Fusion protein, nucleic acid molecule, recombinant vector and application

Abstract

The invention relates to the field of medical detection, in particular to a fusion protein, a nucleic acid molecule, a recombinant vector and application. The invention provides a fusion protein. The fusion protein comprises a P16 nano antibody and horse radish peroxidase. The fusion protein provided by the invention realizes immunohistochemical one-step detection of P16 high-expression cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to fusion proteins, nucleic acid molecules, recombinant vectors and applications. Background Art

[0002] Antibodies are important reagents in in vitro diagnosis. Nanobodies are a cutting-edge technology that has been developed recently. The relevant nanobodies have a small molecular weight and high affinity. Horseradish peroxidase (HRP) is a commonly used enzyme in clinical testing. It can catalyze the color development of substrates and can achieve specific detection when combined with antibodies. The combination of antibodies and HRP is generally achieved through chemical connection, including EDC / NHS method, glutaraldehyde method, etc. The connection efficiency and stability are poor and may affect the function of antibodies or HRP. P16 is a basic gene in the cell cycle, which is directly involved in the regulation of the cell cycle and regulates cell proliferation and division. P16 antibodies can be used for the detection of cancer cells. The commonly used method is a two-step method, which is generally incubated with P16 antibodies and tissue sections, and then HRP-labeled secondary antibodies are used to identify primary antibodies and add substrates for color development. This method is cumbersome to operate, so the one-step method can speed up detection. Summary of the invention

[0003] In view of this, the present invention provides a fusion protein, a nucleic acid molecule, a recombinant vector, a host and applications. The fusion protein provided by the present invention realizes one-step immunohistochemical detection of P16 high-expressing cancer cells.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a fusion protein comprising: P16 nano antibody and horseradish peroxidase.

[0006] In some embodiments of the present invention, in the above fusion protein, the P16 nanobody has:

[0007] (1), the amino acid sequence shown in SEQ ID NO: 1; or

[0008] (2) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or

[0009] (3) A sequence having an identity of 80% or more to the amino acid sequence shown in (1) or (2).

[0010] In some embodiments of the invention, the sequence of SEQ ID NO: 1 is: QVQLVESGGGLVQAGASLRLSCAASGSTYMFSISAMGWYRQAPGKQRELVAAITSGGGDTNYADSVKGRFTISRDRAKNMVYLQMNSLKPEDTAVYYCNFAPGLQSVRSGSWGQGTQVTVSS.

[0011] In some embodiments of the present invention, in the above fusion protein, the horseradish peroxidase has:

[0012] (3) the amino acid sequence shown in SEQ ID NO: 2; or

[0013] (4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (3); or

[0014] (5) A sequence having a homology of 80% or more to the amino acid sequence shown in (3) or (4).

[0015] In some embodiments of the invention, SEQ ID The sequence of NO:2 is: QLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKD SFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNSNS.

[0016] In some embodiments of the present invention, the above-mentioned fusion protein further includes: a connecting peptide.

[0017] In some embodiments of the present invention, in the above fusion protein, the connecting peptide has:

[0018] (6) the amino acid sequence shown in SEQ ID NO: 3; or

[0019] (7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (6); or

[0020] (8) A sequence having an identity of 80% or more to the amino acid sequence shown in (6) or (7).

[0021] In some embodiments of the present invention, the sequence of SEQ ID NO: 3 is: SSSGSG.

[0022] In some embodiments of the present invention, the above fusion protein has:

[0023] (9), the amino acid sequence shown in SEQ ID NO: 4; or

[0024] (10) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (9); or

[0025] (11) A sequence having a homology of 80% or more to the amino acid sequence shown in (9) or (10).

[0026] In some embodiments of the invention, the sequence of SEQ ID NO:4 is: QVQLVESGGGLVQAGASLRLSCAASGSTYMFSISAMGWYRQAPGKQRELVAAITSGGGDTNYADSVKGRFTISRDRAKNMVYLQMNSLKPEDTAVYYCNFAPGLQSVRSGSWGQGTQVTVSSSSSGSGMQLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVS CADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNG NLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNSNSDLGGGGSWSHPQFEKHHHHHH.

[0027] The present invention also provides a nucleic acid molecule encoding the above fusion protein, which has:

[0028] (9), the nucleotide sequence shown in SEQ ID NO: 5; or

[0029] (10) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (9), and having the same or similar function as the nucleotide sequence shown in (9); or

[0030] (11) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (9) or (10).

[0031]

[0032] The present invention also provides a recombinant vector, comprising the above nucleic acid molecule.

[0033] The present invention also provides a host for transforming and / or transfecting the above recombinant vector.

[0034] The present invention also provides a method for preparing the fusion protein, wherein the recombinant vector is used to transform and / or transfect cells, and the fusion protein is obtained after expression and purification.

[0035] The present invention also provides the use of the above fusion protein, the above nucleic acid component, the above recombinant vector, the above host and / or the fusion protein obtained by the above preparation method in preparing a product for detecting cancer cells.

[0036] In some embodiments of the present invention, the above-mentioned cancer cells include: cervical cancer cells.

[0037] The invention provides a fusion protein comprising: P16 nano antibody and horseradish peroxidase.

[0038] The fusion protein provided by the present invention realizes one-step immunohistochemical detection of cancer cells with high P16 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0040] Figure 1 Shown is the aP16HRP staining of paraffin sections of cervical cancer;

[0041] Figure 2 Shown is the aP16HRP staining of paraffin sections of cervical cancer;

[0042] Figure 3 Shown is the aP16HRP staining of paraffin sections of cervical cancer. DETAILED DESCRIPTION

[0043] The invention discloses a fusion protein, a nucleic acid molecule, a recombinant vector and applications.

[0044] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0045] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0046] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0047] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0048] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0049] In Example 1 and Verification Example 1 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0050] The present invention will be further described below in conjunction with embodiments:

[0051] Example 1

[0052] In this embodiment, two genes, namely, the P16 nanobody gene and the HRP gene, are connected through the gene of 6 amino acids SSSGSG. The fusion gene is synthesized by GenScript Gene and expressed in mammalian cells 293F cells after gene synthesis.

[0053] aP16HRP protein expression

[0054] (1) 293F cells were cultured in serum-free medium, suspended and shaken at 37°C and 6% CO2 concentration;

[0055] (2) When the cell density reaches 2*10 6 / mL, transfection was performed;

[0056] (3) Use 1 μg of plasmid and 2 μg of PEI transfection reagent per 1 mL of culture medium. For example, for 50 mL of cell culture, mix 50 μg of plasmid and 100 μg of PEI transfection reagent in 1 mL of PBS buffer, let stand for 20 minutes, then add to the cell culture and continue suspension and shaking culture;

[0057] (4) After 72 hours, the cell culture supernatant was collected and the antibody was purified using protein A filler. Verification Example 1a The function of P16HRP protein was verified by immunohistochemistry

[0058] Experimental plan for effect verification:

[0059] 1. Baking: Place the tissue sections in a 60°C constant temperature oven and bake for 60 minutes before dewaxing;

[0060] 2. Dewaxing: Put the tissue sections into xylene and gradient ethanol in turn, xylene 3 times, each time for 15 minutes; anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, each time for 5 minutes; finally, soak in ultrapure water for 2 minutes;

[0061] 3. Antigen repair: Heat the antigen repair solution (EDTA) in a microwave oven until boiling, add the tissue sections and continue heating for 20 minutes;

[0062] 4. Blocking: After the antigen repair of tissue sections is cooled to room temperature, incubate with nonspecific blocking solution (methanol-H2O2 solution) at room temperature for 15 minutes, and then use 5% BSA diluted in PBS for nonspecific antigen blocking at room temperature for 30 minutes;

[0063] 5. Antibody incubation: Wash with PBS solution 3 times, 2 min each time, and incubate with HRP-fused P16 antibody obtained in Example 1 at room temperature for 1-2 h or at 4°C overnight;

[0064] 6. Elution: Wash with PBS solution 3 times, 2 minutes each time;

[0065] 7. Color development: add freshly prepared DAB working solution, incubate in a wet box, and after staining, soak in PBS for 5 minutes each time, repeat 3 times, remove the residual liquid, and counterstain with hematoxylin for 40 seconds;

[0066] 8. Differentiation: After restaining, rinse thoroughly with tap water, differentiate in 1% hydrochloric acid alcohol solution for a few seconds, and wash the sections again;

[0067] 9. Dehydration and transparency: Place the tissue sections in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene (3 times) in sequence, 3 minutes each time;

[0068] 10. Sealing: After dehydration, wipe off the liquid around the slice, add a drop of neutral gum, cover with a coverslip, and tap the coverslip gently with the blunt end of tweezers to remove bubbles for complete sealing.

[0069] The experimental results are as follows Figure 1 to Figure 3 As shown, Figure 1 to Figure 3 The brown cells are cervical cancer cells, and the blue cells are normal cells. Figure 1 to Figure 3 It can be seen that there is a big difference in color between cervical cancer cells and normal cells, so the aP16HRP fusion protein has both the function of the P16 antibody and the activity of HRP.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that include: P16 nanobody and horseradish peroxidase.

2. The fusion protein according to claim 1, characterized in that The P16 nanobody has: (1), the amino acid sequence shown in SEQ ID NO: 1; or (2) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (1); or (3) A sequence having an identity of 80% or more to the amino acid sequence shown in (1) or (2).

3. The fusion protein according to claim 1 or 2, characterized in that The horseradish peroxidase has: (3) the amino acid sequence shown in SEQ ID NO: 2; or (4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (3); or (5) A sequence having an identity of 80% or more to the amino acid sequence shown in (3) or (4).

4. The fusion protein according to any one of claims 1 to 3, characterized in that Also includes: Connector peptide.

5. The fusion protein according to claim 4, characterized in that The connecting peptide has: (6) the amino acid sequence shown in SEQ ID NO: 3; or (7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (6); or (8) A sequence having an identity of 80% or more to the amino acid sequence shown in (6) or (7).

6. The fusion protein according to any one of claims 1 to 5, characterized in that The fusion protein has: (9), the amino acid sequence shown in SEQ ID NO: 4; or (10) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (9); or (11) A sequence having an identity of 80% or more to the amino acid sequence shown in (9) or (10).

7. A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 6, characterized in that: It has: (9), the nucleotide sequence shown in SEQ ID NO: 5; or (10) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (9), and having the same or similar function as the nucleotide sequence shown in (9); or (11) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (9) or (10).

8. A recombinant vector, characterized in that Comprising the nucleic acid molecule as described in claim 7.

9. A method for preparing a fusion protein, characterized in that: The recombinant vector as claimed in claim 8 is used to transform and / or transfect cells, and the fusion protein is obtained after expression and purification.

10. Use of the fusion protein according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the recombinant vector according to claim 8 and / or the fusion protein obtained by the preparation method according to claim 9 in preparing a product for detecting cancer cells.

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