A recombinant protein of SAA and its preparation method and application

Abstract

The application provides a SAA recombinant protein and a preparation method and application thereof, and the preparation method comprises the following steps: S10, constructing a recombinant plasmid: a gene sequence of an amino acid sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO: 2 is inserted into a pET-28a(+) plasmid which is double-digested by NdeI and XhoI after being double-digested by NdeI and XhoI, so that a recombinant plasmid is obtained; S20, transforming and culturing the recombinant plasmid; S30, protein expression and purification. The application solves the technical problem that the stability of the currently recombinantly expressed SAA protein in vitro is poor, and achieves the technical effect of improving the stability of the SAA recombinant protein in vitro.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a recombinant SAA protein, its preparation method, and its applications. Background Technology

[0002] Serum amyloid protein A (SAA) is an acute-phase reactive protein, a heterogeneous protein belonging to the apolipoprotein family, with a relative molecular weight of approximately 12,000. SAA synthesized in the liver is released into the bloodstream and rapidly binds to HDL. Its metabolism in the body mainly occurs through degradation by proteases in serum, on the cell surface, and within cells, with the liver being the primary site of degradation. SAA produced by extrahepatic cells is mainly degraded by proteases on the cell surface or within cells through intercellular adhesion and endocytosis. SAA is the most sensitive of the acute-phase proteins; as the disease improves and recovers, SAA is the first to decrease and return to normal. Clinical studies worldwide over the past 20 years have demonstrated that SAA is also an excellent indicator for dynamic disease monitoring. For the same patient with the same disease, SAA levels can fluctuate significantly over several days, thus giving SAA high clinical value in evaluating treatment efficacy and prognostic assessment.

[0003] Currently, the main methods used for clinical serum SAA detection include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunorate turbidimetry, and microsphere capture enzyme immunoassay (MEIA). These methods all require the use of different or specific concentrations of SAA antigen as standards to create a standard curve, thereby calculating the SAA content in the sample.

[0004] The problem is that the recombinant SAA protein currently expressed is very prone to aggregation and degradation, and its stability in vitro is very poor. Summary of the Invention

[0005] This invention solves the technical problem that recombinant SAA proteins are prone to aggregation and degradation and have poor in vitro stability, and achieves the technical effect of improving the in vitro stability of recombinant SAA proteins.

[0006] To address the above problems, the present invention provides a recombinant SAA protein having the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2.

[0007] Compared with existing technologies, the technical effects achieved by this technical solution are as follows: by modifying the amino acid sequence of natural SAA, soluble expression is achieved while retaining the activity of natural protein, significantly improving the stability of the modified recombinant SAA protein without affecting its immune activity.

[0008] The present invention also provides the application of the above-mentioned SAA recombinant protein in immunodiagnostics.

[0009] Compared with existing technologies, the technical effects achieved by this technical solution are: SAA recombinant protein has good stability and high immunogenicity in liquid state, thus meeting the requirements for use as a calibrator in clinical diagnosis.

[0010] In one embodiment of the present invention, recombinant SAA protein is used to prepare an SAA detection kit.

[0011] Compared with existing technologies, the technical benefits achieved by this solution are: the recombinant SAA protein exhibits good stability in the liquid state, thus enabling its use in the preparation of SAA detection kits.

[0012] This invention also provides a method for preparing SAA recombinant protein, comprising the following steps: S10: Constructing a recombinant plasmid: The gene sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 or the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2 is digested with NdeI and XhoI double enzymes and then inserted into the NdeI and XhoI double-digested pET-28a(+) plasmid to obtain a recombinant plasmid; S20: Transforming and culturing the recombinant plasmid: The recombinant plasmid is transformed into competent Escherichia coli cells and cultured to obtain an activated strain; the activated strain is expanded and induced to obtain an expression strain; S30: Expression and purification of the protein: The expression strain is cultured and induced, the cell supernatant is collected by centrifugation, and the cell supernatant is purified by nickel column chromatography to obtain SAA recombinant protein.

[0013] In one embodiment of the present invention, the recombinant plasmid is transformed into competent Escherichia coli cells and cultured to obtain an activated strain, comprising the following steps: S21: take a suspension of competent Escherichia coli cells and the recombinant plasmid, mix them well to obtain a bacterial solution; S22: take the bacterial solution onto LB solid medium containing kanamycin and spread it evenly; S23: after the bacterial solution is absorbed by the LB solid medium, culture at a constant temperature to obtain an activated strain.

[0014] In one embodiment of the present invention, the activated strain is expanded and induced to obtain an expression strain, including the following steps: S24: The activated strain is inoculated into LB liquid medium containing kanamycin at a ratio of 1:100, cultured at constant temperature to the logarithmic phase, and induced by IPTG to obtain an expression strain.

[0015] In one embodiment of the present invention, the expression strain is cultured by the following steps: S31: the expression strain is inoculated into LB liquid medium containing kanamycin and cultured under constant temperature and shaking to obtain seed strain; S32: the seed strain is inoculated into LB liquid medium at a ratio of 1:100 and cultured under constant temperature and shaking.

[0016] In one embodiment of the present invention, the induction in S30 and the centrifugation to collect the cell supernatant include the following steps: S33: constant temperature shaking culture until the cell OD600 of S32 reaches 0.6, add IPTG at a concentration of 0.4 mmol / L for induction, and constant temperature shaking culture; S34: induce expression until the cell OD600 decreases, centrifuge, and collect the cell supernatant.

[0017] In one embodiment of the present invention, the temperature for isothermal oscillation culture is 37°C.

[0018] In one embodiment of the present invention, the steps following S34 are: S35: the cell supernatant is purified by nickel column elution with 500mM imidazole elution buffer to obtain purified SAA recombinant protein; S36: dialysis is performed to change the medium and obtain the final SAA recombinant protein.

[0019] Compared with existing technologies, the technical advantages achieved by this solution are as follows: Traditional extraction methods for recombinant SAA protein suffer from numerous drawbacks, including high cost, low yield, easy denaturation of the target protein, and limited material sources. The preparation method provided by this invention is convenient and easy to implement. It enables protein expression in *E. coli* with a short growth cycle, low production cost, high conversion rate, and high expression of the target protein, with expression levels far exceeding those of other expression systems. Furthermore, the material originates from SAA mutants that modify the amino acid sequence of natural SAA; therefore, the recombinant SAA protein obtained through this preparation method not only possesses the immunomodulatory activity of natural SAA but also exhibits excellent stability. Attached Figure Description

[0020] Figure 1 Protein expression electrophoresis provided for embodiments of the present invention Figure 1 .

[0021] Figure 2 Protein expression electrophoresis provided for embodiments of the present invention Figure 2 . Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0023] Example 1:

[0024] This embodiment provides a recombinant SAA protein having the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2.

[0025] SEQ ID NO: 1 is:

[0026] Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg Asp MetTrp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile Gly Ser Asp Lys Tyr PheHis Ala Arg Gly Asn Tyr Asp Ala Ala Lys Arg Gly Pro Gly Gly Val Trp Ala AlaGlu Ala Ile Gly Asp Ala Arg Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala GluAsp Ser Leu Ala Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro AsnHis Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr。

[0027] SEQ ID NO: The sequence of SEQ ID NO: 2 is:

[0028] Arg Ser Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg Asp MetTrp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile Gly Ser Asp Lys Tyr PheHis Ala Arg Gly Asn Tyr Asp Ala Ala Lys Arg Gly Pro Gly Gly Ala Trp Ala AlaGlu Val Ile Gly Asn Ala Arg Glu Asn Ile Gln Arg Phe Phe Gly Arg Gly Ala GluAsp Ser Leu Ala Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro AsnHis Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr。

[0029] In one specific embodiment, based on the amino acid sequence of the SAA protein published on the NCBI website and the characteristics of that sequence, the 52nd amino acid was replaced by Val instead of Ala, the 57th amino acid was replaced by Val instead of Ala, and the 59th amino acid was replaced by Thr instead of Gly, to obtain mutant B1, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0030] In another specific embodiment, based on the amino acid sequence of the SAA protein published on the NCBI website and the characteristics of that sequence, the 59th amino acid was replaced by Thr with Gly, the 60th amino acid was replaced by Asp with Asn, and the 71st amino acid was replaced by His with Arg, to obtain mutant B2, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0031] By modifying the amino acid sequence of natural SAA, soluble expression is achieved while retaining the activity of the natural protein. This significantly improves the stability of the modified recombinant SAA protein without affecting its immune activity.

[0032] Example 2:

[0033] This embodiment provides the application of the SAA recombinant protein of SEQ ID NO: 1 or the SAA recombinant protein of SEQ ID NO: 2 in immunodiagnostics.

[0034] Preferably, recombinant SAA protein is used as a calibrator for SAA immunodiagnostic reagents.

[0035] Preferably, recombinant SAA protein is used to prepare SAA detection kits.

[0036] Specifically, the recombinant SAA protein provided in this embodiment has good stability in the liquid state and can meet the requirements for use as a calibrator in clinical diagnosis, and can be used in the preparation of SAA immunodiagnostic reagent calibrators and SAA detection kits.

[0037] Example 3:

[0038] This embodiment provides a method for preparing recombinant SAA protein, including the following steps:

[0039] S10: Constructing recombinant plasmids: The gene sequence of the amino acid sequence shown in SEQ ID NO: 1 or the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2 is digested with NdeI and XhoI, and then inserted into the pET-28a(+) plasmid digested with NdeI and XhoI to obtain recombinant plasmids.

[0040] S20: Transformation and culture of recombinant plasmids: Transform recombinant plasmids into competent E. coli cells and culture to obtain activated strains; expand the activated strains through culture and induce expression to obtain expression strains;

[0041] S30: Protein expression and purification: The expression strain was cultured, induced, and the cell supernatant was collected by centrifugation. The cell supernatant was purified by nickel column purification to obtain SAA recombinant protein.

[0042] Furthermore, the recombinant plasmid is transformed into competent E. coli cells, and activated strains are obtained through culture, including the following steps:

[0043] S21: Take the Escherichia coli competent cell suspension and recombinant plasmid, mix them well, and obtain the bacterial solution;

[0044] S22: Take the bacterial culture and spread it evenly on LB solid medium containing kanamycin;

[0045] S23: After the bacterial culture is absorbed by LB solid medium, it is cultured at a constant temperature of 37°C to obtain the activated strain.

[0046] Furthermore, the activated strain is cultured on a larger scale and induced to obtain the expression strain, including the following steps:

[0047] S24: The activated strain was inoculated into LB liquid medium containing kanamycin at a ratio of 1:100, cultured at constant temperature to the logarithmic phase, and induced by IPTG to obtain the expression strain.

[0048] Further, the expression strain is cultured, including the following steps: S31: The expression strain is inoculated into LB liquid medium containing kanamycin and cultured at constant temperature with shaking to obtain seed bacteria; S32: The seed bacteria are inoculated into LB liquid medium at a ratio of 1:100 and cultured at constant temperature with shaking.

[0049] Further, the induction in S30, and the centrifugation to collect the cell supernatant, includes the following steps: S33: constant temperature shaking culture until the cell OD600 of S32 reaches 0.6, add IPTG at a concentration of 0.4 mmol / L for induction, and constant temperature shaking culture; S34: induce expression until the cell OD600 decreases, centrifuge, and collect the cell supernatant.

[0050] Furthermore, the temperature for isothermal shaking culture was 37°C.

[0051] Furthermore, S34 is followed by the following steps:

[0052] S35: The cell supernatant was purified by nickel column purification and eluted with 500mM imidazole elution buffer to obtain purified SAA recombinant protein;

[0053] S36: Dialysis and fluid exchange to obtain the final SAA recombinant protein.

[0054] In summary, in one specific embodiment, the method for preparing SAA recombinant protein includes:

[0055] (1) Construction of SAA mutant expression vector

[0056] The SAA mutant gene sequence was double-digested with NdeI and XhoI, and the fragment between XhoI and NdeI in the prokaryotic expression vector pET-28a(+) was replaced to obtain pET-28a(+)-SAAB1 and pET-28a(+)-SAAB2.

[0057] (2) Construction of SAA mutant expression strains

[0058] E. coli competent cells BL-21 (100 μL / cell) were taken from a -80℃ freezer, transformed, and cultured on plates. Single colonies were selected and inoculated into 5 mL of LB medium containing kanamycin, and cultured at 37℃ for 16-18 hours. They were then inoculated at a 1:100 ratio into 50 mL of LB medium containing kanamycin and cultured at 37℃ until the logarithmic growth phase. After IPTG induction for 3 hours, SDS-PAGE electrophoresis was performed. These were the successfully transformed host cells, named pET-28a(+)-SAAB1 / BL21 and pET-28a(+)-SAAB2 / BL21, respectively.

[0059] (3) Expression and purification of SAA mutants

[0060] The obtained positive clones pET-28a(+)-SAAB1 / BL21 and pET-28a(+)-SAAB2 / BL21 were inoculated into LB medium containing kanamycin and cultured at 37°C and 250 rpm for 16-18 hours to obtain seed bacteria.

[0061] The seed bacteria were inoculated into LB medium at a ratio of 1:100 and cultured at 37℃ and 250 rpm until the OD600 reached 0.6. IPTG was added to a final concentration of 0.4 mmol / L and cultured at 37℃ and 250 rpm for 3 hours. Samples were taken every hour for OD600 detection.

[0062] Testing revealed a decrease in bacterial OD600 during the second and third hours of induction, indicating that bacteria could be harvested after one hour of induction. After centrifugation, the bacterial cells were subjected to ultrasonic disruption, and the supernatant was analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 1 As shown.

[0063] For details, see Figure 1M: Marker; 1, 2: pET-28a(+)-SAAB1 / BL21, 3, 4: pET-28a(+)-SAAB2 / BL21, 5: Blank bacteria. Compared with the blank bacteria, it can be seen that both pET-28a(+)-SAAB1 / BL21 and pET-28a(+)-SAAB2 / BL21 contain approximately 13KD of target protein.

[0064] Further purification of the supernatant was performed using a nickel column with an equilibration buffer of 20 mM, pH 7.4 PB, followed by elution with 500 mM imidazole elution buffer (formulation: 20 mM PB + 500 mM imidazole, pH 7.4). After dialysis, the eluted sample was aliquoted and stored in buffer. SDS-PAGE electrophoresis was performed on the dialysis samples, and the results are shown below. Figure 2 As shown.

[0065] For details, see Figure 2 , M: Marker; 1: pET-28a(+)-SAAB1, 2: pET-28a(+)-SAAB2. from Figure 2 The results show that the molecular weight of the protein is consistent with the theoretical size.

[0066] Example 4:

[0067] In one specific embodiment, the stability of the mutant SAA antigen and the control SAA antigen was tested using an in vitro diagnostic kit for SAA.

[0068] The SAA mutant B1 and SAA mutant B2 provided in Experiment 1, along with the natural SAA antigen as a control, were incubated at 37°C for 7 days. The endpoint method was used for detection, and both were analyzed separately using a Hitachi 7180 biochemical analyzer. For ease of detection, two concentrations were used: 100 μg / mL for direct detection and 3 mg / mL for detection after a 1 / 20 dilution. The results are shown in Tables 1 and 2.

[0069] Table 1. Stability testing of SAA mutants

[0070]

[0071]

[0072] Table 2. Stability Test of Natural SAA

[0073]

[0074] As shown in Tables 1 and 2, compared with day 0, after destruction at 37℃, the SAA detection value of the control group decreased significantly and the stability was poor, while the detection value of the SAA mutant after destruction at 37℃ did not fluctuate much and the stability was good.

[0075] Example 5:

[0076] In one specific embodiment, an immunoassay was performed on the SAA mutant.

[0077] Twenty New Zealand rabbits were immunized with SAA mutant B1, SAA mutant B2, and natural protein, respectively. Antiserum was collected five days later for sandwich ELISA evaluation. The coating antibody was MEDIX monoclonal antibody, and the antigen was the natural antigen. The antiserum was serially diluted for detection. The proportion of antibodies with titers in the range of 2,560,000-5,120,000 was statistically analyzed. The results are shown in Table 3.

[0078] Table 3. Statistics on Immunological Titer

[0079] Immunogen Natural protein SAAB1 SAAB2 Proportion 80％ 70％ 85％

[0080] As shown in Table 3, the immune activity of the mutant protein was not significantly affected and it still possessed the immune activity of the natural protein.

[0081] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A recombinant SAA protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A method for preparing recombinant SAA protein, characterized in that, Includes the following steps: S10: Constructing the recombinant plasmid: The gene sequence of the amino acid sequence shown in SEQ ID NO: 1 or the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2 is digested with NdeI and XhoI, and then inserted into the pET-28a(+) plasmid digested with NdeI and XhoI to obtain the recombinant plasmid; S20: Transformation and culture of the recombinant plasmid: Transform the recombinant plasmid into competent Escherichia coli cells and culture to obtain an activated strain; The activated strain was cultured on a large scale and induced to obtain an expression strain; S30: Protein expression and purification: The expressed strain was cultured, induced, and the cell supernatant was collected by centrifugation; The cell supernatant was purified by nickel column chromatography to obtain the SAA recombinant protein.

3. The preparation method according to claim 2, characterized in that, The process of transforming the recombinant plasmid into competent E. coli cells and culturing to obtain an activated strain includes the following steps: S21: Take the Escherichia coli competent cell suspension and the recombinant plasmid, mix them well, and obtain the bacterial solution; S22: Take the bacterial culture and spread it evenly on LB solid medium containing kanamycin; S23: After the bacterial solution is absorbed by the LB solid culture medium, it is cultured at a constant temperature to obtain the activated strain.

4. The preparation method according to claim 2, characterized in that, The process of expanding and inducing the expression of the activated strain to obtain an expression strain includes the following steps: S24: The activated strain was inoculated into LB liquid medium containing kanamycin at a ratio of 1:100, cultured at constant temperature to the logarithmic phase, and induced by IPTG to obtain the expression strain.

5. The preparation method according to claim 2, characterized in that, The culturing of the expressed strain includes the following steps: S31: The expressed strain was inoculated into LB liquid medium containing kanamycin and cultured under constant temperature and shaking to obtain seed strain; S32: Inoculate the seed bacteria into LB liquid medium at a ratio of 1:100 and culture at a constant temperature with shaking.

6. The preparation method according to claim 5, characterized in that, The induction process in S30, followed by centrifugation to collect the cell supernatant, includes the following steps: S33: The cells of S32 were cultured under constant temperature and shaking until the OD600 of the cells reached 0.

6. IPTG at a concentration of 0.4 mmol / L was added for induction, and the cells were cultured under constant temperature and shaking. S34: Induce expression until bacterial OD600 decreases, centrifuge, and collect the cell supernatant.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The temperature for the isothermal oscillation culture was 37°C.

8. The preparation method according to claim 6, characterized in that, The steps following S34 are as follows: S35: The cell supernatant was purified by nickel column chromatography and eluted with 500mM imidazole elution buffer to obtain purified SAA recombinant protein; S36: Dialysis and fluid exchange to obtain the final recombinant SAA protein.

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