阅读说明：本技术 含有aif1细胞因子抗体的小球藻及其制备方法和应用 (Chlorella containing AIF1 cytokine antibody, and its preparation method and application ) 是由 吴信忠 蔡小辉 于 2021-08-10 设计创作，主要内容包括：本发明属于生物技术领域,涉及含有AIF1细胞因子抗体的小球藻及其制备方法和应用。本发明采用纯化出来的近江牡蛎AIF1细胞因子抗体蛋白制品标记FITC荧光标记,以pVEC穿膜肽为载体将AIF1细胞因子抗体蛋白分子导入小球藻,在室温黑暗条件下,进行AIF1细胞因子抗体孵育小球藻,从而获得含有AIF1细胞因子抗体分子物质成分的小球藻种类。用抗体分子免疫荧光标记生物学技术、激光共聚焦显微镜检测、验证小球藻体内含有牡蛎AIF1细胞因子抗体蛋白质成分及其含量。(The invention belongs to the technical field of biology, and relates to chlorella containing AIF1 cytokine antibody, and a preparation method and application thereof. The invention adopts a purified ostrea rivularis AIF1 cytokine antibody protein product to mark FITC fluorescent marker, introduces AIF1 cytokine antibody protein molecules into chlorella by taking pVEC transmembrane peptide as a carrier, and incubates the chlorella with AIF1 cytokine antibody under the dark condition at room temperature, thereby obtaining the chlorella species containing AIF1 cytokine antibody molecular substance components. Detecting and verifying protein components and contents of oyster AIF1 cytokine antibody in chlorella by using antibody molecular immunofluorescence labeling biology technology and laser confocal microscope.)

1. Chlorella containing AIF1 cytokine antibody is provided.

2. The method for producing chlorella containing AIF1 cytokine antibody as claimed in claim 1, wherein purified crassostrea rivularis AIF1 cytokine antibody protein preparation is labeled with FITC fluorescent label, AIF1 cytokine antibody protein molecule is introduced into chlorella using pVEC cell-penetrating peptide as carrier, and the chlorella is incubated with AIF1 cytokine antibody under dark condition at room temperature to obtain chlorella containing AIF1 cytokine antibody.

3. The method of claim 2, wherein: the preparation of the AIF1 cytokine antibody comprises the following steps:

1) taking total RNA of crassostrea rivularis blood lymphocytes as a template, carrying out reverse transcription to obtain a chain cDNA, and cloning to obtain a coding frame nucleotide sequence of an AIF1 gene;

2) cloning the sequence fragment of the coding frame of the AIF1 gene into a pMD19-T vector, carrying out restriction enzyme digestion, and connecting the sequence fragment with a pET-32(a) prokaryotic expression vector subjected to the same enzyme digestion to obtain a recombinant expression plasmid pET-32(a) -AIF 1;

3) positive recombinant plasmid PET-32(a) -AIF1 is transformed into expression host bacterium E.coli Rossetta (DE3), isopropyl thiogalactoside (IPTG) is induced to express, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used for detection;

4) carrying out amplification culture on the positive expression bacterial liquid, and separating and purifying the recombinant protein by using a protein purification kit;

5) the purified AIF1 protein was injected into New Zealand white rabbits at multiple sites to prepare purified polyclonal antibodies.

4. The method of claim 2, wherein: the method for introducing the AIF1 cytokine antibody protein molecule into chlorella comprises the following steps:

1) AIF1 cytokine antibody purification and green fluorescence labeling

Purifying with Protein A Sepharose affinity chromatography column to obtain antiserum total IgG; labeling purified AIF1 polyclonal antibody with NHS-Fluorescein (Pierce, USA);

2) chlorella culture and growth curve drawing

Subculturing chlorella by adopting f/2 culture solution, and carrying out subculturing on sterile chlorella according to the ratio of 1: inoculating 10 proportion of the mixture into a fresh culture medium, and culturing under the following conditions: the temperature is 23 ℃, the illumination intensity is 3000lx, the light-dark period ratio is 12 h: and (4) 12 h. Sampling algae under aseptic condition every 24h, measuring light absorption value at 660nm on an enzyme labeling instrument, drawing a chlorella growth curve, and executing 3 biological repetitions at each time point;

3) chlorella into which AIF1 cytokine antibody is introduced

Chlorella in logarithmic growth phase was collected and centrifuged (400 Xg, 10min), using sterile PBS

Washing Chlorella for 3 times, and adjusting concentration to 10 by counting under microscope using cell counting plate⁶cell/mL, centrifuging and removing supernatant; 100 μ l of AIF1 antibody at a final concentration of 100 μ g/ml was mixed with pVEC at a final concentration of 20 μ M and added to the algal pellet to give a concentration of 10⁶cell/mL; incubating in a dark room at room temperature, respectively incubating for 15min, sampling and placing on a glass slide at 30min, observing and photographing 50% glycerol sealing sheets under a confocal microscope, and setting 3 biological replicates for each experiment.

5. The production method according to any one of claims 2 to 4, wherein: detecting and verifying protein components and contents of oyster AIF1 cytokine antibody in chlorella by laser confocal microscope.

6. Use of the chlorella containing AIF1 cytokine antibody as claimed in claim 1 for preparing immune feed against diseases of fishes and shrimps by infection control.

7. Use according to claim 6, for the preparation of an immunological bait against gram-negative bacteria and rickettsia-like organism (RLO) infections.

8. A bait which is characterized in that: a chlorella containing the AIF1 cytokine antibody as set forth in claim 1.

Technical Field

The invention belongs to the technical field of biology, and relates to chlorella containing AIF1 cytokine antibody, and a preparation method and application thereof.

Background

The blue marine industry is an important source for solving the contradiction between the excess population and the shortage of land resources in the present human, but with the development of the mariculture industry (including shellfish, shrimp and fish culture industry), the outbreak and the popularity of mariculture diseases greatly attack the development of the mariculture industry, and become the bottleneck restriction factor of the development of the industry. Therefore, there is an urgent need to solve the following two significant problems: namely prevention and treatment (or control) of diseases, and the key for solving the problems is as follows: the method is used for solving the problem of rapid detection of epidemic diseases and realizing early diagnosis before outbreak of the diseases; secondly, the problem of the self resistance of the cultured animals is solved, namely, pathogenic infection is avoided, and the mass death of cultured animal groups is reduced. The international application of molecular biology techniques (including molecular immunology) to the prevention and treatment of mariculture animal diseases is the leading development direction of the mariculture disease research at home and abroad at present, and the techniques comprise various molecular and immune rapid detection techniques, various molecular vaccine techniques, various cell or immune factors, genetic engineering techniques and the like. However, at present, no technology and product for preparing disease-resistant immune bait and antibacterial microecological immune preparation in the production of shellfish, shrimp and fish culture industries by adopting high-titer cytokine antibody technology are available at home and abroad.

Our previous studies of AIF1 against the gram-negative bacterial component LPS and Rickettsial (RLO) infection showed that: oyster AIF1(Ca-AIF1) polyclonal rabbit antiserum can obviously inhibit the expression of inflammation-related factor LITAF caused by the stimulation of rickettsia-like organism (RLO) and LPS (LPS + anti-Ca-AIF1 is reduced by 70.95%, 84.18%, 39.49% and 87.53% in 1.5-12 hours, respectively), and RLO + anti-Ca-AIF1 is reduced by 88.57%, 84.80%, 59.36% and 59.16% in 1.5-12 hours, respectively), so that the Ca-AIF1 polyclonal antiserum has the effect of effectively inhibiting the inflammatory response caused by RLO infection and gram-negative bacteria. For another example, the Ca-AIF1 antiserum can obviously inhibit RLO infection and cell apoptosis and cell necrosis caused by gram-negative bacteria and obviously increase the cell survival rate, and the number of the living cells of the RLO + anti-Ca-AIF1 experimental group and the number of the living cells of the LPS + anti-Ca-AIF1 experimental group are respectively increased by 51 percent and 49 percent; while the late apoptotic cell rate decreased by about 50% and 18%, respectively.

Disclosure of Invention

In view of the deficiencies in the prior art, the invention provides an antibody acclimatized and cultured chlorella containing AIF1 cytokine and a preparation method thereof.

Chlorella containing AIF1 cytokine antibody is provided.

A preparation method of chlorella containing AIF1 cytokine antibody comprises adopting purified Crassostrea gigas AIF1 cytokine antibody protein product to label FITC fluorescent label, introducing AIF1 cytokine antibody protein molecule into chlorella by taking pVEC transmembrane peptide as carrier, and incubating chlorella with AIF1 cytokine antibody under dark condition at room temperature to obtain chlorella containing AIF1 cytokine antibody.

Wherein, the preparation of the AIF1 cytokine antibody comprises the following steps:

1) taking total RNA of crassostrea rivularis blood lymphocytes as a template, carrying out reverse transcription to obtain a chain cDNA, and cloning to obtain a coding frame nucleotide sequence of an AIF1 gene;

2) cloning the sequence fragment of the coding frame of the AIF1 gene into a pMD19-T vector, carrying out restriction enzyme digestion, and connecting the sequence fragment with a pET-32(a) prokaryotic expression vector subjected to the same enzyme digestion to obtain a recombinant expression plasmid pET-32(a) -AIF 1;

3) positive recombinant plasmid PET-32(a) -AIF1 is transformed into expression host bacterium E.coli Rossetta (DE3), isopropyl thiogalactoside (IPTG) is induced to express, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used for detection;

4) carrying out amplification culture on the positive expression bacterial liquid, and separating and purifying the recombinant protein by using a protein purification kit;

5) the purified AIF1 protein was injected into New Zealand white rabbits at multiple sites to prepare purified polyclonal antibodies.

The method for introducing the AIF1 cytokine antibody protein molecule into chlorella comprises the following steps:

purification and green fluorescence labeling of AIF1 cytokine antibody

1.1AIF1 cytokine polyclonal antibody purification

The antiserum was purified to total IgG using Protein a Sepharose affinity column (solibao, china) according to the instructions. The specific method comprises the following steps: binding buffer (0.15M NaCl, 20mM Na) was used₂HPO₄pH 7.0), loading onto a chromatographic column, washing with about 10 column volumes of Binding buffer (pH 7.0) to remove impurities, desorbing with elution buffer (100mmol/L glycine, pH3.0) and collecting the protein, neutralizing the collected protein with 1.0mol/L Tris-HCl (pH 8.0) immediately, and storing at-20 ℃.

1.2 fluorescent labeling of purified AIF1 cytokine polyclonal antibody

The purified AIF1 polyclonal antibody was labeled with NHS-Fluorescein (Pierce, USA) as follows: 1ml of purified AIF1 polyclonal antibody with a concentration of 7.88mg/ml was transferred to a 1.5ml sterile centrifuge tube, and 37. mu.l of NHS-Fluorescein fluorescent solution with a concentration of 10mg/ml was added and reacted at 4 ℃ for 24 hours. After the reaction, the reaction product was put into a dialysis bag (Viskease, USA) and dialyzed in 1L PBS (4 degrees), unbound fluorescein was removed, the dialysate was changed for 3 times, and the coupling product was taken out after dialysis was completed (the dialysate outside the dialysis solution was a transparent colorless solution). After dilution by 5 times, absorbance values of A280 and A493 are respectively measured to detect the concentration of the labeled protein and the fluorescence labeling rate.

2. Chlorella culture and growth curve drawing

The chlorella used in the experiment is given as a gift by Guangxi Zhuang autonomous region Marine research institute, and f/2 culture solution (225mg/L NaNO3, 5mg/L KH2PO4, 100mg/L NaHCO3, 200 μ g/L vitamin B) is adopted₁₂200 ug/L vitamin B₁And,) subculturing, and mixing sterile chlorella according to the ratio of 1: inoculating 10 proportion of the mixture into a fresh culture medium, and culturing under the following conditions: the temperature is 23 ℃, the illumination intensity is 3000lx, the light-dark period ratio is 12 h: and (4) 12 h. Sampling algae at intervals of 24h under aseptic condition, measuring absorbance at 660nm on enzyme labeling instrument, and drawing chlorella growth curveLine, 3 biological replicates were performed per time point.

Introduction of AIF1 cytokine antibody into Chlorella

Chlorella in logarithmic growth phase was collected and centrifuged (400 Xg, 10min), and the chlorella was washed 3 times with sterile PBS and the concentration was adjusted to 10 by counting under a microscope using a cell counting plate⁶cell/mL, and centrifuging to remove the supernatant. 100 μ l of AIF1 antibody at a final concentration of 100 μ g/ml was mixed with pVEC at a final concentration of 20 μ M and added to the algal pellet to give a concentration of 10⁶cell/mL. Incubating in a dark room at room temperature, respectively incubating for 15min, sampling and placing on a glass slide at 30min, observing and photographing 50% glycerol sealing sheets under a confocal microscope, and setting 3 biological replicates for each experiment.

Further, the protein components and the content of oyster AIF1 cytokine antibody in chlorella are detected and verified by a laser confocal microscope.

In addition, the invention also provides application of the chlorella containing AIF1 cytokine antibody in preparing immune disease-resistant bait for preventing and treating shellfish and fish and shrimp diseases.

The application is to prepare immune feed for resisting gram-negative bacteria and rickettsia-like organism (RLO) infection.

The invention also provides bait, which comprises chlorella containing AIF1 cytokine antibody.

The invention adopts a purified ostrea rivularis AIF1 cytokine antibody protein product to mark FITC fluorescent marker, introduces AIF1 cytokine antibody protein molecules into chlorella (remark: the chlorella is a main alga for breeding oyster larvae and fish and shrimp baits) by taking pVEC transmembrane peptide as a carrier, and incubates the chlorella with AIF1 cytokine antibody under the dark condition at room temperature, thereby obtaining the chlorella species containing AIF1 cytokine antibody molecular substance components. Detecting and verifying protein components and contents of oyster AIF1 cytokine antibody in chlorella by using antibody molecular immunofluorescence labeling biology technology and laser confocal microscope. Has the following characteristics:

1. the oyster proinflammatory cytokine AIF1 protein is prepared and purified by adopting a gene cloning technology.

2. AIF1 antibody protein molecules (or preparations) against gram-negative bacteria and Rickettsial (RLO) disease were prepared and purified.

3. The research of introducing AIF1 cytokine antibody molecules into chlorella and the domestication and mixed culture of the chlorella are carried out by adopting the purified antibody protein molecules (or products) of the crassostrea rivularis AIF1 cytokine.

4. Detecting and verifying protein components and contents of a crassostrea rivularis AIF1 cytokine antibody in chlorella by adopting a molecular biology technology (comprising a molecular immunology technology, a protein separation and purification technology, an antibody molecular immunofluorescence labeling biology technology, a laser confocal microscope and the like).

The invention obtains a technology for introducing AIF1 cytokine antibody protein molecules into chlorella, contains oyster AIF1 cytokine antibody domesticated and cultured chlorella, obtains the chlorella domesticated and cultured by crassostrea rivularis AIF1 cytokine antibody and is used for preparing immune bait for resisting gram negative bacteria and rickettsia-like organism (RLO) infection, and realizes important technical innovation on disease delivery administration modes.

Drawings

Figure 1AIF1 antibody titer assay, wherein 1A: ELISA method, 1B: western Blot.

FIG.2 confocal assay of the introduction of AIF1 cytokine into Chlorella vulgaris, wherein 2A: FITC-labeled AIF antibody; 2B: pVEC incubated FITC antibody for 15 min; 2C: pVEC incubated FITC antibody for 30 min.

FIG. 3 is a schematic diagram of the mechanism of the cell-penetrating peptide pVEC for mediating the entry of AIF1 antibody into Chlorella vulgaris.

Detailed Description

Firstly, preparing and purifying oyster proinflammatory cytokine AIF1 protein and preparation of cytokine antibody thereof by adopting gene cloning technology

1. Taking total RNA of crassostrea rivularis blood lymphocytes as a template, carrying out reverse transcription to obtain a chain cDNA, and cloning to obtain a coding frame nucleotide sequence of the AIF1 gene.

2. The sequence fragment of the coding frame of the AIF1 gene was cloned into pMD19-T vector (Takara, Japan), digested with restriction enzymes, and ligated with pET-32(a) prokaryotic expression vector (Novagen, USA) digested with the same enzymes, to obtain recombinant expression plasmid pET-32(a) -AIF 1.

3. The positive recombinant plasmid PET-32(a) -AIF1 was transformed into expression host bacterium E.coli Rossetta (DE3) (all-purpose gold, China), isopropyl thiogalactoside (IPTG) (Sangon, Canada) for inducible expression, and detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

4. And (3) carrying out amplification culture on the positive expression bacterial liquid, and separating and purifying the recombinant protein by using a protein purification kit.

5. The purified AIF1 protein was injected into New Zealand white rabbits at multiple sites to prepare purified polyclonal antibodies.

The specific operation steps are as follows:

1. crassostrea rivularis blood lymphocyte RNA extraction and cDNA one-strand amplification

Extracting total RNA of Concha Ostreae blood lymphocyte, and usingOne-Step gDNA Removal and cDNA Synthesis SuperMix (all-type gold, China) were reverse transcribed into cDNA single strand.

2. Obtaining full-length sequence of crassostrea rivularis AIF1 coding frame nucleotide

Specific primers (AIF 1F: CGC) were designed based on the AIF1(HM749971.1) gene sequence in NCBI databaseGGATCCATGTCGGTGGACTTCAAAG，AIF1R：CCGCTCGAGTGGCAGATCGGAGAGGGATTTC, underlinedThe restriction sites are respectivelyBamHI and XhoI), using Hi-Fi enzyme(s) ((II)HS DNA Polymerase, TaKaRa, japan) clone the full-length sequence of the coding frame nucleotides of AIF 1. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, 35 cycles (denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s), and extension at 72 ℃ for 10 min. PCR amplification was performed with the PMD-19T vector, E.coli DH 5. alpha. (Takara, Japan) was transformed, and several clones were selected for PCR identification and further sequencing identification.

3. Construction of PET-32(a) -AIF1 expression vector

The positive clones identified in step 2 were amplified to extract plasmids, which were digested with BamHI and XhoI restriction enzymes (Takara, Japan) and ligated to the corresponding sites of the pET-32a vector digested with the same restriction enzymes, and transformed into E.coli DH5 α (Tiangen, China). Positive clones were PCR-screened and sequenced to obtain the expression vector pET-32(a) -AIF1 with correct coding frame.

4. Expression of recombinant proteins

Amplifying and culturing the positive clone identified in the step 3 to extract a recombinant plasmid pET-32(a) -AIF1, transforming the recombinant plasmid into expression host bacterium E.coli Rosseta (DE3) competence, and screening positive clones by using colony PCR. The positive bacteria are enlarged and cultured to OD according to the ratio of 1:100₆₀₀When the concentration is 0.4-0.5, adding IPTG with the final concentration of 1mmol/L, continuing to culture for 4 hours, and centrifuging to collect host bacteria. The bacteria were sonicated (300W, 20min, 2s sonication, 3s intervals) and the supernatant and pellet were centrifuged and subjected to SDS-PAGE.

5. Purification and quantification of recombinant proteins

And (3) carrying out amplification culture on the positive expression bacteria, carrying out ultrasonic treatment according to the step 4, separating and purifying an expression product by using a Ni-NTA affinity chromatography column (full-scale gold, China), and carrying out electrophoretic identification by adopting 12% SDS-PAGE.

The purified protein was quantified using a Bradford protein quantification kit (Tiangen Biochemical technology Co., Ltd., China).

6. preparation of pET32a-AIF1 polyclonal Rabbit antiserum

After protein quantification, a proper amount of protein is taken, 2 healthy New Zealand white rabbits (male rabbits) weighing about 2kg are selected for polyclonal antibody preparation, purified pET32a-AIF1 is used as an antigen, an adjuvant with the same volume is added into the antigen for full emulsification, and then the rabbits are immunized by adopting multi-point back subcutaneous injection for four times.

(1) Before immunization, 3-5ml of normal serum was collected from the ear vein as a negative control in the detection of antibodies.

(2) Shearing off rabbit hair on the back of the rabbit with scissors, and performing subcutaneous multipoint injection on the back after alcohol disinfection.

(3) Primary immunization: taking about 500 mu g of antigen, adding equal volume of complete Freund's adjuvant, fully emulsifying and injecting, selecting 8-10 points on the back, and injecting about 0.1ml of antigen per point;

(4) and (3) second immunization: after the interval of 14 days, the antigen amount is 500 mug, and the same volume of incomplete Freund's adjuvant is added for full emulsification and injection, and the method is the same as the above;

(5) third and fourth immunizations: after the interval of 14 days, the same method is carried out, after the fourth immunization, 1ml of blood is collected from the ear vein, serum is separated, the antibody titer of the immune serum is detected by a two-phase agar diffusion method, and the blood can be discharged only when the titer reaches more than 1: 16;

(6) separating serum: and 7d after the fourth immunization, collecting blood from carotid artery and separating serum after the antibody titer meets the requirement. Subpackaging and storing at-80 deg.C.

7. AIF1 cytokine polyclonal antibody titer assay

7.1ELISA (enzyme-linked immunosorbent assay) method for detection

The method for measuring the specific antibody titer of AIF1 in the immune rabbit serum by using an indirect ELISA method comprises the following specific steps:

(1) antigen coating: diluting the antigen to 1-10 mu g/ml by using a coating buffer solution, adding the diluted antigen into an ELISA plate according to 100 mu l/hole, covering a membrane, and standing at 4 ℃ overnight.

(2) Washing: taking out the enzyme label plate the next day, pouring out the liquid in the hole, and placing the plate on paper for drying; TTBS was added at 300. mu.l/well, left at room temperature for 3min, shaken dry, and washed 3 times in total.

(3) And (3) sealing: add blocking solution 200. mu.l/well to the ELISA plate, cover the membrane, and block at 37 ℃ for 1 h.

(4) And (3) storage: pouring off the sealing liquid without beating, putting the sealed enzyme label plate into a self-sealing bag, and storing at-20 ℃ for later use. (note: making antigen marks on the ELISA plate; holding the ELISA plate with gloves.)

(5) Primary antibody incubation: diluting primary antibody with blocking solution, setting blank control (blocking solution) and negative control (non-immune blood supernatant) at a dilution gradient of 1:1000, 1:3000,1:9000,1:27000,1:81000 and 1:243000, and mixing the two solutions in the gradient dilution process; after dilution, 100. mu.l of primary anti-dilution solution is added into the ELISA plate, and incubation is carried out for 1h at 37 ℃.

(6) After incubation, the primary antibody was removed, and TTBS was added thereto at 300. mu.l/well, and the mixture was allowed to stand at room temperature for 3min and washed 3 times in total.

(7) And (3) secondary antibody incubation: the secondary antibody (1: 1000) was diluted with blocking solution, and after washing, 100. mu.l/well of the secondary antibody dilution was added and incubated at 37 ℃ for 1 h.

(8) After the incubation, the cells were washed with TTBS at a concentration of 300. mu.l/well for 3min each time, and then rinsed dry for 4 times.

(9) Washing, placing the enzyme label plate in a dark room, adding 150 mu l/hole TMB, adding 50 mu l/hole 2M H after gradient blue appears₂SO4, terminating the reaction. (Note: it is convenient to observe the color change, a piece of white paper can be arranged under the enzyme label plate, the sequence of adding TMB and adding H should be added₂SO4, in consistent order. )

(10) And (4) placing the ELISA plate in an ELISA reader to detect the absorbance of 450 nm.

(11) And (5) storing data, photographing the ELISA plate and data printing paper, and recording.

7.2Western Blot (immunoblot) method of detection

The antibody titer was determined by diluting AIF1 polyclonal rabbit antiserum at a certain ratio, according to the method of Sambrook et al (Sambrook J, Russell D W. molecular Cloning: A Laboratory Manual,3rd ed. New York: Cold Spring Harbor Laboratory Press,2001), as follows:

(1) SDS-PAGE electrophoresis: starting 90V electrophoresis, and adjusting the voltage to 150V after running under the concentrated gel until the electrophoresis is finished. (Note: Point Predyeing Marker)

(2) Film shearing: and (3) cutting the PVDF membrane with the same size as the glue, marking the hole order and the Marker position, activating for 1-2min by using methanol, then putting the PVDF membrane into a membrane transfer Buffer, and putting two pieces of filter paper and a transfer clamp into the Buffer together. (Note: Buffer box closed to avoid methanol volatilization)

(3) Film transfer: after the electrophoresis is finished, the gel is carefully taken down, the agarose gel and the concentrated gel are cut off, then the gel is placed in a Buffer, a transfer clamp is opened (the black surface faces downwards), a piece of wet filter paper is placed firstly, the gel is placed on the filter paper, then the membrane is covered on the gel, then another piece of filter paper is covered, and finally the transfer clamp is covered and placed in the Buffer for clamping. The transfer tank is installed, and electrophoresis is carried out in ice-water bath for 2 hours under a constant current of 180 mA.

(4) And (3) sealing: pouring a part of prepared confining liquid into the plastic box, taking out the PVDF membrane after electrophoresis, activating with methanol for 1-2min, drying, placing the membrane into the plastic box, and sealing the plastic box on a shaking table at 37 ℃ for 1 h.

(5) And (3) incubation: taking 1ml of the sealing solution by using a 1.5ml centrifuge tube, adding corresponding primary antibody and secondary antibody (generally 1 mu l of each primary antibody and secondary antibody, specifically according to the antibody titer), fully and uniformly mixing, placing the membrane in a plastic bag, adding the antibody diluent, sealing, and placing on a shaking bed for incubation at 37 ℃ for 1 h. (Note: PVDF membrane is in a wet state as always as possible)

(6) Washing: after incubation, the membranes were put into a plastic box and an appropriate amount of TTBS solution (submerged membrane) was added and washed on a shaker for 3 times, 10min each.

(7) Gel imaging system exposure: preparing color developing solution, wherein each film needs 200 mul of solution A and solution B (used in preparation), sucking water of the PVDF film, then uniformly adding the color developing solution on the PVDF film, putting the PVDF film into an imaging instrument, and setting exposure time to develop strips.

Secondly, the AIF1 cytokine antibody protein molecule is introduced into the chlorella

Purification and green fluorescence labeling of AIF1 cytokine antibody

1.1AIF1 cytokine polyclonal antibody purification

The total IgG of the antiserum was purified by Protein A Sepharose affinity column (Solebao, China) according to the protocol. The specific method comprises the following steps: binding buffer (0.15M NaCl, 20mM Na) was used₂HPO₄pH 7.0), loading onto a chromatographic column, washing with about 10 column volumes of Binding buffer (pH 7.0) to remove impurities, desorbing with elution buffer (100mmol/L glycine, pH3.0) and collecting the protein, neutralizing the collected protein with 1.0mol/L Tris-HCl (pH 8.0) immediately, and storing at-20 ℃.

1.2 fluorescent labeling of purified AIF1 cytokine polyclonal antibody

The purified AIF1 polyclonal antibody was labeled with NHS-Fluorescein (Pierce, USA) as follows: 1ml of purified AIF1 polyclonal antibody with a concentration of 7.88mg/ml was transferred to a 1.5ml sterile centrifuge tube, and 37. mu.l of NHS-Fluorescein fluorescent solution with a concentration of 10mg/ml was added and reacted at 4 ℃ for 24 hours. After the reaction, the reaction product was put into a dialysis bag (Viskease, USA) and dialyzed in 1L PBS (4 degrees), unbound fluorescein was removed, the dialysate was changed for 3 times, and the coupling product was taken out after dialysis was completed (the dialysate outside the dialysis solution was a transparent colorless solution). After dilution by 5 times, absorbance values of A280 and A493 are respectively measured to detect the concentration of the labeled protein and the fluorescence labeling rate.

2. Chlorella culture and growth curve drawing

The chlorella used in the experiment is given as a gift by Guangxi Zhuang autonomous region Marine research institute, and f/2 culture solution (225mg/L NaNO3, 5mg/L KH2PO4, 100mg/L NaHCO3, 200 μ g/L vitamin B) is adopted₁₂200 ug/L vitamin B₁And,) subculturing, and mixing sterile chlorella according to the ratio of 1: inoculating 10 proportion of the mixture into a fresh culture medium, and culturing under the following conditions: the temperature is 23 ℃, the illumination intensity is 3000lx, the light-dark period ratio is 12 h: and (4) 12 h. And (3) taking algae samples under the aseptic condition every 24h, measuring the light absorption value at 660nm on an enzyme labeling instrument, drawing a chlorella growth curve, and performing 3 biological repetitions at each time point.

Introduction of AIF1 cytokine antibody into Chlorella

Chlorella in logarithmic growth phase was collected and centrifuged (400 Xg, 10min), and the chlorella was washed 3 times with sterile PBS and the concentration was adjusted to 10 by counting under a microscope using a cell counting plate⁶cell/mL, and centrifuging to remove the supernatant. 100 μ l of AIF1 antibody at a final concentration of 100 μ g/ml was mixed with pVEC at a final concentration of 20 μ M and added to the algal pellet to give a concentration of 10⁶cell/mL. Incubating in a dark room at room temperature, respectively incubating for 15min, sampling and placing on a glass slide at 30min, observing and photographing 50% glycerol sealing sheets under a confocal microscope, and setting 3 biological replicates for each experiment.

The method comprises the following specific steps:

1.AIF1 cytokine polyclonal antibody purification

The AIF1 polyclonal antibody was purified using Protein A Sepharose affinity column. The specific method comprises the following steps: protein A Sepharose was subjected to column packing using Binding buffer(0.15M NaCl，20mM Na₂HPO₄pH 7.0), the prepared rabbit anti-AIF 1 polyclonal antibody serum was adjusted to pH 7.0, loaded onto a Protein A Sepharose affinity chromatography column, washed with 10 column volumes of Binding buffer (pH 7.0), and finally eluted with 100mmol/L glycine (pH3.0) and collected in a 1.5ml centrifuge tube, and the collected Protein was immediately neutralized with 1.0mol/L Tris-HCl (pH 8.0) and stored at-20 ℃.

2. Purified AIF1 cytokine antibody titer assay

The prepared rabbit anti-AIF 1 polyclonal antibody titer is higher than 1:243000 and is detected by ELISA and Western methods (figure 1).

3.AIF1 cytokine polyclonal antibody fluorescent label

1ml of purified AIF1 polyclonal antibody with the concentration of 7.88mg/ml is transferred to a 1.5ml sterile centrifuge tube, 37. mu.l of NHS-Fluorescein fluorescent solution with the concentration of 10mg/ml is added, and the reaction is carried out for 24h at 4 ℃. After the reaction, the reaction product was put into a dialysis bag (Viskease, USA) and dialyzed in 1L PBS (4 degrees), unbound fluorescein was removed, the dialysate was changed for 3 times, and the coupling product was taken out after dialysis was completed (the dialysate outside the dialysis solution was a transparent colorless solution). After 5-fold dilution, A280-1.066 and A493-0.715 were determined, where 493nm is the specific absorption peak of FITC and the molar extinction coefficient of FITC is 70000M^-1cm^-1The molar concentration of FITC in the product was calculated to be 5.11X 10^-5M; furthermore, FITC also absorbs at 280nm, and the absorbance is 30% at 493nm, so that the absorbance of the AIF1 antibody is (1.066-0.715 × 0.3) × 5 ═ 4.26, and the molar extinction coefficient of the antibody is 210000M^-1cm^-1And calculating to obtain the product with the antibody molar concentration of 2.03 multiplied by 10^-5M, the mass concentration of the antibody is 3.04mg/ml, the FITC labeling rate is FITC: antibody 2.52: 1.

4. introduction of fluorescent-labeled AIF1 cytokine polyclonal antibody into chlorella

Chlorella in logarithmic growth phase was collected and centrifuged (400g, 10min), and the chlorella was washed 3 times with sterile PBS and adjusted to 10 concentration using a cell counting plate⁶cell/ml. Transferring 100. mu.L of the algae solution to a 1.5mL sterile centrifuge tube, centrifuging at room temperature 400g to 1And (3) removing supernatant and collecting precipitate after 0 min. 100 μ L PBS was taken into a 1.5mL centrifuge tube, and FITC-labeled AIF1 antibody and pVEC cell-penetrating peptide were added to give final concentrations of 100 μ g/mL and 20 μ M, respectively. The mixture is added to the algal precipitate to suspend the algae. Incubating in a dark room at room temperature, sampling and placing on a glass slide after incubating for 15min and 30min respectively, and observing and photographing a 50% glycerol seal under a confocal microscope. As can be seen from fig.2, when the fluorescence-labeled AIF1 antibody was incubated with chlorella for 15min, only individual chlorella (autofluorescence) contained green fluorescence (FITC-labeled AIF1 antibody) (fig.2 a). When the incubation time reaches 30min, a large amount of green fluorescence (FIG.2B) in chlorella can be seen. It was shown that the polyclonal antibody to AIF1 was successfully introduced into Chlorella vulgaris. FIG. 3 is a schematic diagram of the mechanism of cell-penetrating peptide pVEC for mediating the entry of AIF1 antibody into Chlorella vulgaris.

Finally, it should also be noted that the above list is only a few specific embodiment frameworks of the present invention. It is obvious that the present invention is not limited to the above embodiments, and all modifications directly derivable or suggested to a person skilled in the art from the present disclosure should be considered as within the scope of protection of the present invention.

Sequence listing

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