CN114671938A

CN114671938A - Micropterus salmoides Nesfatin-1 protein and prokaryotic expression method and application thereof

Info

Publication number: CN114671938A
Application number: CN202210392371.3A
Authority: CN
Inventors: 苏时萍; 刘帆; 刘鑫鑫; 陈芳芳; 李西雷; 张君
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-06-28

Abstract

The invention discloses a neofatin-1 protein of micropterus salmoides, a prokaryotic expression method and application thereof. The invention expresses the Micropterus salmoides Nesfatin-1 protein by using an escherichia coli prokaryotic expression system for the first time, and a large amount of soluble recombinant protein is obtained by purifying the protein by using a nickel column affinity chromatography, wherein the concentration reaches 1.05 mg/mL; the titer of the polyclonal antibody detected by an ELISA method is more than 1: 204800, the specificity of the polyclonal antibody detected by a Western blot method is good, and the antibody can specifically identify the Nesfatin-1 protein in liver and pancreas of micropterus salmoides verified by a tissue immunofluorescence method. The method realizes the in vitro expression of the Micropterus salmoides Nesfatin-1 protein, prepares the polyclonal antibody with high titer and good specificity, can completely meet the requirements of related experiments, and provides a powerful tool for deeply researching the biological function of the Micropterus salmoides Nesfatin-1 protein.

Description

Micropterus salmoides Nesfatin-1 protein and prokaryotic expression method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a neofatin-1 protein of micropterus salmoides, a prokaryotic expression method and application thereof.

Background

In recent years, the weever culture mode is changed from the mode of feeding small iced fresh fish to the mode of artificial compound feed, and due to the influence of the culture mode, the feed formula and the culture water environment, the fat content of cultured weever can be greatly deposited, so that organism tissues and organs are damaged, the growth of the fish is inhibited, and even death is caused. Research shows that the liver function of the largemouth bass is easily injured when fat reaches a certain level.

Nesfatin-1 is an anorexic peptide consisting of 82 amino acids encoded by the nuclear nexin 2(NUCB2) gene. Studies show that Nesfatin-1 is a functional core fragment of NUCB2, mainly participates in energy metabolism regulation, and has wide biological functions, such as (i) anorexia is caused by central action and peripheral blood brain barrier and calcium ion channel mechanisms: ② passing T type Ca²⁺Channel elicits DMNV neuronal Ca²⁺The internal flow stimulates the efferent vagus nerve in the body, inhibits gastric acid secretion and weakens gastric motility; reducing blood sugar, inhibiting gluconeogenesis rate-limiting enzyme in liver, thereby improving the sensitivity of organism to insulin; and regulating fat metabolism through suppressing appetite, energy consumption, and inhibiting differentiation of fat cells. Central nervous system response stress; sixthly, the propagation is mainly involved in the initiation of the developmental stage. The natural Nesfatin-1 protein exists in various organ tissues, the content of the natural Nesfatin-1 protein in the liver is the most abundant, but the technical difficulty of separating and purifying the Pericarpus salmoides Nesfatin-1 protein in the tissues is higher, and the chemical synthesis cost is overhigh, so that the preparation of the recombinant Pericarpus salmoides Nesfatin-1 protein by using a gene engineering technology is an effective way for solving the problems. Most of the antibodies in the market are antibodies against human, rat and mouse Nesfatin-1, but no antibody with high titer and high specificity for Perciformis micropterus salmoides Nesfatin-1 protein is reported at present.

Most of recombinant proteins induced and expressed by an escherichia coli prokaryotic expression system exist in an inclusion body form, while soluble proteins are only 13.3% -23% of total expression proteins, and cannot express a large amount of soluble proteins.

Disclosure of Invention

The invention aims to provide a neofatin-1 protein of micropterus salmoides, a prokaryotic expression method and application thereof.

The invention also aims to provide a method for preparing the polyclonal antibody with high titer and good specificity by using the recombinant Nesfatin-1 protein produced by a prokaryotic expression system.

In order to achieve the object, in a first aspect, the invention provides a micropterus salmoides Nesfatin-1 protein, which comprises or consists of the following amino acid sequence:

i) as shown in SEQ ID NO: 1; or

ii) an amino acid sequence obtained by connecting a label at the N end and/or the C end of the i); or

iii) protein with same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence of i) or ii).

In a second aspect, the invention provides a nucleic acid molecule for encoding a neofatin-1 protein of micropterus salmoides, and the sequence is shown as SEQ ID NO: 2, respectively.

In a third aspect, the invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

In a fourth aspect, the invention provides a prokaryotic expression method of Nesfatin-1 protein of black fungus, which comprises the following steps:

(1) extracting the total RNA of liver and pancreas tissues of micropterus salmoides;

(2) designing primers Nesfatin-1-F and Nesfatin-1-R for amplifying Nesfatin-1 gene of micropterus salmoides;

(3) amplification by RT-PCR: reverse transcribing the total RNA into cDNA, and performing PCR amplification by using the primers Nesfatin-1-F and Nesfatin-1-R by using the cDNA as a template;

(4) constructing the PCR amplification product obtained in the step (3) into a prokaryotic expression vector, and transforming Escherichia coli by using the obtained recombinant expression vector to obtain a transformant;

(5) IPTG inducing expression of target protein and centrifuging to collect thallus;

(6) and (3) ultrasonically breaking the bacterial cells, and purifying the target protein by using a nickel affinity chromatography column containing a His label.

Preferably, the nucleotide sequences of the primers Nesfatin-1-F and Nesfatin-1-R in the step (2) are respectively as follows (SEQ ID NO: 3-4):

Nesfatin-1-F：5’-CCCGAATTC(EcoR I restriction site) ATGGTGCCCATCAGTATGG-3'

Nesfatin-1-R：5’-CCGCTCGAG(Xho I cleavage site) GCTACAACTCATCCAGTTTTG-3'

Preferably, the prokaryotic expression vector in step (4) is a pET series vector, such as pET-32a (+), pET-28a, pET-30a, etc., more preferably pET-32a (+).

Preferably, the final concentration of IPTG in step (5) is 0.2 mmol/L.

Preferably, in step (6), the nickel affinity chromatography column is eluted by 10, 40, 80 and 100mmol/L imidazole in sequence, and the eluted target protein is renatured by dialysate.

The dialysate is: 140mmol/L of sodium chloride, 2.7mmol/L of potassium chloride, 10mmol/L of disodium hydrogen phosphate dodecahydrate and 1.8mmol/L of potassium dihydrogen phosphate, and the pH value is 8.0.

In a fifth aspect, the invention provides an anti-micropterus salmoides Nesfatin-1 protein specific antibody, which comprises a polyclonal antibody and a monoclonal antibody.

The polyclonal antibody is prepared by taking the Micropterus salmoides Nesfatin-1 protein as an immunogen and assisting an adjuvant to immunize experimental animals.

The monoclonal antibody is prepared by taking the Micropterus salmoides Nesfatin-1 protein as an immunogen, assisting an adjuvant to immunize an experimental animal and adopting a hybridoma technology or a DNA recombination technology.

In a sixth aspect, the invention provides application of the antibody in preparation of a detection reagent or a kit for detecting neofatin-1 protein of micropterus salmoides.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention expresses the Nesfatin-1 protein by using an escherichia coli prokaryotic expression system for the first time, the molecular weight is about 30kDa, the induced expression time is 4h, the optimal concentration is 0.2mmol/L, and the large-scale soluble expression of the Nesfatin-1 protein of the micropterus salmoides is successfully realized; the relatively pure soluble recombinant protein is obtained by utilizing a nickel column affinity chromatography separation method, and the concentration reaches 1.05 mg/mL; the titer of the polyclonal antibody detected by an ELISA method reaches more than 1: 204800, the specificity of the polyclonal antibody detected by a Western blot method is good, and the antibody can specifically identify the Nesfatin-1 protein in liver and pancreas of micropterus salmoides verified by a tissue immunofluorescence method. The method realizes the in vitro expression of the micropterus salmoides Nesfatin-1 protein and the preparation of the polyclonal antibody with high titer and good specificity, can completely meet the requirements of related experiments, and provides a powerful tool for the deep research of the biological function of the micropterus salmoides Nesfatin-1 protein.

Drawings

FIG. 1 is a diagram showing an electrophoresis of RT-PCR amplified micropterus salmoides NUCB2/Nesfatin-1 gene in the preferred embodiment of the present invention, wherein M is DNA marker; PCR amplification map; 2. and (5) negative control.

FIG. 2 is an electrophoresis chart of the expression result of Nesfatin-1 recombinant plasmid of IPTG inducer at different concentrations in the preferred embodiment of the present invention, M: pre-dyeing a protein Marker; 1: negative control; 2-6: inducing with 0.2, 0.4, 0.6, 1mmol/L IPTG; 7: empty vector control.

FIG. 3 is an electrophoretogram of an expression form of a target protein in a preferred embodiment of the present invention, M: pre-staining a protein Marker; 1: supernatant fluid; 2: and (4) precipitating.

FIG. 4 is an electrophoretogram of a purified recombinant protein according to a preferred embodiment of the present invention, M: pre-staining a protein Marker; 1: penetrating liquid; 2-5: 10. 40, 80 and 100mmol/L imidazole eluent.

FIG. 5 shows Western blot identification results of a polyclonal antibody against Nesfatin-1 recombinant protein in a preferred embodiment of the invention, wherein M: pre-staining a protein Marker; 1: and (3) purifying the fusion protein.

FIG. 6 shows the fluorescence localization of Nesfatin-1 protein in liver and pancreas of Lateolabrax japonicus using polyclonal antibody according to the preferred embodiment of the present invention, wherein A: staining nuclei with DAPI; b: detecting Nesfatin-1 protein by tissue immunofluorescence; c: and (5) superposition effect.

Detailed Description

The invention provides prokaryotic expression of a micropterus salmoides NUCB2/Nesfatin-1 gene and a preparation method of a polyclonal antibody thereof. Designing a specific primer of the largemouth bass NUCB2/Nesfatin-1 gene to amplify cDNA thereof; the amplified product and a pET-32a (+) vector are subjected to double enzyme digestion by EcoR I and Xho I and then are connected and transformed to a competent DH5 alpha cell to construct a prokaryotic expression recombinant vector (named as pET-32 a-Nesfatin-1); the recombinant plasmid pET-32a-Nesfatin-1 is transformed into BL21 bacteria (DE3), is induced and optimally expressed by isopropyl-beta-D-thiogalactoside (IPTG), successfully expresses a large amount of soluble protein of the Nesfatin-1 of the largemouth bass, and is separated by an affinity chromatography to obtain relatively pure recombinant protein; the purified recombinant protein is used for immunizing Balb/c mice for 3 times, the indirect ELISA detection antibody titer is very high, and the Western blot identification antibody specificity is strong. The method successfully induces the in vitro expression of the Percocephalus salmoides Nesfatin-1 protein and prepares the Percocephalus salmoides Nesfatin-1 polyclonal antibody with high titer and strong specificity.

The invention adopts the following technical scheme:

the invention provides a prokaryotic expression method of a micropterus salmoides NUCB2/Nesfatin-1 gene, which comprises the following steps:

taking liver and pancreas tissues of micropterus salmoides, and extracting total RNA of the liver and pancreas by using a Trizol reagent;

designing a primer in the step (2):

Nesfatin-1-F：5’-CCCGAATTC(EcoR I cleavage site) ATGGTGCCCATCAGTATGG-3';

Nesfatin-1-R：5’-CCGCTCGAG(Xho I cleavage site) GCTACAACTCATCCAGTTTTG-3';

step (3) amplification of RT-PCR: the total RNA was reverse-transcribed into cDNA, and a 50. mu.l PCR amplification system was prepared using the cDNA as a template.

Step (4) constructing a recombinant expression vector: recovering the PCR amplification product obtained in the step (3) by using an agarose gel recovery kit, respectively carrying out enzyme digestion on the recovered product and an escherichia coli pET-32a (+) vector, connecting enzyme digestion products, transforming the connection product to escherichia coli DH5 alpha competent cells containing ampicillin resistance, screening and identifying to obtain a correctly constructed recombinant vector pET-32 a-Nesfatin-1;

extracting the recombinant plasmid in the step (5): shaking the recombinant DH5 alpha strain obtained in the step (4) after ampicillin resistance selection to OD₆₀₀When the value reaches 0.6, extracting the plasmid to obtain a pET-32a-Nesfatin-1 recombinant plasmid;

and (6) expression and form identification of Nesfatin-1 protein: transforming the pET-32a-Nesfatin-1 recombinant plasmid obtained in the step (5) into a competent cell BL21, carrying out IPTG induction culture and optimizing induction conditions, carrying out ultrasonic cell disruption after successful expression, and identifying the expression form of the Nesfatin-1 protein of the micropterus salmoides;

and (7) purifying the Nesfatin-1 protein: purification of Nesfatin-1 protein by using His-tagged nickel column affinity chromatography

Step (8) preparation of anti-Nesfatin-1 protein polyclonal antibody: the purified Nesfatin-1 protein is used as an antigen, Balb/c mice are immunized for 3 times for 6 weeks, and blood is taken and serum is separated to obtain the anti-Nesfatin-1 protein polyclonal antibody.

Further, the PCR amplification system in the step (3) is as follows: 2 × Taq Mix 25 μ l; 10 μ M of chiRF 3-R2.5 μ l; 2.5. mu.l of 10. mu.M chiRF 3-F; 8 mul of sterile ultrapure water; mu.l of cDNA.

Further, the amplification procedure used in the PCR amplification system of step (3): pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 10 min.

Further, the PCR amplification product of the NUCB2/Nesfatin-1 gene and the pET-32a (+) vector in the step (4) are subjected to enzyme digestion by using EcoR I and Xho I endonucleases respectively.

Further, in the step (6), 3. mu.l of the recombinant plasmid was added to 50. mu.l of the competent expression strain BL21, mixed, placed on ice for 30min, heat-shocked at 42 ℃ for 90s, rapidly kept on ice for 2min, added to 450. mu.l of LB medium, shake-cultured at 37 ℃ and 220r/min for 45 min, inoculated into LB solid medium containing Amp, inverted at 37 ℃ for overnight culture, single colonies were picked up and overnight cultured in LB medium containing Amp, and inoculated into LB medium containing Amp at a ratio of 1: 100, cultured until the mixture became OD₆₀₀At 0.6, IPTG was added to a final concentration of 0.2mmol/L, an induction time of 4h at 37 ℃ and 220 r/min.

Further, the expression form of the recombinant Nesfatin-1 protein in the step (6) is identified as a ultrasonication method, wherein the ultrasonication time is 5s, the gap time is 7s, the total time is 25min, the power is 35%, and the ultrasonication method is analyzed by SDS-PAGE electrophoresis.

Further, the method for purifying the protein in the step (7) is a nickel column affinity chromatography method, and imidazole and small biological molecular impurities are removed by using dialysate after purification.

Further, in the step (8), the method for preparing the polyclonal antibody by the recombinant protein comprises the steps of firstly detecting the protein concentration by using a BCA protein concentration determination method; secondly, mixing and emulsifying the purified recombinant protein and equivalent Freund's complete adjuvant, injecting the mouse into multiple points subcutaneously with the injection amount of the protein being 100ug, immunizing three times, taking blood after the first two times of immunization every two weeks, finally-every other week, taking blood after the third time of immunization-week, and collecting serum.

Further, step (8) further comprises validation of the polyclonal antibody: ELISA, Western blot, tissue immunofluorescence and the like.

The transcript of NUCB2 includes three proteins, Nesfatin-1, Nesfatin-2 and Nesfatin-3, wherein the function of Nesfafin-2 and Nesfatin-3 is unknown.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 prokaryotic expression of Micropterus salmoides NUCB2/Nesfatin-1 Gene

1. Experimental materials: the micropterus salmoides are purchased from Anhui Zhang forest fishery, Inc., pET-32a plasmid is stored in the laboratory, escherichia coli DH5 alpha and Rosetta (DE3) competence, Trizol, reverse transcription kit, 2 xTaq Mix, gel recovery kit, T4 DNA ligase, restriction endonuclease, ampicillin and LB solid culture medium.

2. The experimental method comprises the following steps:

taking fresh liver and pancreas tissues of the largemouth bass, extracting total RNA of the liver and pancreas by using Trizol reagent, and carrying out reverse transcription by using a reverse transcription kit to obtain cDNA.

Specific primers were designed using primer6.0 software based on the micropterus salmoides NUCB2a sequence published on GenBank (accession number XM _ 038700643.1):

the sequence of the upstream primer is as follows: 5' -CCCGAATTCATGGTGCCCATCAGTATGG-3', downstream primer sequence: 5' -CCGCTCGAGGCTACAACTCATCCAGTTTTG-3', the EcoR I/Xho I cleavage sites are underlined, and the primers were synthesized by Shanghai Sangni Biotech Ltd.

Amplification by RT-PCR: amplifying by using cDNA as a template, wherein the reaction system (50 mu l) is 2 XTaq Mix 25 mu l; 10 μ M of chiRF 3-R2.5 μ l; 2.5. mu.l of 10. mu.M chiRF 3-F; 8 mul of sterile ultrapure water; mu.l of cDNA. Amplification procedure applied in PCR amplification system: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 10 min. The PCR product was verified by electrophoresis on a 1.0% agarose gel.

Constructing a recombinant expression vector: recovering the target fragment by using a gel recovery kit, carrying out double enzyme digestion on the recovered product and a pET-32a (+) vector for 2h at 37 ℃ by using EcoR I and Xho I respectively, carrying out electrophoresis separation on the enzyme digestion product by using 1% agarose gel, purifying and recovering by using the gel recovery kit, connecting Fast T4 DNA Ligase at 16 ℃ according to a certain proportion (the molar ratio of the plasmid to the fragment is 3: 7), and connecting the Nesfatin-1 gene between the EcoR I (GAATTC) -Xho I (CTCGAG) sites of the pET-32a (+) vector.

Transformation of the recombinant vector into DH5 α competent cells: mu.l of the recombinant vector is added into 50. mu.l of the competent expression strain DH5 alpha, mixed and placed on ice for 30min, heat shock is carried out for 90s at 42 ℃, the mixture is quickly kept stand on ice for 2min, 450. mu.l of LB culture medium is added, shaking culture is carried out for 45 min at 37 ℃ and 220r/min, and the mixture is inoculated into LB solid culture medium containing Amp and inverted overnight culture is carried out at 37 ℃.

Selecting a single colony, screening positive clones by PCR, carrying out amplification culture on the positive clones, then sending the positive clones to Hipposony biotechnology limited company for sequencing, inoculating the positive clones which are correctly identified to an LB culture medium containing ampicillin, and culturing overnight to extract a recombinant plasmid pET-32 a-Nesfatin-1.

Expression of the protein of interest: the recombinant plasmid pET-32a-Nesfatin-1 is transformed into BL21(DE3) competent cells, inoculated into LB culture medium containing aminobenzene antibiotics, and subjected to shaking culture of bacterial liquid OD at 37 ℃ and 200r/min₆₀₀When the value reaches 0.6, 1ml of bacterial liquid is taken as an uninduced control group, the rest bacterial liquid is added with IPTG with the final concentration of 0.2, 0.4, 0.6, 0.8 and 1.0mmol/L for induction expression, and the bacterial bodies are centrifugally collected after the induction at 37 ℃ for 4 hours. Meanwhile, BL21 bacteria transformed with pET-32a vector are used as negative control, all bacteria solutions are respectively added into SDS Loading Buffer and boiled in boiling water at 100 ℃ for 10min, 10 mu l of the mixture is taken out after centrifugation at 8000rpm for SDS-PAGE electrophoresis, and the detection is carried out after Coomassie brilliant blue staining.

3. Test results

The RT-PCR amplification results shown in FIG. 1: the size of the gene NUCB2/Nesfatin-1 of the largemouth bass obtained by amplification is 246 bp; the recombinant vector connected with the pET-32a (+) vector is transformed into DH5a competent cells, and the sequencing result of the screened positive clone is shown in SEQ ID NO: 2, respectively.

FIG. 2 shows the result of IPTG concentration induced expression of pET-32a-Nesfatin-1 protein, the relative molecular weight of the fusion protein is 30kDa, which is consistent with the expected result, and the optimal final concentration of induction concentration is 0.2 mmol/L.

Example 2 detection, separation, purification and identification of expression form of Percocephalum larvum Nesfatin-1 protein

1. Test materials: isopropyl-beta-D-thiogalactoside (IPTG) and BCA protein concentration determination kit.

2. Detecting the expression form of the recombinant Nesfatin-1 protein: example 1 after confirming that the target protein is expressed, after inducing a large amount of expressed protein with the optimum induction concentration of IPTG, the cells are collected by centrifugation at 8000rpm and 4 ℃, washed and centrifuged repeatedly with PBS (pH7.4), blown and mixed uniformly, placed in an ultra-low temperature refrigerator for repeated freeze thawing for 3 times, and an ultrasonic crusher is set up: the ultrasonic time is 5s, the gap time is 7s, the total time is 25min, and the power is 35%. After ice-bath ultrasonication, respectively collecting supernatant and precipitate, preparing samples, performing SDS-PAGE electrophoresis, and analyzing by a gel imaging system after Coomassie brilliant blue staining and decoloring.

3. Separating, purifying and identifying the recombinant Nesfatin-1 protein: purifying target protein by His protein-tagged nickel column affinity chromatography, washing the nickel column with pure water and PBS for 6 times, respectively, injecting the crushed supernatant into the nickel column, sealing, placing in a shaking table, and incubating on ice for 1 h. The transudate was collected, and the target protein was collected using 10, 40, 80, and 100mmol/L imidazole and analyzed by SDS-PAGE. Renaturing the eluted target protein by using dialysate, replacing the dialysate every 12h at 4 ℃, and storing the purified Nesfatin-1 protein in a low-temperature refrigerator at-80 ℃ for later use. The protein concentration after purification is determined by using a BCA protein concentration determination kit.

4. Test results

As a result of the expression pattern of the fusion protein shown in FIG. 3, the fusion protein was mainly present in the supernatant and was soluble.

The result of the purification of the fusion protein shown in FIG. 4 by affinity chromatography, purified Perciformis Latifolia Nesfatin-1 protein is obtained; the protein concentration measured by BCA method is 1.05mg/mL, and the protein is preserved at-80 ℃ for use as antigen.

Example 3 preparation of polyclonal antibody of Micropterus salmoides Nesfatin-1 protein and evaluation of antibody titer and specificity

1. Test materials: SPF-grade Balb/C mice, Freund complete adjuvant, Freund incomplete adjuvant, TBST, goat anti-mouse IgG marked by HRP, PVDF membrane, TMB color developing solution, DAB color developing solution, goat anti-mouse IgG marked by FITC and DAPI color developing solution.

2. Preparation of polyclonal antibody: the purified Nesfatin-1 fusion protein is used for immunizing BALB/c mice with 6 weeks of age. Mixing the fusion protein and Tween-80 (Tween-80) in a volume ratio of 24: 1 to obtain an aqueous phase; mixing the white oil and Span-80 (Span-80) according to a volume ratio of 24: 1 to form an oil phase; mixing the water phase and the oil phase according to the volume ratio of 1: 3, adding 30 mul VAS immunopotentiator, and emulsifying into immunogen for multipoint subcutaneous injection by full shaking. Boosting the immunity for 1 time every two weeks, totally boosting the immunity for 3 times, taking blood after the last boosting immunity for one week, standing for 1h at room temperature, separating serum, subpackaging, and storing in a low-temperature refrigerator at-80 ℃.

3. And (3) evaluating the specificity:

ELISA detection of polyclonal antibody titer: diluting the purified Nesfatin-1 fusion protein to a 100ng/mL coated enzyme label plate by using a carbonate coating solution, incubating for 2h at 37 ℃, washing by using TBST after patting dry, taking the serum to be detected as a primary antibody, diluting from 1: 200 to 1: 409600, taking goat anti-mouse IgG marked by HRP as a secondary antibody, developing the color by using a Tetramethylaniline (TMB) solution, and measuring OD (optical density) by using an enzyme label analyzer₄₅₀The value is obtained. OD of detection well and negative control well₄₅₀The dilution at which the ratio of the values is not less than 2.1 is the antibody titer. And simultaneously setting the serum of the non-immunized mouse as a negative control and the PBS buffer solution as a blank control, repeating each sample for three times, and taking an average value of results.

Detecting the specificity of the polyclonal antibody by Western blot: the purified Nesfatin-1 fusion protein is sampled and transferred to a PVDF membrane (300mA constant current transfer membrane for 60min) by SDS-PAGE gel electrophoresis. After washing once with TBST, PVDF membrane was blocked with 5% skimmed milk powder at room temperature for 2h, and then washed with TBST for 5min 2 times. The antibody serum was diluted 1: 1000 and used as a primary antibody and incubated overnight at 4 ℃ in a horizontal shaker. Washing with TBST for 4 times (5 min each time), adding goat anti-mouse IgG labeled with HRP (horse radish peroxidase) diluted at 1: 1000 as a secondary antibody, incubating at room temperature for 2h, washing with TBST for 4 times (5 min each time), and developing with DAB developing solution.

c. And (3) immune tissue fluorescence detection: randomly selecting 3 micropterus salmoides, fixing the liver with 4% paraformaldehyde, performing paraffin embedding and slicing, putting the embedded slice into a constant-temperature oven at 60 ℃ for 6h, and naturally cooling for 30min to room temperature. Dewaxing by dimethylbenzene, rehydrating by gradient alcohol, putting into 0.1mol/L citric acid repair liquid with pH of 6.0, heating for repairing fully, and naturally cooling to room temperature. And (3) dripping the DPBS covering tissue into the repaired section, dripping 10% of closed serum covering tissue, and closing for 30min at room temperature. Adding anti-Nesfatin-1 serum, incubating overnight at 4 ℃, then adding a FITC labeled secondary antibody (diluting the antibody at a ratio of 1: 150) in a dark place, washing with PBS buffer solution for 3 times, adding DAPI, dyeing for 10min at room temperature, washing with PBS buffer solution for 5 times, each time for 5min, sealing, observing with a laser confocal microscope, and photographing and recording.

4. Test results

The results of ELISA detection of polyclonal antibody titer shown in Table 1 show that the ratio of the polyclonal antibody diluted 1: 204800 still reaches 6, indicating that the antibody titer is greater than 1: 204800.

TABLE 1

FIG. 5 shows the result of detecting the specificity of polyclonal antibody by Western blot, which detects that the size of single protein band is about 30kDa, which is consistent with the size of fusion protein induction result.

FIG. 6 shows the results of the localization of Nesfatin-1 protein in liver and pancreas tissues of Lateolabrax japonicus, the antibody can specifically identify the Nesfatin-1 protein in liver and pancreas of Lateolabrax japonicus, and the Nesfatin-1 protein is dispersedly distributed in liver and pancreas, and pancreatic cells around blood vessels in liver and pancreas tissues show strong positive reaction.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<120> Micropterus salmoides Nesfatin-1 protein and prokaryotic expression method and application thereof

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Claims

1. The protein of the neofatin-1 of the largemouth bass is characterized by comprising or consisting of the following amino acid sequences:

i) as shown in SEQ ID NO: 1; or

ii) an amino acid sequence obtained by connecting a tag to the N-terminal and/or C-terminal of i); or

2. A nucleic acid molecule encoding the protein of claim 1.

3. Biological material containing a nucleic acid molecule according to claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

4. A method for prokaryotic expression of a protein according to claim 1, characterized in that it comprises the following steps:

(2) primers Nesfatin-1-F and Nesfatin-1-R designed for amplification of the nucleic acid molecule according to claim 2;

(3) amplification by RT-PCR: reverse transcribing the total RNA into cDNA, and performing PCR amplification by using the primers Nesfatin-1-F and Nesfatin-1-with the cDNA as a template;

5. The method as claimed in claim 4, wherein the nucleotide sequences of the primers Nesfatin-1-F and Nesfatin-1-R in the step (2) are respectively shown in SEQ ID NO: 3 and 4.

6. The method according to claim 4, wherein the prokaryotic expression vector in step (4) is a pET series vector, preferably pET-32a (+), pET-28a, pET-30 a.

7. The method of claim 4, wherein the final concentration of IPTG in step (5) is 0.2 mmol/L.

8. The method of claim 4, wherein in step (6), the nickel affinity chromatography column is eluted by 10, 40, 80 and 100mmol/L imidazole in sequence, and the eluted target protein is renatured by dialysate;

the dialysate is: 140mmol/L sodium chloride, 2.7mmol/L potassium chloride, 10mmol/L disodium hydrogen phosphate dodecahydrate, 1.8mmol/L potassium dihydrogen phosphate, and pH 8.0.

9. The anti-micropterus salmoides Nesfatin-1 protein specific antibody is characterized by comprising a polyclonal antibody and a monoclonal antibody;

the polyclonal antibody is prepared by taking the protein as the immunogen of claim 1 and immunizing experimental animals with adjuvant;

the monoclonal antibody is prepared by taking the protein of claim 1 as an immunogen and assisting an adjuvant to immunize an experimental animal by adopting a hybridoma technology or a DNA recombination technology.

10. The use of the antibody of claim 9 in the preparation of a detection reagent or kit for the neofatin-1 protein of micropterus salmoides.