Transgenic expression cassette, recombinant eukaryotic expression vector, eukaryotic cell, NS1 protein and preparation method and application thereof

Abstract

The invention provides a transgenic expression cassette, a recombinant eukaryotic expression vector, a eukaryotic cell, an NS1 protein and a preparation method and application thereof, and relates to the technical field of biology. The transgenic expression cassette provided by the invention is matched with an eukaryotic expression system, efficient secretory expression of subtypes in NS1 protein 4 is successfully realized, the obtained NS1 protein retains native conformation and glycosylation modification, the antigen specificity is obviously superior to that of a prokaryotic expression product, and the transgenic expression cassette can be used for preparation of an NS1 antibody and research and development of an NS1 protein detection product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a transgenic expression cassette, a recombinant eukaryotic expression vector, eukaryotic cells, NS1 protein, its preparation method, and its application. Background Technology

[0002] Dengue fever is a mosquito-borne infectious disease caused by the dengue virus, which has four subtypes (DENV-I to DENV-IV). The NS1 protein is a glycoprotein secreted by dengue-infected cells and is required for viral replication. The NS1 protein is an important antigenic marker of the dengue virus, detectable in the blood of patients in the early stages of infection, and is a key target for diagnosing dengue fever. Currently, NS1 protein preparation mainly uses prokaryotic expression systems, but these methods suffer from incomplete protein folding and glycosylation loss, leading to insufficient antigen specificity. Furthermore, traditional methods struggle to simultaneously and efficiently prepare all four subtypes of NS1 protein, limiting the development of broad-spectrum detection reagents.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The primary objective of this invention is to provide a transgenic expression cassette that enables efficient secretory expression of the NS1 protein, thereby addressing the aforementioned technical problems.

[0005] The second objective of this invention is to provide a recombinant eukaryotic expression vector.

[0006] A third objective of this invention is to provide a eukaryotic cell.

[0007] The fourth objective of this invention is to provide a method for preparing NS1 protein.

[0008] The fifth objective of this invention is to provide an NS1 protein.

[0009] The sixth objective of this invention is to provide the application of the NS1 protein prepared by the above preparation method in the preparation of NS1 antibodies.

[0010] To achieve the above objectives, the following technical solution is adopted:

[0011] In a first aspect, the present invention provides a transgenic expression cassette, comprising, in sequence, a promoter, a coding sequence for a signal peptide, a coding sequence for an NS1 protein, and a terminator;

[0012] The nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1;

[0013] The NS1 protein includes NS1 protein type I, NS1 protein type II, NS1 protein type III, or NS1 protein type IV; the coding sequence of NS1 protein type I is shown in SEQ ID NO.2; the coding sequence of NS1 protein type II is shown in SEQ ID NO.3; the coding sequence of NS1 protein type III is shown in SEQ ID NO.4; and the coding sequence of NS1 protein type IV is shown in SEQ ID NO.5.

[0014] As a further technical solution, the coding sequence of the NS1 protein and the terminator also include a His tag sequence.

[0015] As a further technical solution, the promoter is a cytomegalovirus (CMV) enhancer promoter;

[0016] The terminator is the BGH poly(A) signal.

[0017] Secondly, the present invention provides a recombinant eukaryotic expression vector comprising the aforementioned transgenic expression cassette.

[0018] As a further technical solution, the backbone of the recombinant eukaryotic expression vector is the pcDNA3.1 vector.

[0019] Thirdly, the present invention provides a eukaryotic cell comprising the transgenic expression cassette or the recombinant eukaryotic expression vector described herein.

[0020] As a further technical solution, the eukaryotic cells include HEK-293F cells.

[0021] Fourthly, the present invention provides a method for preparing NS1 protein, wherein the eukaryotic cells are cultured and then separated and purified to obtain NS1 protein.

[0022] Fifthly, the present invention provides an NS1 protein prepared using the aforementioned preparation method.

[0023] Sixthly, the present invention provides the application of the NS1 protein prepared by the above preparation method in the preparation of NS1 antibodies.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The transgenic expression cassette provided by this invention, in conjunction with a eukaryotic expression system, has successfully achieved efficient secretory expression of the NS1 protein subtype 4. The obtained NS1 protein retains its native conformation and glycosylation modification, and its antigen specificity is significantly better than that of the prokaryotic expression product. It can be used for the preparation of NS1 antibodies and the development of NS1 protein detection products. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This is the result of SDS-PAGE testing. Detailed Implementation

[0028] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] In a first aspect, the present invention provides a transgenic expression cassette, comprising, in sequence, a promoter, a coding sequence for a signal peptide, a coding sequence for an NS1 protein, and a terminator;

[0030] The signal peptide can guide the secretion of the expressed recombinant protein into the cell culture medium, facilitating purification. The nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1:

[0031] Gccaccatggagaccgacacctgctgctgtgggtgctgctgctgtgggtgcccggctccaccgga (SEQ ID NO. 1).

[0032] The NS1 protein includes NS1 protein type I, NS1 protein type II, NS1 protein type III, or NS1 protein type IV; the coding sequence of NS1 protein type I is shown in SEQ ID NO.2:

[0033]

[0034] The coding sequence of the NS1 protein type II is shown in SEQ ID NO.3:

[0035]

[0036] The coding sequence of the NS1 protein type III is shown in SEQ ID NO.4:

[0037]

[0038] The coding sequence of the NS1 protein type IV is shown in SEQ ID NO.5:

[0039]

[0040] This invention improves the expression efficiency of NS1 protein in eukaryotic cells by codon optimization of the NS1 protein sequence in the expression cassette.

[0041] In some preferred embodiments, a His tag sequence is also included between the coding sequence of the NS1 protein and the terminator.

[0042] By setting a His tag, the isolation and homozygosity of the NS1 protein can be facilitated.

[0043] In some preferred embodiments, the promoter is a cytomegalovirus (CMV) enhancer promoter;

[0044] The terminator is the BGH poly(A) signal.

[0045] Secondly, the present invention provides a recombinant eukaryotic expression vector comprising the aforementioned transgenic expression cassette.

[0046] After the expression vector is introduced into recipient cells, it enables the expression of NS1 protein.

[0047] This invention does not impose specific limitations on the eukaryotic expression vector. In some optional embodiments, the backbone of the recombinant eukaryotic expression vector is a pcDNA3.1 vector.

[0048] Thirdly, the present invention provides a eukaryotic cell comprising the transgenic expression cassette or the recombinant eukaryotic expression vector described herein.

[0049] This eukaryotic cell can efficiently express the NS1 protein.

[0050] In some alternative embodiments, the eukaryotic cells include, but are not limited to, HEK-293F cells, or other eukaryotic cells known to those skilled in the art.

[0051] Fourthly, the present invention provides a method for preparing NS1 protein, wherein the eukaryotic cells are cultured and then separated and purified to obtain NS1 protein.

[0052] This preparation method is simple and efficient, and yields a high amount of NS1 protein.

[0053] In some alternative embodiments, the separation and purification method includes nickel column affinity chromatography purification.

[0054] Fifthly, the present invention provides an NS1 protein prepared using the aforementioned preparation method.

[0055] This NS1 protein retains its native conformation and glycosylation modification, and its antigen specificity is significantly better than that of the prokaryotic expression product. It can be used for the preparation of NS1 antibodies and the development of NS1 protein detection products.

[0056] Sixthly, the present invention provides the application of the NS1 protein prepared by the above preparation method in the preparation of NS1 antibodies.

[0057] The NS1 protein provided by this invention retains its native conformation and glycosylation modification, and its antigen specificity is significantly better than that of the prokaryotic expression product. It can be used to prepare NS1 antibodies, and the prepared NS1 antibodies can be used for the research and development of NS1 protein detection products.

[0058] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0059] Example 1: Construction of expression vector

[0060] (1) Based on the conserved sequences of the four subtypes of DENV NS1 gene in GenBank, codon optimization was performed and full-length genes were synthesized (nucleic acid sequences are shown in SEQ ID NO.2-5); expression cassettes were constructed by combining the four optimized NS1 gene sequences with signal peptides and 8×His tags (the sequences are, in order, promoter (cytomegalovirus enhancer promoter), signal peptide, NS1 gene sequence, 8×His tag and terminator (BGH polyadenylate signal)).

[0061] (2) The gene was inserted into the pcDNA3.1 vector by double digestion of the expression cassette with HindIII and XbaI to construct a total of 4 recombinant plasmids: pcDNA3.1-NS1-I, pcDNA3.1-NS1-II, pcDNA3.1-NS1-III and pcDNA3.1-NS1-IV. Sequencing was used to verify the correctness.

[0062] Example 2: Protein Expression and Purification

[0063] (1) Transfect HEK-293F cells with plasmid and collect the supernatant after culturing for 6 days;

[0064] (2) The supernatant was filtered through a 0.22 μm filter membrane and loaded into a Ni-NTA column. It was washed with PBS containing 20 mM imidazole and eluted with 100 mM imidazole. After dialysis, it was concentrated to 1 mg / ml by ultrafiltration.

[0065] Example 3: Antigen Detection

[0066] (1) SDS-PAGE analysis of the above antigens revealed bands of type I / II / III / IV NS1 antigens. The purified protein molecular weight was approximately 45-50 kDa, consistent with the theoretical value. Figure 1 );

[0067] SDS-PAGE testing:

[0068] (2) ELISA detection: The NS1 antigen was coated with serially diluted NS1 antigen, and the primary anti-dengue NS1 monoclonal antibody (Guangzhou Feipeng Biotechnology, catalog number DN-REAB-G2-006) was added for incubation. After washing, the secondary antibody (HRP-labeled goat anti-mouse IgM antibody, Shanghai Sangon Biotech, catalog number D110103-0100) was added for incubation. The results showed that all four antigens could specifically bind to the antibody (Table 1).

[0069] Table 1 ELISA test data

[0070]

[0071] As can be seen from the results in Table 1, the NS1 antigens of types I / II / III / IV prepared in this invention can all bind well to NS1 antibodies as detected by ELISA.

[0072] Example 4: Development of a colloidal gold immunochromatographic test strip

[0073] Using the four NS1 antigens mentioned above as markers, colloidal gold particles were labeled, and a colloidal gold immunochromatographic strip for detecting DENVNS1 antibodies was constructed.

[0074] (1) Labeling of colloidal gold: Colloidal gold was labeled using the above-mentioned type I / II / III / IV NS1 antigens.

[0075] S1 First, a colloidal gold solution with a particle size of about 70-150 nm was selected, and the pH of the colloidal gold solution was adjusted to about 7.8 using a 0.1 mol / L K2CO3 solution.

[0076] S2. DENV NS1-I / II / III / IV type NS1 antigens were added to colloidal gold solution at an antigen:colloidal gold mass ratio of 1:7.5, respectively. The mixtures were mixed, incubated for 3 min, and then 1% PEG20000 was added.

[0077] S3 Centrifuge the above solution at 20000g and 4℃ for 30min, and remove the supernatant.

[0078] S4 was resuspended in 0.4 mg / mL PEG 20000 solution, centrifuged to remove the supernatant, and then 0.2 mg / mL sodium azide was added to the same solution (0.4 mg / mL PEG 20000) and stored at 4 °C.

[0079] (2) Making marking pads

[0080] S1 treats and impregnates SB08 glass fibers with gold-labeled antigens at the same concentration.

[0081] After drying S2 overnight, the cutting width should be approximately 0.5cm.

[0082] S3 uses glass fibers treated with the four gold-labeled antigens mentioned above as conjugate pads to construct immunochromatographic test strips. The immunochromatographic test strip uses an immunogold platform and includes a sample pad, a conjugate pad, and a test pad according to the chromatographic direction. The test pad has a test line and a control line. The conjugate pad is coated with colloidal gold-labeled NS1 antigen and mouse IgG antibody. There are two test lines, coated with goat anti-human IgG antibody and goat anti-human IgM antibody, respectively. The control line is coated with goat anti-mouse IgG antibody.

[0083] (3) Detecting actual samples

[0084] Serum samples were added in increments of 5 μL + 80 μL buffer, and whole blood samples were added in increments of 10 μL + 80 μL buffer. Results were interpreted and recorded after 15 minutes using a color chart.

[0085] In all experimental cases, three or more parallel samples were used for testing, and the results were taken as the colorimetric values ​​or average of more than 95% of the parallel samples. The colorimetric values ​​of the test line and control line were referenced from the colorimetric card, which was printed with color blocks from light to dark. The color blocks were measured and calibrated annually according to the national metrological technical specification JJF1232-2009. For quantitative or precision-related colorimetric results, the colorimetric card readings were used. The colorimetric card reading ranged from 1 to 10, with higher numbers indicating darker colors. A reading of 3.5 or higher was considered a visible line. The varying shades of the T-line in subsequent tables are represented using this reading method. "X-" indicates a reading within the range of X-0.25, and "X+" indicates a reading within the range of X+0.25. Unless otherwise specified, C-line readings are ≥8, and in the following tables, these are all T-line readings. "1" indicates a negative result.

[0086] Set up the following experimental groups:

[0087] Table 2

[0088]

[0089]

[0090] Experimental group 5 contained commercially available prokaryotic expression DENV NS1 antigen (Shenzhen Heavy Chain Biotechnology Co., Ltd., recombinant dengue type 1 NS1 antigen, catalog number HP806-1); Experimental group 6 contained dengue virus type 1 E protein (Dongkang Biotechnology, catalog number A030711). The dengue virus E antigen can be used together with the NS1 antigen to detect IgG and IgM antibodies in dengue virus infected individuals to prevent missed detection.

[0091] The test results for the positive samples (serum from different dengue virus infected individuals, samples 1 to 10) are shown in the table below:

[0092] Table 3

[0093]

[0094] As can be seen from the table above, NS1 antigen of types I / II / III / IV were detected in all positive samples.

[0095] The comparison between experimental group 5 and experimental groups 1-4 shows that the detection sensitivity of the NS1-I / II / III / IV type NS1 antigen provided by this invention on the colloidal gold test strip is significantly higher than that of commercially available prokaryotic expression DENV NS1 antigen.

[0096] The comparison between experimental group 6 and experimental groups 1-4 shows that the NS1-I / II / III / IV type NS1 antigen provided by this invention is complementary to DENV E. This indicates that the dengue virus E antigen can be used in combination with the NS1 antigen provided by this invention for the detection of IgG and IgM antibodies in dengue virus infected individuals, preventing missed detection.

[0097] The test results for negative samples (samples 1 to 10 are serum samples from individuals not infected with the dengue virus) are shown in the table below:

[0098] Table 4

[0099]

[0100] The DENV NS1 II group had slight false positives, which disappeared after the dosage was reduced.

[0101] In summary, the four dengue virus subtypes NS1 proteins provided in this invention can meet the requirements for use in constructing colloidal gold immunochromatographic test strips, and have good sensitivity and specificity.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A transgenic expression cassette, characterized in that, It includes, in sequence, the promoter, the coding sequence of the signal peptide, the coding sequence of the NS1 protein, and the terminator; The nucleic acid sequence of the signal peptide is shown in SEQ ID NO.1; The NS1 protein includes NS1 protein type I, NS1 protein type II, NS1 protein type III, or NS1 protein type IV; the coding sequence of NS1 protein type I is shown in SEQ ID NO.2; the coding sequence of NS1 protein type II is shown in SEQ ID NO.3; the coding sequence of NS1 protein type III is shown in SEQ ID NO.4; and the coding sequence of NS1 protein type IV is shown in SEQ ID NO.

5.

2. The transgenic expression cassette according to claim 1, characterized in that, The coding sequence of the NS1 protein and the terminator also include a His tag sequence.

3. The transgenic expression cassette according to claim 1, characterized in that, The promoter is a cytomegalovirus enhancer promoter; The terminator is the BGH polyadenylate signal.

4. A recombinant eukaryotic expression vector, characterized in that, It includes the transgenic expression cassette as described in any one of claims 1-3.

5. The eukaryotic expression vector according to claim 4, characterized in that, The backbone of the recombinant eukaryotic expression vector is the pcDNA3.1 vector.

6. A eukaryotic cell, characterized in that, It comprises the transgenic expression cassette according to any one of claims 1-3 or the recombinant eukaryotic expression vector according to claim 4 or 5.

7. The eukaryotic cell according to claim 6, characterized in that, The eukaryotic cells include HEK-293F cells.

8. A method for preparing NS1 protein, characterized in that, The NS1 protein is prepared by culturing the eukaryotic cells described in claim 6 or 7 and then separating and purifying them.

9. An NS1 protein, characterized in that, It was prepared using the preparation method described in claim 8.

10. The application of the NS1 protein prepared by the method described in claim 8 in the preparation of NS1 antibodies.

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