Exopalaemon carinicauda AMPK alpha protein polyclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an exopalaemon carinicauda AMPK alpha protein polyclonal antibody as well as a preparation method and application thereof, and the preparation method of the exopalaemon carinicauda AMPK alpha protein polyclonal antibody comprises the following steps: constructing a recombinant expression vector pET-22b-AMPK alpha containing an AMPK alpha target gene segment as shown in SEQ ID NO.1; the recombinant expression vector is transformed into escherichia coli, induction expression of fusion protein is carried out, AMPK alpha recombinant protein is obtained after collection and purification, and the recombinant protein contains the AMPK alpha recombinant protein with the amino acid sequence shown as SEQ ID NO.2; carrying out immune treatment by taking the AMPK alpha recombinant protein as an antigen, and purifying to obtain the exopalaemon carinicauda AMPK alpha protein polyclonal antibody. The exopalaemon carinicauda AMPK alpha polyclonal antibody prepared by the method is high in titer, can specifically recognize exopalaemon carinicauda gill tissue AMPK alpha, and can be well applied to biological immunodetection of exopalaemon carinicauda.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry and molecular immunity, and in particular relates to a polyclonal antibody of AMPKα protein of white shrimp (Pseudocalis cyprinid) and a preparation method and application thereof. Background Art

[0002] AMPK (AMP-activated protein kinase) is an AMP-dependent protein kinase that plays an important role in regulating energy metabolism in eukaryotic cells. AMPK can sense changes in intracellular energy levels and regulate the body's energy balance by activating ATP synthesis pathways and inhibiting ATP consumption pathways. The structure of AMPK in eukaryotic cells is relatively conservative and consists of three heterologous subunits: α, β, and γ. Among them, the α subunit, as a catalytic subunit, is the determining factor for AMPK activation.

[0003] As a "regulator" of energy metabolism, AMPK is involved in the response and regulation of aquatic organisms to environmental stress. For example, under salinity stress, aquatic animals consume a lot of energy to resist the stressful environment. Therefore, by analyzing the changes in the expression of AMPKα subunits, we can gain a deeper understanding of the changes in energy metabolism and regulatory mechanisms when the body responds to different environmental stresses.

[0004] White shrimp is one of the important economic shrimp aquaculture varieties in the eastern coastal areas of China and is a euryhaline shrimp. Due to the wide range of salinity adaptability of white shrimp, more and more researchers have begun to try to introduce white shrimp into inland low-salinity waters for aquaculture, increase the utilization rate of low-salinity waters in inland areas, and have achieved good results. However, studies have found that white shrimp consumes a lot of energy for osmotic regulation under environmental stress such as low salt, which in turn affects its normal growth and development. Therefore, the study of the energy regulation mechanism of shrimp under salinity stress has important theoretical and practical significance for the breeding of white shrimp and healthy aquaculture in saline-alkali waters. AMPKα subunit protein antibodies are of great significance for in-depth research on AMPK function and changes in energy metabolism of white shrimp under environmental stress such as low salt. However, at present, there are very few AMPKα antibodies on the market, and they are mainly targeted at vertebrates, with low specificity in white shrimp, which seriously hinders the study of the energy metabolism regulation mechanism of shrimp under different environmental stresses. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide a method for preparing polyclonal antibodies against AMPKα protein of white shrimp. The present invention firstly expresses and obtains AMPKα protein antigen of white shrimp, obtains AMPKα recombinant protein through a prokaryotic expression system, and uses it as an antigen to immunize animals to obtain polyclonal antibodies with good specificity.

[0006] The present invention also provides an application of polyclonal antibodies against AMPKα protein of white shrimp.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the method for preparing a polyclonal antibody of AMPKα protein of white shrimp of the present invention comprises the following steps:

[0008] (1) constructing a recombinant expression vector containing the AMPKα target gene fragment as shown in SEQ ID NO.1;

[0009] (2) transforming the recombinant expression vector into competent cells and inducing the expression of fusion protein, and collecting and purifying the AMPKα recombinant protein;

[0010] (3) The AMPKα recombinant protein was used as an antigen for animal immunization, and the antiserum was collected and separated. After purification, the polyclonal antibody of the AMPKα protein of the white shrimp was obtained.

[0011] Furthermore, in step (1) of the method for preparing the polyclonal antibody, the AMPKα target gene fragment shown in SEQ ID NO.1 is recombined between the double restriction sites of the pET-22b(+) vector.

[0012] Furthermore, the method for inducing the expression of the fusion protein in step (2) of the method for preparing the polyclonal antibody is: the constructed recombinant expression strain is selected and cultured in LB liquid culture medium, and IPTG is added to culture after the bacterial liquid concentration OD600 is 0.6-0.8 to induce the expression of the fusion protein for 4-16 hours.

[0013] Furthermore, the animal subjected to immunization in step (3) of the method for preparing the polyclonal antibody is a New Zealand white rabbit.

[0014] Furthermore, the animal immunization method in step (3) of the polyclonal antibody preparation method is:

[0015] (1) First immunization: The purified AMPKα recombinant protein was thoroughly mixed with Freund's complete adjuvant and the target antigen was immunized subcutaneously at multiple points in New Zealand white rabbits;

[0016] (2) Secondary immunization: Two weeks after the first immunization, the purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then immunized subcutaneously at multiple points in New Zealand white rabbits;

[0017] (3) Three immunizations: One week after the second immunization, three immunizations were performed. The purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then multiple subcutaneous immunizations were performed on New Zealand white rabbits.

[0018] (4) Four immunizations: Two weeks after the third immunization, the fourth immunization was performed. The purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then immunized subcutaneously at multiple points in New Zealand white rabbits.

[0019] Furthermore, 7-10 days after the fourth immunization of the animal in step (3) of the animal immunization method, blood of the test animal is collected and serum is collected to obtain antiserum.

[0020] Furthermore, the purification in step (3) of the polyclonal antibody preparation method is the purified antibody obtained by purification using a SulfoLinkCoupling Resin chromatography column.

[0021] The polyclonal antibody of AMPKα protein of white shrimp of the present invention is prepared by the preparation method of the polyclonal antibody of AMPKα protein of white shrimp of the present invention.

[0022] The invention discloses an application of the polyclonal antibody in detecting the expression of AMPKα protein in different tissues of white shrimp.

[0023] Furthermore, the tissue is gill tissue.

[0024] The present invention obtains the AMPKα gene sequence and protein sequence of the white shrimp for the first time, and obtains the AMPKα recombinant protein antigen through prokaryotic expression for the first time. The polyclonal antibody against AMPKα of the white shrimp is obtained for the first time using the recombinant protein. At present, the specificity of the existing AMPKα polyclonal antibody on the market in white shrimp is poor, and the polyclonal antibody obtained by the present invention has good specificity.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] 1. The polyclonal antibody of AMPKα of white shrimp prepared by this method has high titer and high sensitivity, can specifically identify AMPKα in the gill tissue of white shrimp, and can be well applied to biological immunoassay of white shrimp.

[0027] 2. The present invention fills the gap in anti-AMPKα polyclonal antibodies of white shrimp, and lays a foundation for the study of energy metabolism of white shrimp.

[0028] 3. The present invention uses the prokaryotically expressed and purified AMPKα polypeptide as an immunogen, immunizes New Zealand rabbits four times, collects the blood of the immunized rabbits to prepare antiserum, and then purifies the antiserum by a purification column method to obtain an anti-Caudata AMPKα polyclonal antibody, which is then tested for titer by ELISA, and the specificity of the antibody is tested by Western Blot. The method for preparing antibodies is simple, rapid, low-cost, and has good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The SDS-PAGE and Western Blot results of the recombinant expression strain of the present invention before and after induction expression, wherein M represents the standard protein maker, wherein lane 1: uninduced sample; lanes 2-6: induced samples; lane 7: recombinant protein Western Blot detection results;

[0030] Figure 2 This is the SDS-PAGE result of the purified recombinant protein in the present invention, wherein M represents the standard protein maker, and lane S represents the purified recombinant protein;

[0031] Figure 3 This is a Western Blot test result of the anti-white shrimp polyclonal antibody in the present invention, wherein M represents the standard protein maker, lane 1 is the target band, the size of the target band is between 55-72kda, and the specificity is good;

[0032] Figure 4 This is a comparison diagram of the Western Blot results of detecting AMPKα in the gill tissue of white shrimp using the anti-white shrimp polyclonal antibody of the present invention and commercial antibodies purchased on the market. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0035] White shrimp: from Ganyu Jiaxin Aquaculture Company in Lianyungang City;

[0036] New Zealand white rabbit: 4 months old, weighing about 2.5 kg;

[0037] The commercial antibody AMPKα1 / 2 (WL02254, Wanlei Biotech) was used in the control experiment to detect the expression of AMPKα in the gill tissue of the white shrimp.

[0038] Example 1

[0039] Recombinant vector construction and protein expression and purification

[0040] (1) Total RNA from gill tissue of white shrimp was extracted using an RNA extraction kit (UNIQ-10 Column Trizol Total RNA Isolation Kit, Shanghai Biotech, catalog number: B511321-0100). The extraction steps are described in the product manual.

[0041] (2) using the total RNA of the white shrimp obtained in step (1) as a template, reverse transcription was performed to synthesize cDNA according to the instructions of the reverse transcription kit (HiScript III RT SuperMix for qPCR);

[0042] (3) Using the cDNA obtained in step (2) as a template, PCR amplification was performed to obtain the target gene fragment of AMPKα of white shrimp, wherein the primer sequences used for PCR amplification are as follows:

[0043] Upstream primer: 5'-CG GGATCC ATGGAGATAAGTCAAGGACAGGGCC-3' (the underline indicates the BamHI restriction site)

[0044] Downstream primer: 5'-GG GGTACC TTAACGTGCAAGTTCGGTGATTAAA-3' (the underline indicates the KpnI restriction site);

[0045] (4) purifying the amplified product obtained in step (3) and then performing double digestion with restriction endonucleases BamHI and KpnI. After purification, the digestion product is connected with the expression vector pET-22b(+) that has also been double digested with restriction endonucleases to obtain the recombinant plasmid pET-22b-AMPKα;

[0046] (5) The successfully constructed pET-22b-AMPKα recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells to obtain a recombinant expression strain containing the sequence shown in SEQ ID NO.1;

[0047] (6) The constructed recombinant expression strain was picked into LB liquid medium and cultured at 37°C for 4 h. When the bacterial solution concentration OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM and cultured at 37°C and 220 rpm for 4 h to induce the expression of the fusion protein. After induction, the bacterial solution was sampled and subjected to SDS-PAGE electrophoresis and Western blot detection. The detection results are shown in FIG. Figure 1 As shown, the recombinant expression strain was induced normally, and the target gene fragment carried by the vector was expressed normally and the size was consistent with the expectation;

[0048] (7) According to step 6, the recombinant expression strain was expanded to 1L of bacterial solution in LB medium, induced at 37°C for 4h, and the bacterial solution concentration OD600 was 0.6-0.8. The cells were collected by centrifugation, rinsed twice with PBS and resuspended, and ultrasonically disrupted. The precipitate was dissolved with Tris-8M Urea buffer (8M urea, 100mM Tris pH8.5), centrifuged at 10,000rpm for 10 minutes, and the supernatant was collected for Ni column affinity chromatography purification. The protein eluted with imidazole (concentration of 250mM) was collected to obtain the purified AMPKα recombinant protein as an antigen. After sequencing, its amino acid sequence is shown in SEQ ID N0.2. The purified protein was analyzed by SDS-PAGE electrophoresis. The test results are shown in Figure 2 As shown: the target protein is well expressed, the bands are clear, the size of the target protein is consistent with the predicted results, and the purity is high.

[0049] Example 2

[0050] Preparation of AMPKα polyclonal antibody

[0051] The AMPKα recombinant protein prepared in Example 1 was used as an antigen to immunize New Zealand white rabbits. The whole process required four immunizations. The specific process was as follows:

[0052] 1. Animal immunization

[0053] (1) First immunization: 0.5 mg of the purified recombinant protein was mixed with an equal volume of Freund's complete adjuvant, emulsified by ultrasonication, and injected subcutaneously at multiple points in New Zealand white rabbits;

[0054] (2) Secondary immunization: Two weeks after the first immunization, 0.5 mg of the purified recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant, emulsified, and emulsified by ultrasonication. The mixture was then injected subcutaneously at multiple points in New Zealand white rabbits.

[0055] (3) Three immunizations: One week after the second immunization, three immunizations were performed. 0.5 mg of the purified recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. The mixture was then emulsified by ultrasonication and injected subcutaneously at multiple points in New Zealand white rabbits. Serum was collected one week later to preliminarily determine the antibody specificity.

[0056] (4) Four immunizations: Two weeks after the third immunization, four immunizations were performed. 0.5 mg of the purified recombinant protein was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. The mixture was then emulsified by ultrasonication and injected subcutaneously at multiple points into New Zealand rabbits. Serum was collected one week later.

[0057] 2. Serum antibody titer detection

[0058] (1) Antigen coating and blocking: dilute the antigen to 1 μg / ml with CBS coating buffer (0.05 M carbonate buffer, pH 9.6), coat each well with 100 μl of antigen in a 96-well plate, and coat overnight at 4°C; wash the plate 5 times with PBST the next day, block with 5% milk powder-PBS at room temperature for 1 h, and wash the plate again 5 times with PBST;

[0059] (2) Incubation with primary antibody: Serum was serially diluted with PBS (pH 7.4), with an initial dilution ratio of 1:2000 and a final dilution ratio of 1:128000. PBS (pH 7.4) was set as a negative control. The cells were added to a 96-well plate and incubated at 37°C for 1 h.

[0060] (3) Incubation with secondary antibody: Wash the plate 5 times with PBST, dilute the secondary antibody (goat anti-rabbit IgG-HRP, RRID: AB_2313567) at 1:10000 with 5% milk powder-PBS, add 100 μl of the diluted secondary antibody to each well, and incubate at 37°C for 40 min.

[0061] (4) Color development and reading: Wash the plate 5 times with PBST, add 100 μl of TMB working solution to each well, incubate at 37°C for 5 min, add 50 μl of stop solution to each well, and measure the absorbance at OD450 with a microplate reader. The serum antibody titer obtained in this experiment was above 1:128,000 (Table 1).

[0062] Table 1 New Zealand rabbit serum antibody titer test table

[0063] Dilution ratio Absorbance 1 / 2000 2.1499 1 / 4000 1.9387 1 / 8000 1.6387 1 / 16000 1.2588 1 / 32000 0.8435 1 / 64000 0.5285 1 / 128000 0.3046 Negative control 0.0552

[0064] 3. Antibody Purification

[0065] (1) Preparation of antigen affinity column: Take 3 mg of antigen and add it to a 2 ml equilibrated SulfoLink Coupling Resin chromatography column (thermo, 20402, containing blocking buffer and coupling eluent). Oscillating reaction at room temperature for 1 hour. After standing at room temperature for 15 minutes, wash the filler with coupling solution. Then add 2 times the column volume of blocking solution, oscillate and react at room temperature for 30 minutes, and stand and incubate for 30 minutes. After draining the blocking solution, add 10 times the column volume of washing solution, store in 2 times the column volume of sealing solution, and store at 4°C.

[0066] (2) Antibody affinity purification: Take 10-12 ml of the rabbit serum prepared in this example, centrifuge (8000 r / min, 20 min) and filter (made of PES, pore size 0.45 um needle filter), take 10 ml for antigen affinity column (antigen affinity column prepared in the previous step), collect the flow-through, repeat the column loading once, rinse with 5 times the volume of the column bed equilibrium buffer, add eluent, and collect the eluted antibodies; repeat this step until no more antibodies are eluted in the eluent. The eluted antibodies were dialyzed into PBS, and the dialyzate was ultrafiltered and concentrated with an ultrafiltration tube (sartorius, 50KD, VS2032) to obtain the polyclonal antibody of AMPKα of white shrimp. The concentrated and purified antibodies were measured according to the serum antibody titer detection method, and the titer was 1 / 128000 (Table 2)

[0067] Table 2 ELISA titer test results of purified and concentrated antibodies

[0068] Dilution ratio Absorbance 1 / 2000 2.4971 1 / 4000 2.6492 1 / 8000 2.7788 1 / 16000 2.4163 1 / 32000 2.2725 1 / 64000 1.7342 1 / 128000 0.8801 Comparison 0.0504

[0069] 4 Antibody specificity detection and its application in white shrimp

[0070] The antibody specificity was detected by Western blot, and the prepared antibody was used to detect the expression of AMPKα in the gill tissue of white shrimp. The specific detection method was as follows: RIPA lysis buffer was used to extract the total protein of the gill tissue of white shrimp. SDS-PAGE gel was prepared according to the standard method, 50ug of the total protein of the gill tissue of white shrimp was taken, and 5μL of protein loading buffer was added. After high-temperature denaturation at 95℃ for 5min, the mixture was mixed and centrifuged, and then placed on ice. The denatured protein product was added to the gel wells, and different molecular weight proteins were separated by 120V constant voltage electrophoresis. Semi-dry transfer was used, and the 0.45um pore size PVDF membrane was activated with methanol in advance and then fully soaked with absorbent filter paper in the transfer buffer. The protein was adsorbed to the PVDF membrane at 25V constant voltage for 45min, and the blocking solution (blocking solution was 5% bovine serum albumin) was used to block the membrane on a shaker at room temperature for 1h. The antibody of AMPK-α was used as the primary antibody, diluted to 1:1000 with diluent, and incubated overnight at 4℃. The membrane was washed 3 times with 1×TBST, and HRP-labeled goat anti-rabbit secondary antibody (Aibotek AS014) (1:1000) was added for incubation for 1 h, and then the membrane was washed 3 times with 1×TBST. The immunoblot results were obtained after DAB color development. Figure 3 As shown, M represents the standard protein maker, lane 1 is the target band, the size of the target band is between 55-72kda, the Western Blot result is consistent with the predicted size, and the prepared AMPKα polyclonal antibody has good specificity.

[0071] The same detection method as above was used, except that the AMPK-α antibody was replaced with the commercial antibody AMPKα1 / 2 as the primary antibody, and the other processes remained unchanged. Figure 4 As shown, the left figure is the detection result of the AMPK-α antibody of the present invention, and the right figure is the detection result of the commercial antibody, which shows that the antibody obtained in the present invention can specifically detect the expression level of AMPKα protein in the tissue of the white shrimp, while the commercial antibody cannot detect it.

Claims

1. A method for preparing polyclonal antibodies against AMPKα protein of white shrimp, characterized in that: The steps include: (1) constructing a recombinant expression vector containing the AMPKα target gene fragment as shown in SEQ ID NO.1; (2) transforming the recombinant expression vector into competent cells and inducing the expression of fusion protein, and collecting and purifying the AMPKα recombinant protein; (3) The AMPKα recombinant protein was used as an antigen for animal immunization, and the antiserum was collected and separated. After purification, the polyclonal antibody of the AMPKα protein of the white shrimp was obtained.

2. The method for preparing polyclonal antibodies against AMPKα protein of white shrimp according to claim 1, characterized in that: In step (1), the AMPKα target gene fragment shown in SEQ ID NO.1 is recombined between the double restriction sites of the pET-22b(+) vector.

3. The method for preparing polyclonal antibodies against AMPKα protein of white shrimp according to claim 1, characterized in that: The method for inducing the expression of the fusion protein in step (2) is: picking the constructed recombinant expression strain and culturing it in LB liquid culture medium, adding IPTG to culture when the bacterial liquid concentration OD600 is 0.6-0.8, and inducing the expression of the fusion protein for 4-16 hours.

4. The method for preparing polyclonal antibodies against AMPKα protein of white shrimp according to claim 1, characterized in that: The animal subjected to the immunization treatment in step (3) is a New Zealand white rabbit.

5. The method for preparing polyclonal antibodies against AMPKα protein of white shrimp according to claim 1, characterized in that: The animal immunization treatment method in step (3) is: (1) First immunization: The purified AMPKα recombinant protein was thoroughly mixed with Freund's complete adjuvant and the target antigen was immunized subcutaneously at multiple points in New Zealand white rabbits; (2) Secondary immunization: Two weeks after the first immunization, the purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then immunized subcutaneously at multiple points in New Zealand white rabbits; (3) Three immunizations: One week after the second immunization, three immunizations were performed. The purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then multiple subcutaneous immunizations were performed on New Zealand white rabbits. (4) Four immunizations: Two weeks after the third immunization, the fourth immunization was performed. The purified AMPKα recombinant protein was fully emulsified and mixed with Freund's incomplete adjuvant, and then immunized subcutaneously at multiple points in New Zealand white rabbits.

6. The method for preparing polyclonal antibodies against AMPKα protein of Litopenaeus cyprinid according to claim 5, characterized in that: 7-10 days after the fourth immunization of the animal in step (3), blood is collected from the test animals and serum is collected to obtain antiserum.

7. The method for preparing polyclonal antibodies against AMPKα protein of Litopenaeus cyprinid according to claim 1, characterized in that: The purification in step (3) is to obtain the purified antibody using a SulfoLink Coupling Resin chromatography column.

8. A polyclonal antibody against AMPKα protein of white shrimp, characterized in that: The polyclonal antibody is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the polyclonal antibody according to claim 8 in detecting the expression of AMPKα protein in different tissues of Litopenaeus cyprinid.

10. The use according to claim 9, characterized in that: The tissue is gill tissue.

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