 1) PSA activation: Mix metalloproteinase and PSA at a mass ratio of 1:20, then add activation buffer (50 mM Tris-HCl, 10 mM CaCl2, 0.15 M NaCl, pH 7.8), react at 37°C for 5 min, and immediately add EDTA to a final concentration of 25 mM to terminate the reaction.

[0095] 2) Synthesis of PSA-ACT complex: ACT and activated PSA are mixed at a molar ratio of 2:1, and then reaction buffer is added. The mixture is reacted in a water bath at 37°C for 2 hours to obtain crude PSA-ACT containing ACT and PSA monomers.

[0096] (2) Purification of PSA-ACT complex

[0097] 1) DEAE ion exchange chromatography purification: Solution A used for purification was 10 mM Tris-HCl, pH 7.1, and Solution B was 10 mM Tris-HCl, 1 M NaCl, pH 7.1. Linear elution with salt ion concentrations removed unreacted PSA monomers.

[0098] 2) Hydrophobic chromatography purification: Solution A used for purification was 10 mM Tris-HCl, 2 M NaCl, pH 7.1; Solution B was 10 mM Tris-HCl, pH 7.1. Linear elution with salt ion concentration removed unreacted ACT monomers.

[0099] High-purity recombinant PAS-ACT protein was finally obtained using a two-step chromatography technique, such as... Figure 3 As shown, the recombinant PAS-ACT has a molecular weight of 74 kDa (under reduction conditions) and a purity of over 80%.

[0100] Example 4

[0101] This invention also discloses the application of the recombinant PSA-ACT protein prepared by the above method in a prostate cancer diagnostic kit.

[0102] Immunogenicity and antigenic titer of recombinant PSA-ACT protein were detected using a double-antibody sandwich method. First, the recombinant PSA-ACT antigen was serially diluted, and then the luminescence values ​​of the recombinant PSA-ACT antigen and its corresponding antibody were measured at each dilution. The results are shown in Table 1. The luminescence value of the recombinant PSA-ACT antigen diluted 10,000 times was approximately 35,900, and the luminescence value of diluted 100,000 times was approximately 4,100, with an effective concentration of 58,000 ng / mL. Furthermore, antigen correlation assays are shown in Table 2. Correlation curves were plotted based on Table 2. Figure 4 As shown, the correlation is >0.999, indicating that the recombinant PSA-ACT antigen and the two antibodies have a strong affinity, which fully meets the requirements for its application in prostate cancer diagnostic kits.

[0103] Table 1. PSA-ACT Antigen Titer Determination

[0104]

[0105] Table 2. PSA-ACT Antigen Correlation Assay

[0106]

[0107] The recombinant PSA-ACT protein obtained in this invention has many advantages compared with the naturally derived PSA-ACT protein:

[0108] 1) The production process of recombinant proteins can be carried out in a strictly controlled laboratory environment, avoiding potential pathogen contamination. Natural proteins may be contaminated by viruses, bacteria, or other pathogens; 2) The production of recombinant proteins can be precisely controlled to ensure the consistency of each batch; 3) Although the initial cost of R&D and production of recombinant proteins may be higher, once the production process is established, its production cost will be lower than that of natural proteins; 4) Recombinant proteins can be produced on a large scale using cell culture technology, while the acquisition of natural proteins is usually limited by the availability of biological sources.

[0109] This invention comprehensively considers the advantages of both prokaryotic and eukaryotic expression systems. Combining the Expi293F and BL21 expression systems not only improves recombinant protein expression efficiency but also saves production costs. Expi293F is a suspension culture cell line derived from human embryonic kidney cells (HEK293) and is widely used in mammalian cell expression systems. First, Expi293F can perform mammalian-specific post-translational modifications, ensuring the expressed protein possesses biological activity and function. Second, Expi293F exhibits high transfection efficiency for various transfection methods, facilitating gene introduction and expression. Finally, Expi293F cells are suitable for large-scale bioreactor production, meeting the demands of industrial production. However, compared to prokaryotic expression systems, Expi293F has higher culture costs and more stringent requirements for culture media and growth conditions. BL21, as a common prokaryotic expression host, is widely used in the production of recombinant proteins. First, the BL21 strain lacks Lon and OmpT proteases, which reduces the degradation of recombinant proteins and increases protein yield. Second, BL21 can grow rapidly in ordinary culture media, shortening the production cycle. Finally, the BL21 expression system is simple to operate, inexpensive, and suitable for large-scale production. However, BL21 lacks the post-translational modification mechanisms found in eukaryotic cells, which may pose some technical challenges when processing complex proteins. This study combines two expression systems to provide an efficient and scalable solution for the production of recombinant protein complexes.

[0110] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A method for preparing a recombinant PSA-ACT complex protein, characterized in that, Includes the following steps: Obtain the PSA gene fragment and the ACT gene fragment respectively; Gene fragments are ligated into the starting vector to form corresponding linkers; The linkers are transformed and screened to obtain the corresponding recombinant plasmids; The recombinant plasmids were transformed / transfected and induced to express to obtain ACT fermentation broth and PSA fermentation broth; The fermentation broth was subjected to separation and affinity chromatography purification to obtain PSA recombinant protein and ACT recombinant protein; The PSA recombinant protein was activated by metalloproteinases to obtain the active PSA recombinant protein; The active PSA recombinant protein and ACT recombinant protein were mixed and reacted to obtain the crude PSA-ACT complex. The crude PSA-ACT complex was separated and purified by ion exchange chromatography and hydrophobic chromatography to obtain high-purity and high-activity recombinant PSA-ACT complex protein.

2. The method for preparing a PSA-ACT complex recombinant protein according to claim 1, characterized in that, The method for obtaining the PSA gene fragment is as follows: The PSA amino acid sequence was retrieved from the UniProt database. The first 17 amino acids were the signal peptide. The PSA amino acid sequence was obtained by removing the first 17 amino acids. The PSA gene fragment was synthesized based on the PSA amino acid sequence.

3. The method for preparing a PSA-ACT complex recombinant protein according to claim 2, characterized in that, The amino acid sequence of the PSA is shown in SEQ ID NO:1; the nucleotide sequence of the PSA gene fragment is shown in SEQ ID NO:

2.

4. The method for preparing a PSA-ACT complex recombinant protein according to claim 1, characterized in that, The method for obtaining the ACT gene fragment is as follows: The ACT amino acid sequence was retrieved from the UniProt database. The first 23 amino acids were the signal peptide. The ACT amino acid sequence was obtained by removing the first 23 amino acids. The ACT gene fragment was synthesized based on the ACT amino acid sequence.

5. The method for preparing a PSA-ACT complex recombinant protein according to claim 4, characterized in that, The amino acid sequence of the ACT is shown in SEQ ID NO:3; the nucleotide sequence of the ACT gene fragment is shown in SEQ ID NO:

4.

6. The method for preparing a PSA-ACT complex recombinant protein according to claim 1, characterized in that, The starting vector for the PSA gene fragment is pcDNA 3.4G, and the starting vector for the ACT gene fragment is pET34.

7. The method for preparing a PSA-ACT complex recombinant protein according to claim 1, characterized in that, The recombinant plasmids were pcDNA3.4G-PSA and pET34-ACT, respectively.

8. The method for preparing a PSA-ACT complex recombinant protein according to claim 7, characterized in that, Transformation / transfection and expression induction of recombinant plasmids include: The pET34-ACT recombinant plasmid was transformed into BL21(DE3) for induced expression; The pcDNA3.4G-PSA recombinant plasmid was transfected into Expi293F to induce expression.

9. The use of the PSA-ACT complex recombinant protein prepared by the preparation method according to any one of claims 1 to 8 in a prostate cancer diagnostic kit.

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