Nucleic acid fragments, recombinant vectors, recombinant cells, and recombinant procalcitonin antigen, their preparation methods and applications

Abstract

This invention discloses nucleic acid fragments, recombinant vectors, recombinant cells, and recombinant procalcitonin antigen, as well as their preparation methods and applications, relating to the field of procalcitonin preparation. The amino acid sequence of the recombinant procalcitonin antigen is shown in SEQ ID NO: 1, or the amino acid sequence of the recombinant procalcitonin antigen is obtained by attaching a protein purification tag to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1. The recombinant procalcitonin antigen provided by this invention has higher expression levels and purity.

Description

Technical Field

[0001] This invention relates to the field of procalcitonin preparation, and particularly to nucleic acid fragments, recombinant vectors, recombinant cells and recombinant procalcitonin antigen, as well as their preparation methods and applications. Background Technology

[0002] Procalcitonin (PCT) is a non-hormonally active procalcitonin glycoprotein, a precursor peptide of calcitonin (CT). It originates from a single-copy gene located on chromosome 11 (11p15, 4) (i.e., procalcitonin is encoded by the Calc-I gene on chromosome 11, with a coding region of 426 bp). This gene consists of 2800 base pairs, containing 6 exons and 5 introns. After transcription, it is translated into preprocalcitonin in the rough endoplasmic reticulum of parafollicular cells of the thyroid gland. Preprocalcitonin comprises three parts: the N-terminal 84 amino acids, active calcitonin, and calcitonin protein. Under the action of endogenous peptidase, the nPro-CT terminus of the preprocalcitonin is cleaved to generate 116 amino acids of procalcitonin, with a molecular weight of approximately 13 kDa. Procalcitonin and calcitonin share an identical 32-amino acid sequence (positions 60–91).

[0003] Procalcitonin can be used as a raw material in the development of procalcitonin diagnostic kits, but the expression level of procalcitonin prepared by existing methods is low, which hinders its large-scale application.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide nucleic acid fragments, recombinant vectors, recombinant cells and recombinant procalcitonin antigen, as well as their preparation methods and applications, in order to solve the problem of low expression levels of procalcitonin prepared by existing methods.

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

[0007] In a first aspect, the present invention provides a recombinant procalcitonin antigen, wherein the amino acid sequence of the recombinant procalcitonin antigen is as shown in SEQ ID NO: 1, or the amino acid sequence of the recombinant procalcitonin antigen is an amino acid sequence obtained by attaching a protein purification tag to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a nucleic acid fragment comprising a nucleotide sequence encoding the recombinant procalcitonin antigen as described above.

[0009] Optionally, the nucleotide sequence of the nucleic acid fragment is as shown in SEQ ID NO: 2.

[0010] A third aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains the nucleic acid fragments of the present invention as described above.

[0011] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell contains the recombinant vector of the present invention as described above.

[0012] A fifth aspect of the present invention provides a method for preparing recombinant procalcitonin antigen, comprising the following steps:

[0013] The nucleotide sequence encoding the recombinant procalcitonin antigen of the present invention as described above is inserted into the plasmid to obtain the recombinant plasmid;

[0014] The recombinant plasmid was transformed into competent E. coli cells, and then an inducer was added to induce expression. After purification, the recombinant procalcitonin antigen was obtained.

[0015] Optionally, the step of transforming the recombinant plasmid into competent E. coli cells and then adding an inducer to induce expression to obtain the recombinant procalcitonin antigen specifically includes:

[0016] The recombinant plasmid was transformed into E. coli competent cells to obtain recombinant competent cells;

[0017] The recombinant competent cells were seeded in LB liquid medium, and then an inducer was added to induce expression, resulting in a bacterial culture.

[0018] After centrifuging the bacterial culture, the bacterial cells were collected, resuspended, and then sonicated. The supernatant was collected, purified, and the recombinant procalcitonin antigen was obtained.

[0019] Optionally, the plasmid is a pET series plasmid, and the Escherichia coli competent cells are Escherichia coli BL21(DE3) competent cells.

[0020] Optionally, the induction temperature is 30°C, and the inducer includes isopropyl-β-D-thiogalactoside;

[0021] The purification process involves first performing His-tag nickel column purification, followed by anion purification.

[0022] In a sixth aspect, the present invention provides the use of the recombinant procalcitonin antigen as described above or the recombinant procalcitonin antigen prepared by the preparation method described above in the preparation of calibrators and / or quality control materials for procalcitonin antigen immunoassay kits.

[0023] Beneficial effects: This invention extracts the amino acid sequence UniProtKB-P01258 found on the Uniprot website, and then replaces lysine at position 37 with arginine and tyrosine at position 71 with cysteine ​​to obtain the recombinant procalcitonin antigen with the amino acid sequence shown in SEQ ID NO: 1. Alternatively, after further attaching a protein purification tag to the N-terminus or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, it becomes the amino acid sequence of the recombinant procalcitonin antigen. The recombinant procalcitonin antigen provided by this invention has higher expression levels and purity compared to the procalcitonin antigen before the amino acid substitution. Attached Figure Description

[0024] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis results of the product in Example 1.

[0025] Figure 2 This is a diagram showing the SDS-PAGE electrophoresis results of the product in Example 2. Detailed Implementation

[0026] This invention provides nucleic acid fragments, recombinant vectors, recombinant cells, and recombinant procalcitonin antigen, as well as their preparation methods and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0028] This invention provides a recombinant procalcitonin antigen, wherein the amino acid sequence of the recombinant procalcitonin antigen is as shown in SEQ ID NO: 1, or the amino acid sequence of the recombinant procalcitonin antigen is an amino acid sequence obtained by attaching a protein purification tag to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0029] This invention extracts the amino acid sequence UniProtKB-P01258 (as shown in SEQ ID NO: 3) found on the Uniprot website. Then, based on the extracted amino acid sequence, the 37th lysine is replaced with arginine, and the 71st tyrosine is replaced with cysteine, resulting in the recombinant procalcitonin antigen with the amino acid sequence shown in SEQ ID NO: 1. Alternatively, a protein purification tag (such as a His tag, etc., to facilitate the purification of the recombinant procalcitonin antigen) can be further attached to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 1, forming the amino acid sequence of the recombinant procalcitonin antigen. This recombinant procalcitonin antigen has a higher expression level and purity than the procalcitonin antigen before the amino acid substitution, and is easier to purify. In this embodiment of the invention, the expression level of the recombinant procalcitonin antigen is increased by amino acid substitution.

[0030] This invention also provides a nucleic acid fragment, wherein the nucleic acid fragment contains a nucleotide sequence encoding the recombinant procalcitonin antigen as described above.

[0031] In some embodiments, the nucleotide sequence of the nucleic acid fragment is as shown in SEQ ID NO: 2.

[0032] To facilitate the purification of recombinant procalcitonin antigen, in this embodiment, a His tag is attached to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 to obtain the amino acid sequence of the recombinant procalcitonin antigen. The nucleotide sequence encoding this recombinant procalcitonin antigen is then shown in SEQ ID NO: 2. Using this nucleic acid fragment is beneficial for the purification of the recombinant procalcitonin antigen.

[0033] This invention also provides a recombinant vector containing the nucleic acid fragment described above in this invention. The recombinant vector includes a plasmid and a nucleic acid fragment (nucleotide sequence as shown in SEQ ID NO: 2) inserted into the plasmid. The plasmid can be a pET series plasmid, specifically a pET28a(+) plasmid, but is not limited thereto.

[0034] pET series plasmids are currently the most widely used recombinant protein expression vectors, offering advantages such as low cost and high expression levels. During vector construction, the target fragment is cloned into the vector's phage T7 transcription-translation system, and then expressed under the induction of host T7 RNA polymerase after transformation. T7 RNA polymerase possesses a powerful and specific ability to initiate the expression of T7 promoter genes; when fully induced, it can convert almost all of the host's own expression into the expression of the target gene.

[0035] This invention also provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described above in this invention. The recombinant cell includes Escherichia coli cells and the recombinant vector located within the Escherichia coli cells.

[0036] This invention also provides a method for preparing recombinant procalcitonin antigen, wherein the method for preparing recombinant procalcitonin antigen includes the following steps:

[0037] S1. Insert the nucleotide sequence encoding the recombinant procalcitonin antigen as described in the embodiments of the present invention into the plasmid to obtain the recombinant plasmid;

[0038] S2. The recombinant plasmid is transformed into competent Escherichia coli cells, and then an inducer is added to induce expression, thereby obtaining the recombinant procalcitonin antigen.

[0039] The preparation method provided by this invention is simple in steps and low in cost. It can improve the expression level and purity of recombinant procalcitonin antigen, increase the yield of recombinant procalcitonin antigen, and facilitate the large-scale application of recombinant procalcitonin antigen.

[0040] In step S1, in some embodiments, the plasmid is the pET28a(+) plasmid.

[0041] Specifically, in this embodiment, the nucleotide sequence shown in SEQ ID NO: 2 is inserted into the pET28a(+) plasmid to obtain a recombinant plasmid.

[0042] In step S2, in some embodiments, the step of transforming the recombinant plasmid into competent E. coli cells and then adding an inducer to induce expression to obtain the recombinant procalcitonin antigen specifically includes:

[0043] S21. The recombinant plasmid is transformed into Escherichia coli competent cells to obtain recombinant competent cells;

[0044] S22. The recombinant competent cells are seeded in LB liquid medium, and then an inducer is added to induce expression, thereby obtaining bacterial culture.

[0045] S23. After centrifuging the bacterial solution, collect the bacterial cells, resuspend the bacterial cells, sonicate them, collect the supernatant, purify them, and obtain the recombinant procalcitonin antigen.

[0046] In some embodiments, the competent Escherichia coli cells are Escherichia coli BL21(DE3) competent cells.

[0047] In some embodiments, the induction temperature is 30°C, and the inducer includes isopropyl-β-D-thiogalactoside (IPTG).

[0048] In step S23, in some embodiments, the ultrasonic disruption is performed in an ice bath;

[0049] The ultrasonic fragmentation process involves a total of 6 ultrasonic waves, with a 1-minute pause between each ultrasonic wave.

[0050] During each ultrasound session, the ultrasound lasts for 2 seconds and is paused for 3 seconds, for a total time of 5 minutes.

[0051] In some embodiments, the purification involves first performing His-tag (histidine-tagged) nickel column purification, followed by anion exchange purification. This two-step purification process yields recombinant procalcitonin antigen with high purity.

[0052] The embodiments of the present invention also provide the application of the recombinant procalcitonin antigen as described above or the recombinant procalcitonin antigen prepared by the preparation method described above in the preparation of calibrators and / or quality control products for procalcitonin antigen immunoassay kits.

[0053] The present invention will be further described below through specific embodiments.

[0054] Example 1

[0055] The amino acid sequence UniProtKB-P01258 (as shown in SEQ ID NO: 3, where the first 1-25 amino acids form a signal peptide with a disulfide bond between 85C and 91C) found on the Uniprot website was truncated to obtain the amino acid sequence 26-141, which is the truncated amino acid sequence (i.e., the mature PCT amino acid sequence, the corresponding procalcitonin antigen is denoted as PCT2, and the nucleotide sequence of the PCT2 gene is shown in SEQ ID NO: 4). Then, based on the truncated amino acid sequence (i.e., the mature PCT amino acid sequence), lysine at position 37 was replaced with arginine, and tyrosine at position 71 was replaced with cysteine, resulting in the amino acid sequence shown in SEQ ID NO: 1. A His tag was then added to its N-terminus, resulting in the amino acid sequence shown in SEQ ID NO: 5, which is the amino acid sequence of the recombinant procalcitonin antigen (denoted as PCT1). The nucleotide sequence of the PCT1 gene is shown in SEQ ID NO: 2.

[0056] The PCT1 gene (nucleotide sequence shown in SEQ ID NO: 2) and PCT2 gene (nucleotide sequence shown in SEQ ID NO: 4) were synthesized using chemical synthesis methods and sequenced by Shanghai Sangon Biotech. Restriction sites (NcoI and XhoI) were designed, and pET28a(+) was used as the expression vector to obtain the PCT1 and PCT2 recombinant plasmids.

[0057] The PCT1 recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells to obtain recombinant competent cells. The recombinant competent cells were seeded into 100 mL of LB liquid medium, and then 0.05 mmol / L, 0.1 mmol / L, and 0.25 mmol / L of IPTG were added respectively. The cells were incubated overnight at 250 rpm and 30 °C (i.e., expression time > 12 h) to induce recombinant protein expression and obtain PCT1 bacterial culture.

[0058] Collect the above PCT1 bacterial culture, centrifuge at 3000 rpm for 20 min, and collect the precipitated PCT1 bacterial cells and supernatant (i.e., the supernatant before PCT1 disruption).

[0059] PCT1 cells were resuspended in a 1 / 10 volume of resuspension buffer (containing 20 mM PB (phosphate buffer) and 0.5 M NaCl). The cells were then sonicated 6 times using a cell sonicator, with a 1-minute pause between each sonication to avoid foaming. The sonication parameters were 2 seconds per sonication followed by a 3-second pause, for a total of 5 minutes. The sonication temperature was 0°C (ice bath), and the sonication probe was kept at least 1 cm below the surface of the resuspended cells. The supernatant (i.e., the PCT1 cell supernatant after disruption) was then collected and analyzed for solubility using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

[0060] The PCT2 recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. The PCT2 supernatant before and after lysis was obtained using the same method described above, and the solubility was analyzed by SDS-PAGE electrophoresis.

[0061] The results are as follows Figure 1 As shown, lanes 1 to 3 are electrophoretic bands of the supernatant after PCT2 disruption (lanes 1 to 3 are PCT2 cells obtained under IPTG conditions of 0.05 mmol / L, 0.1 mmol / L, and 0.25 mmol / L, respectively); lane 4 is the supernatant of PCT2 before disruption; lane 5 is the protein molecular weight standard (marker); lanes 6 to 8 are electrophoretic bands of the supernatant after PCT1 disruption (lanes 6 to 8 are PCT1 cells obtained under IPTG conditions of 0.05 mmol / L, 0.1 mmol / L, and 0.25 mmol / L, respectively); lane 9 is the supernatant of PCT1 before disruption.

[0062] Electrophoresis showed that PCT1 had a wider band than PCT2, indicating higher purity. Concentration determination revealed that PCT1 had a higher concentration than PCT2. The electrophoresis results also showed that most of the bacterial protein after sonication was in the supernatant, including recombinant procalcitonin antigen. The recombinant procalcitonin antigen protein was expressed in a soluble manner.

[0063] Example 2

[0064] Under the optimized conditions (IPTG concentration 0.05 mmol / L, bacterial OD600 between 0.5 and 0.6 during induction, induction temperature 30℃, induction time greater than 12 h, and rotation speed 250 rpm), large-scale induced expression of recombinant protein was performed. PCT1 was purified using a His-tag nickel column, and the purified product was collected and anion purified. The purity of the sample obtained after two-step purification reached 100%. After boiling, the sample was identified by SDS-PAGE electrophoresis and PCT detection kit. The purification steps are shown in Table 1.

[0065] Table 1. Purification Steps

[0066]

[0067] The results are as follows Figure 2 As shown, the meanings of the different lanes are as follows:

[0068] Lane 1: marker (protein molecular weight standard); Lane 2: PCT1 loading; Lane 3: PCT1 flow-through; Lane 4: PCT1-Na0.1 (i.e., elution condition 2); Lane 5: PCT1-Na0.2 (i.e., elution condition 3); Lane 6: PCT1-Na0.5 (i.e., elution condition 4); Lane 7: PCT-Na0.7 (i.e., elution condition 5); Lane 8: PCT-Na1 (i.e., elution condition 6).

[0069] After purification with different Na ion concentrations, electrophoresis results showed that PCT1 had the highest purity under elution conditions 4 (20 mM PB, 0.5 M NaCl).

[0070] The PCT1 prepared in Example 2 (anion purification was performed under elution condition 4) was tested:

[0071] (1) Stability test

[0072] a. Prepare solutions of PCT1 with different concentrations (PCT1 concentrations of 0 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL and 110 ng / mL, respectively), and label them as PCT-1-1, PCT-1-2, PCT-1-3, PCT-1-4, PCT-1-5, PCT-1-6 and PCT-1-7, respectively;

[0073] The above solution was placed at a temperature of 2-8℃, and its light intensity was measured on days 7, 14, 21, 28 and 42. The results are shown in Table 2.

[0074] Table 2. Data results of samples placed at 2-8℃ for different numbers of days.

[0075]

[0076] In Table 2, CV represents the coefficient of variation for repeatability.

[0077] b. Prepare solutions of PCT1 with different concentrations (PCT1 concentrations of 0 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL and 110 ng / mL, respectively), and label them as PCT-1-1, PCT-1-2, PCT-1-3, PCT-1-4, PCT-1-5, PCT-1-6 and PCT-1-7, respectively;

[0078] The above solution was placed at a temperature of 37°C, and its light intensity was measured on the 7th, 14th, and 21st days. The results are shown in Table 3.

[0079] Table 3. Data results of samples placed at 37℃ for different numbers of days.

[0080]

[0081] In Table 3, CV represents the repeatability coefficient of variation.

[0082] c. Prepare solutions of PCT1 with different concentrations (PCT1 concentrations of 0 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL and 110 ng / mL, respectively), and label them as PCT-1-1, PCT-1-2, PCT-1-3, PCT-1-4, PCT-1-5, PCT-1-6 and PCT-1-7, respectively;

[0083] After freeze-drying, its light intensity value was measured;

[0084] The freeze-dried powder was then placed at temperatures of 2-8℃ and -20℃, and its photoluminescence value was tested at 7, 14, 21, 28 and 42 days, respectively. The results are shown in Table 4.

[0085] Table 4. Results of reconstitution of lyophilized powder samples after different number of days at 2-8℃ and -20℃.

[0086]

[0087]

[0088] In Table 4, CV represents the repeatability coefficient of variation.

[0089] It is evident that PCT1 exhibits good stability and reconstitution stability.

[0090] (2) Assignment accuracy test

[0091] PCT1 was prepared at different concentrations as primary calibrators (numbered PCT-1, PCT-2, PCT-3, PCT-4, PCT-5, PCT-6, and PCT-7, respectively). Then, at another time, PCT1 was prepared at different concentrations as new primary calibrators (numbered PCT-1-1, PCT-1-2, PCT-1-3, PCT-1-4, PCT-1-5, PCT-1-6, PCT-1-7, PCT-2-1, PCT-2-2, PCT-2-3, PCT-2-4, PCT-2-5, PCT-2-6, and PCT-2-7, respectively). The light intensity values ​​were measured to test the accuracy of the assigned values. The results are shown in Table 5.

[0092] Table 5. Test data on the accuracy of PCT1 assignment

[0093]

[0094]

[0095] Wherein, the fitting deviation = (fitting concentration - theoretical concentration) / theoretical concentration.

[0096] It is evident that PCT1 exhibits good assignment stability.

[0097] (3) PCT1 was prepared as a master calibrator and then clinically tested. The clinical samples used for testing were positive serum. The results are shown in Table 6.

[0098] Table 6. PCT Clinical Trial Data

[0099]

[0100] The qualification requirements are as follows:

[0101] Compared with qualified batches of reagents (i.e., competing reagents):

[0102] a. For clinical samples with <0.046 ng / mL, the absolute deviation (i.e., the absolute deviation between the full point regression value of the master calibration 1 and the full point assignment value of the qualified batch, and the absolute deviation between the full point regression value of the master calibration 2 and the full point assignment value of the qualified batch) is within the range of [-0.005, 0.005] ng / mL;

[0103] b. For clinical samples with concentrations of 0.046–0.25 ng / mL, the absolute deviation (absolute deviation between the full-point regression value of the master calibration 2 and the full-point assignment value of the qualified batch) is within the range of [-0.01, 0.01] ng / mL;

[0104] c. ≥0.25ng / mL, with the relative deviation (i.e., the ratio of the difference between the full-point return value of the main calibration 1 and the full-point assignment value of the qualified batch to the full-point assignment value of the qualified batch) within ±5%;

[0105] d. The proportion of clinical samples meeting the above requirements is ≥95% of all samples.

[0106] The clinical test results above show that the PCT1 master calibrator test data are basically no different from those of competing products, meeting the qualification requirements, with a clinical compliance rate of ≥95%. Furthermore, the above clinical data also demonstrates that PCT1 is reactive and its function remains unchanged compared to competing products (or PCT2).

[0107] As can be seen, after using PCT1 to prepare the master calibrator, the stability and accuracy of the assignment meet the requirements, with a clinical concordance rate of ≥95%. The recombinant procalcitonin antigen prepared by this invention can be used in the preparation of calibrators and quality control products in procalcitonin antigen immunoassay kits, and can be applied in the field of immunoluminescence diagnostic reagents, which can significantly reduce R&D costs and avoid the risks caused by the shortage of raw materials.

[0108] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant procalcitonin antigen, characterized in that, The amino acid sequence of the recombinant procalcitonin antigen is as shown in SEQ ID NO: 1, or the amino acid sequence of the recombinant procalcitonin antigen is the amino acid sequence obtained by attaching a protein purification tag to the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the recombinant procalcitonin antigen as described in claim 1.

3. A nucleic acid fragment, characterized in that, The nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid fragment as described in any one of claims 2-3.

5. A recombinant cell, characterized in that, The recombinant cells contain the recombinant vector as described in claim 4.

6. A method for preparing recombinant procalcitonin antigen, characterized in that... It includes the following steps: The nucleotide sequence encoding the recombinant procalcitonin antigen of claim 1 is inserted into the plasmid to obtain the recombinant plasmid; The recombinant plasmid was transformed into competent Escherichia coli cells, and then an inducer was added to induce expression, thereby obtaining the recombinant procalcitonin antigen.

7. The preparation method according to claim 6, characterized in that, The specific steps of transforming the recombinant plasmid into competent E. coli cells, followed by inducing expression with an inducer to obtain the recombinant procalcitonin antigen include: The recombinant plasmid was transformed into E. coli competent cells to obtain recombinant competent cells; The recombinant competent cells were seeded in LB liquid medium, and then an inducer was added to induce expression, resulting in a bacterial culture. After centrifuging the bacterial culture, the bacterial cells were collected, resuspended, and then sonicated. The supernatant was collected, purified, and the recombinant procalcitonin antigen was obtained.

8. The preparation method according to claim 6, characterized in that, The plasmid is a pET series plasmid, and the Escherichia coli competent cells are Escherichia coli BL21(DE3) competent cells.

9. The preparation method according to claim 6, characterized in that, The induction temperature is 30°C, and the inducer includes isopropyl-β-D-thiogalactoside. The purification process involves first performing His-tag nickel column purification, followed by anion purification.

10. The use of the recombinant procalcitonin antigen of claim 1 in the preparation of calibrators and / or quality control materials for the procalcitonin antigen immunoassay kit.

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