CN113679827A

CN113679827A - Construction method of mstn self-gene vaccine based on epinephelus coioides

Info

Publication number: CN113679827A
Application number: CN202110879530.8A
Authority: CN
Inventors: 杨慧荣; 夏俊; 杨炎; 杨金增
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2021-11-23

Abstract

The invention relates to a construction method of mstn self-gene vaccine based on Epinephelus coioides, which comprises the following steps: designing exogenous genes, carrying out enzyme digestion, ligation reaction and transformation to obtain positive strains; and (3) carrying out mass culture and purification on plasmids, concentrating the plasmids, adjusting the concentrations to be 500 ng/mu L, 1000 ng/mu L and 2000 ng/mu L respectively, and then storing at-80 ℃ to obtain the mstn autologous nucleic acid vaccine.

Description

Construction method of mstn self-gene vaccine based on epinephelus coioides

Technical Field

The invention relates to the field of genetic engineering, in particular to a construction method of an mstn self-gene vaccine based on epinephelus coioides.

Background

Myostatin (mstn) is a negative regulator gene that inhibits the proliferation and growth of muscle cells, and belongs to the TGF- β family of members, and is also known as gdf-8. It has been found that inhibition or mutation inactivation of the gene expression can block the inhibitory effect of Mstn on the proliferation and growth of muscle cells, cause extensive muscle proliferation and significantly reduce body surface fat deposition, and produce a phenomenon of muscle hypertrophy characterized by a significant increase in the number of muscle fibers and a significant increase in body weight, the so-called "double muscle trait".

The Mstn genetic engineering vaccine is prepared and used for immunization, and then an anti-Mstn antibody is produced for a long time by utilizing an own immune system, and the antibody completely has the capacity of specially binding and inhibiting Mstn, so that the inhibition of the Mstn on the muscle cell growth can be relieved. At present, the method for active immunization by using mstn mainly focuses on animal husbandry such as cattle, sheep, pigs and the like, and there are few reports of mstn in the aquatic product field, especially in the breeding of epinephelus coioides.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a construction method of an mstn self-gene vaccine based on epinephelus coioides.

In order to achieve the purpose, the invention adopts a technical scheme that: a construction method of mstn self-gene vaccine based on Epinephelus coioides is characterized by comprising the following steps:

s1 exogenous gene design: the exogenous gene is 'KOZAK sequence-encephalitis B virus signal peptide-hepatitis B virus surface core antigen-SS-SS-Th epitope-Linker connecting sequence-epinephelus coioides mstn mature peptide gene sequence', and is synthesized and cloned to T load;

s2 enzyme digestion: carrying out double enzyme digestion on the recombinant plasmid in S1 and the pcDNA3.1+ vector at 37 ℃ by using TAKARA Nhe I enzyme and Xho I enzyme respectively, and then carrying out fragment recovery by using a DNA fragment purification kit;

s3 ligation: connecting the vector fragment in the S2 with the exogenous gene fragment by T4 DNA ligase, and connecting the reaction system at 16 ℃ for 8 h;

s4 transformation: taking JM109 competent cells out of an ultralow temperature refrigerator at minus 80 ℃, placing the JM109 competent cells on ice for dissolving, adding successive products in S3, adding an LB liquid culture medium without resistance, culturing in a biochemical box, and performing PCR (polymerase chain reaction) verification to obtain a positive strain;

mass culture and purification of S5 plasmid: culturing the positive strain obtained in S5 in LB liquid culture medium containing corresponding resistance, obtaining plasmid solution under the action of lysozyme, and adding the plasmid solution into a purifier to obtain purified plasmid solution;

concentration of S6 plasmid: the plasmid concentration obtained in S5 is 500 ng/. mu.L, after the plasmid concentration, the concentration is respectively adjusted to 500 ng/. mu.L, 1000 ng/. mu.L, 2000 ng/. mu.L, then the plasmid is preserved at-80 ℃, and the mstn self-nucleic acid vaccine is obtained.

In a preferred embodiment of the present invention, the recombinant plasmid and vector double digestion system of the foreign gene further comprises: 1.0 μ L of Nhe I, 1.0 μ L of Xho I, less than or equal to 2.0 μ g gene/plasmid, 2.0 μ L of 10 XBuffer, no more than 20.0 μ L of ddH₂0, 20.0 μ L Total Volume. In a preferred embodiment of the present invention, the method for recovering the fragments using the DNA fragment purification kit in S2 further comprises the steps of:

s201, adding 3 times volume of Buffer DC into PCR reaction solution, and then uniformly mixing;

s202, arranging Spin columns in the kit on a Collection Tube;

s203, transferring the solution of the S201 into a Spin Column, centrifuging for 1min at 12000rpm and room temperature, and removing the filtrate;

s204, adding 700 mu L of Buffer WB into Spin Column, centrifuging at 12000rpm at room temperature for 30S, and removing the filtrate;

s205 repeats operation S204;

s206, placing Spin Column on the Collection Tube, and centrifuging for 1min at 12000rpm and room temperature;

s207, taking a new 1.5mL centrifuge tube, arranging Spin Column on the centrifuge tube, adding about 30 mu L of precipitation Buffer at the center of the Spin Column membrane, and standing for 1min at room temperature;

the DNA was eluted by centrifugation at S20812000 rpm for 1min at room temperature.

In a preferred embodiment of the present invention, the reaction system of the successive reactions in S3 is as follows: 6.0. mu.L of exogenous gene fragment, 6.0. mu.L of vector fragment, 1.0. mu. L T4 DNA ligase, 1.0. mu.L of 10 XT 4 DNA Ligation Buffer, and 10.0. mu.L of Total Volume.

In a preferred embodiment of the present invention, the transformation process of S4 further comprises the following steps:

s401 taking out JM109 competent cells from an ultra-low temperature refrigerator at-80 ℃, dissolving the cells on ice, adding all the 10 mu L of the ligation products, and blowing and beating the cells for several times by using a pipette;

s402, incubating on ice for about 45min, transferring to a 42 ℃ water bath kettle, thermally shocking for 90S, immediately stopping taking out, and standing on ice for 10 min;

s403, adding 400 mu L of LB liquid culture medium without resistance, and carrying out shake culture at 37 ℃ and 200rpm for 60 min;

s4044000 rpm, centrifuging for 5min, taking out, discarding the upper layer liquid, leaving about 100 μ L of liquid, and blowing uniformly by using a pipette;

s405, uniformly dripping 100 mu L of liquid onto a flat plate containing resistance, coating and dispersing the liquid by using a coating rod, wherein the coating rod needs to be sterilized at high temperature before use and can be used when the temperature is lowered to room temperature, and the whole process is operated in a super clean bench;

s406, placing the flat plate in a biochemical incubator at 37 ℃, placing the flat plate on the front surface for 1h, and then inverting the flat plate overnight;

S407A single white colony on a plate is selected, added into a 1.5mL centrifuge tube filled with 1mL LB liquid medium containing resistance, blown and beaten for several times by a pipette gun, and shake-cultured at the conditions of 200rpm and 37 ℃.

S4083-4 h, taking the culture bacterial liquid for PCR verification;

s409, performing nucleic acid gel electrophoresis on the reaction product, and selecting a bacterial solution with a positive electrophoresis result.

In a preferred embodiment of the present invention, the reaction system in S408 further comprises: 5.0. mu.L of PCR mix, 3.5. mu.L of Primer F/R, 1.0. mu.L of bacterial solution,0.5μL ddH₂o, 11.0 μ L Total Volume, reaction program: 3min at 94 ℃; 35 cycles: 30s at 94 ℃; 1min at 55 ℃; 72 ℃ for 10min, 4 ℃ infinite.

In a preferred embodiment of the present invention, the mass culture and purification of the S5 plasmid further comprises:

s501, inoculating the positive bacteria to 6L of LB liquid culture medium containing corresponding resistance, and culturing overnight in a shaking table at the temperature of 37 ℃ and the speed of 200 rpm;

s502, subpackaging the overnight cultured bacterial liquid into 6 50mL centrifuge tubes, centrifuging for 10min at 4000g, and discarding the supernatant;

s503, adding 40mL of Solution I into the precipitate of each tube, violently shaking until the precipitate is completely dissolved, and subpackaging into 200mL glass bottles;

s504, adding 4mL of lysozyme solution into each glass bottle, and gently mixing the lysozyme solution and the lysozyme solution;

s505, adding 80mL of Solution II into a wide-mouth bottle, taking up the glass bottle, slightly and quickly reversing the glass bottle for a plurality of times to mix the content, shaking the glass bottle, and placing the glass bottle in ice water for ice bath for 30 min;

s506, adding Solution III after the Solution is clear, and slightly shaking to generate a large amount of flocculent precipitates;

s507, placing the mixture in ice water for ice bath for 10min, filtering the mixture on 8 layers of gauze, and taking all clear solution;

s508 washing the hollow fiber filtration system AKTA Flux: opening AKTA Flux, washing the instrument by 50mL of 0.5mol/L NaOH, then adding 50mL of 10% alcohol for washing, and finally adding 50mL of pure water for washing;

s509, using a sieve pore CFP-4-E-2U, adding the obtained plasmid solution into a purifier, setting parameters as Mixer 40, purifying Feed50, and storing all the purified solution;

s510 washing the machine again as in step S508 above;

s511, the sieve pores are changed into UFP-300-C-2U, parameters are set as Mixer 40 and Feed50 in the same way, the solution obtained in S510 is purified continuously, the solution is purified to half of the original amount, and the obtained plasmid solution is stored.

In a preferred embodiment of the present invention, the step of concentrating the plasmid in S6 further comprises:

s601, adding a certain volume of plasmid solution, adding 1/10 volumes of sodium acetate into the plasmid solution, and fully and uniformly mixing, wherein the concentration of the sodium acetate is 3mol/L, and the pH value is 5.2, so that the final concentration is 0.3 mol/L;

s602, adding ice precooling absolute ethyl alcohol with the volume 2 times that of the plasmid solution, uniformly mixing the mixture again, and standing the mixture for 20min at the temperature of minus 20 ℃;

s603, centrifuging at 12000g for 10min at room temperature, carefully removing supernatant, and sucking off all liquid drops on the wall pipe;

s604, adding 70% ethanol with the capacity of 1/2 centrifuge tubes, centrifuging for 2min at 12000g, carefully removing supernatant, and removing all liquid drops on the wall tubes by suction;

s605 placing the uncapped centrifuge tube on an experiment table at room temperature to volatilize the residual liquid to be dry;

s606, adding a proper amount of deionized water to dissolve the plasmid precipitate;

s607 the concentration of the plasmid after concentration was determined, and the plasmid was stored at-80 ℃ after adjusting the concentration to 500 ng/. mu.L, 1000 ng/. mu.L, or 2000 ng/. mu.L, respectively.

In a preferred embodiment of the invention, the mstn self-gene vaccine is further used for immunizing epinephelus coioides.

The invention solves the defects in the background technology, and has the following beneficial effects:

(1) the vaccine construction technical route is simple and effective, the operability is strong, and the immune effect of the vaccine is good.

(2) After the mstn self-gene vaccine prepared by the application is used for immunizing Epinephelus coioides, the growth speed and the weight gain rate of an experimental group with the injection dose of 200 mu g and a control group have significant difference, and the weight gain speed of the experimental group with the injection dose of 100 mu g and the control group has significant difference; during the period from the third injection to the sampling, the growth rate and the weight gain rate of the experimental group injected with the dose of 100 mug were significantly different from those of the control group.

(3) In this application, the muscle of Epinephelus coioides is sectioned in paraffin and HE stained, and photographed under a microscope (a, b, c, d). The average relative cross-sectional area of the single muscle fiber of the control group and the single muscle fiber of the three experimental groups are calculated by the image processing of Adobe Photoshop CS6 software, and compared with the control group, the average relative cross-sectional area of the muscle fiber of the experimental group is larger than that of the control group in general, and the relative cross-sectional area of the average muscle fiber is increased along with the increase of the immune dose of the experimental group, and the significance of the experimental groups with the injection dose of 100 mug and 200 mug compared with the control group is found by statistical analysis.

(4) In the application, the experimental fish after the culture is finished is subjected to muscle sampling, and the tissue RNA is extracted and is subjected to reverse transcription to prepare a cDNA template. The expression quantity of mstn signal paths such as smad3, myod, myog, p21 and mrf4 and related genes downstream of the mstn signal paths is detected by RT-qPCR. After immunization of mstn autologous nucleic acid vaccine, mRNA expression levels of smad3 and mrf4 of three dose experimental groups are in a descending trend compared with a control group, and statistical analysis shows that the smad3 is not significantly different when the dose is 50 mu g and 100 mu g, the smad3 is significantly different when the dose is 200 mu g, the smad 4 is not significantly different when the dose is 50 mu g and 200 mu g; the mRNA expression levels of myod, myog and p21 all increased, but significant differences were observed at doses of 200. mu.g.

Drawings

The invention is further explained below with reference to the figures and examples;

FIG. 1 is a graph showing the growth rate of the body length of Epinephelus coioides;

FIG. 2 is a graph showing the growth rate of the body length of Epinephelus coioides;

FIG. 3 is a graph of the body weight gain rate of Epinephelus coioides;

FIG. 4 is a graph of the body weight gain of Epinephelus coioides;

FIG. 5 staining of paraffin sections HE of myofibers of control group;

FIG. 6A 50. mu.g group muscle fiber paraffin section HE staining;

FIG. 7 immunization of 100. mu.g groups of muscle fiber paraffin sections HE staining;

FIG. 8 shows HE staining patterns of paraffin sections of 200. mu.g immunized groups of muscle fibers;

FIG. 9 is a graph of the average relative area of individual muscle fibers;

FIG. 10 is an expression profile of smad 3;

FIG. 11 is an expression level map of p 21;

FIG. 12 is a map of the expression level of myog;

FIG. 13 is a map of expression level of myod;

FIG. 14 expression level map of 14 mrf 4.

Detailed Description

The present invention will now be described in further detail with reference to examples.

Example 1: the embodiment discloses a construction method of an mstn self-gene vaccine based on Epinephelus coioides, which comprises the following specific steps:

firstly, scheme design:

(1) designing an exogenous gene: KOZAK sequence-encephalitis B virus signal peptide-hepatitis B virus surface core antigen-SS-SS-Th epitope-Linker connecting sequence-fish mstn mature peptide gene sequence.

(2) Design description:

firstly, the addition of a KOZAK sequence is required in a pcDNA3.1+ vector specification, and can improve the expression of foreign proteins;

② adding signal peptide of encephalitis B virus to guide foreign protein correctly and improve protein biological efficiency;

the added Th (helper T cell) is positioned in the hepatitis B surface core antigen gene to improve the immunogenicity of the protein; other documents are referred to for their contents;

adding hepatitis B surface core antigen gene to make the mstn mature peptide exogenous, because the fish immune system can only identify non-self protein and produce antibody, if only the mstn mature peptide gene is added without Japanese encephalitis virus core antigen, it is impossible to excite the immune system to produce antibody;

adding SS (somatostatin) gene because SS and mstn are both the gene for inhibiting muscle growth;

sixthly, the Linker connecting sequence is added because the biological function of the mstn mature peptide protein can be influenced if the hepatitis B surface core antigen protein is tightly connected with the mstn mature peptide protein, the mstn mature peptide protein can be moved as if two people walk side by side and need to be separated by a certain distance, and the hepatitis B surface core antigen protein and the mstn mature peptide protein are difficult to walk if the hands and the feet of the two people are bound together. Therefore, a harmless connection sequence for human and livestock is added, the two proteins are connected together, and certain space is provided for the two proteins to exert respective biological functions.

Seventhly, after the whole exogenous gene is connected in series, codon optimization is needed to achieve the purpose of optimal expression in the fish body so as to improve the expression quantity.

(3) After exogenous gene 'exogenous signal peptide + hepatitis B virus surface core antigen + mstn mature peptide' is synthesized by hybridization in the Shanghai, the exogenous gene 'exogenous signal peptide + hepatitis B virus surface core antigen + mstn mature peptide' is cloned to pcDNA3.1+ vector, and finally recombinant plasmid and positive bacteria are obtained.

Second, enzyme digestion and fragment recovery

(1) Double enzyme digestion: carrying out double enzyme digestion on the exogenous gene recombinant plasmid and the vector at 37 ℃ by using TAKARA Nhe I enzyme and Xho I enzyme respectively according to the following systems:

TABLE 1 double digestion system of foreign gene recombinant plasmid and vector

(2) The DNA fragment purification kit (TaKaRa) was used for fragment recovery, and the procedures were as follows according to the instructions:

adding 3 times volume of Buffer DC (100 mu L if the amount of the Buffer DC to be added is less than 100 mu L) into PCR reaction solution (or other enzymatic reaction solution), and then uniformly mixing;

secondly, arranging Spin columns in the kit on a Collection Tube;

transferring the solution obtained in the first operation to Spin Column, centrifuging at 12000rpm at room temperature for 1min, and removing the filtrate;

fourthly, 700 mu L of Buffer WB is added into Spin Column, centrifuged for 30s at 12000rpm and room temperature, and the filtrate is discarded;

fifthly, repeating the operation step IV;

sixthly, placing Spin Column on the Collection Tube, and centrifuging for 1min at 12000rpm and room temperature;

seventhly, taking a new centrifugal tube with the volume of 1.5mL, arranging Spin Column on the centrifugal tube, adding about 30 mu L of Elution Buffer in the center of the Spin Column membrane, and standing for 1min at room temperature (note: the Elution Buffer is heated to 60 ℃ for use);

(iii) centrifuge at 12000rpm at room temperature for 1min to elute DNA.

III, ligation reaction

The vector fragment and the foreign gene fragment were ligated by T4 DNA ligase in the following protocol T4 DNA ligase, and the reaction system was prepared as follows and ligated at 16 ℃ for 8 h:

TABLE 2 mstn and Carrier ligation reaction System

Fourthly, transformation

(1) JM109 competent cells were taken out from an ultra-low temperature freezer at-80 deg.C, and dissolved on ice, and then 10. mu.L of the above ligation product was added thereto and blown with a pipette several times;

(2) incubating on ice for about 45min, transferring to 42 deg.C water bath, thermally shocking for 90s, immediately stopping taking out, and standing on ice for 10 min;

(3) adding 400 μ L of LB liquid medium without resistance, shaking and culturing at 37 deg.C and 200rpm for 60min (in this step, ultra-clean bench ultraviolet is turned on, and the plate containing resistance is preheated in biochemical box);

(4) centrifuging at 4000rpm for 5min, taking out, removing upper layer liquid, leaving about 100 μ L of liquid, and blowing with liquid-transferring gun;

(5) uniformly dripping 100 mu L of the liquid on a flat plate containing resistance, coating and dispersing the liquid by using a coating rod, wherein the coating rod needs to be sterilized at high temperature before use and can be used when the temperature is lowered to room temperature, and the whole process is operated in a super clean bench;

(6) placing the plate in a biochemical incubator at 37 ℃, placing the plate on the front surface for 1h, and then inverting the plate overnight;

(7) a single white colony on the plate was picked, added to a 1.5mL centrifuge tube containing 1mL LB liquid medium containing resistance, blown up several times with a pipette gun, and cultured with shaking at 37 ℃ at 200 rpm.

(8) After 3-4h, taking the culture solution for PCR verification, wherein the reaction system is as follows:

TABLE 3 bacterial liquid verification reaction system

(9) Reaction procedure: 3min at 94 ℃; 35 cycles: 30s at 94 ℃; 1min at 55 ℃; 72 ℃ for 10min, 4 ℃ infinite.

(10) And (3) carrying out nucleic acid gel electrophoresis on the reaction product, and selecting the bacterial liquid with a positive electrophoresis result and sending the bacterial liquid to sequencing.

Fifthly, mass culture and purification of plasmids

(1) Inoculating the positive bacteria into 6L LB liquid culture medium containing corresponding resistance, and shaking at 37 deg.C and 200rpm for overnight culture;

(2) subpackaging the overnight cultured bacterial liquid into 6 centrifugal tubes with 50mL, centrifuging for 10min at 4000g, and discarding the supernatant;

(3) adding 40mL of Solution I into the precipitate of each tube, shaking vigorously until the precipitate is completely dissolved, and subpackaging into 200mL glass bottles;

(4) adding 4mL of lysozyme solution into each glass bottle, and gently mixing the lysozyme solution and the lysozyme solution;

(5) adding Solution II 80mL into a wide-mouth bottle, taking up a glass bottle, slightly and quickly reversing the glass bottle for a plurality of times to mix the content, shaking the glass bottle, and placing the glass bottle in ice water for ice bath for 30 min;

(6) when the Solution is clear, adding Solution III, and slightly shaking to generate a large amount of flocculent precipitate;

(7) ice-bath for 10min in ice water, filter on 8 layers of gauze, and take the whole clear solution.

(8) Washing the hollow fiber filtration system AKTA Flux: the AKTA Flux was turned on, and the instrument was washed with 50mL NaOH (0.5mol/L), followed by 50mL 10% ethanol and finally 50mL pure water.

(9) The resulting plasmid solution was loaded into a purifier using a mesh CFP-4-E-2U, setting the parameters (Mixer 40, Feed 50). Purifying, and storing all the purified solution.

(10) The machine was washed again as in (8) above.

(11) The screen holes were changed to UFP-300-C-2U, and the parameters were also set to (Mixer 40, Feed 50). The purification of the just obtained solution was continued, the solution was purified to half of the original amount, and the obtained plasmid solution was stored.

Sixthly, plasmid concentration

The extracted plasmids were all measured to be at a concentration of about 500 ng/. mu.L, and the plasmids were concentrated to achieve the desired plasmid concentration. The concentration method comprises the following steps:

(1) adding a certain volume of plasmid solution, adding 1/10 volume of sodium acetate (3mol/L, pH 5.2) into the plasmid solution, and mixing well to make the final concentration 0.3 mol/L;

(2) adding 2 times of plasmid solution volume of ice to precool anhydrous ethanol, mixing uniformly again, and standing in-20 deg.C environment for 20 min;

(3) centrifuging at 12000g for 10min at room temperature, carefully removing the supernatant, and aspirating all droplets from the wall tube;

(4) adding 70% ethanol with the capacity of 1/2 centrifuge tube, centrifuging at 12000g for 2min, carefully removing supernatant, and removing all liquid drops on the wall tube by suction;

(5) placing the uncapped centrifuge tube on a laboratory table at room temperature to volatilize the residual liquid to dryness;

(6) adding a proper amount of deionized water to dissolve the plasmid precipitate;

(7) the concentration of the plasmid after concentration was measured, and the concentration was adjusted to 500 ng/. mu.L, 1000 ng/. mu.L, or 2000 ng/. mu.L, respectively, and then stored at-80 ℃.

500 ng/muL, 1000 ng/muL, 2000 ng/muL mstn self-gene vaccine is obtained.

Example two: immunization

Taking 180 tails of epinephelus coioides with the body length of about 13cm, grouping and immunizing according to the following table, wherein each group comprises 3 parallel fries, each parallel fry comprises 15 fries, and the injection volume is 100 mu L:

TABLE 4

Grouping	Concentration (ng/. mu.L)
		Experimental group (1)	500
Experiment set 2	1000
		Experiment group III	2000
Control group	Physiological saline

Each group was immunized 3 times with 2 weeks interval, and after the third immunization, the culture was sampled 3-4 weeks later.

Study on influence of mstn autologous nucleic acid vaccine on body weight and body length of Epinephelus coioides

(1) During the period, weighing and measuring the full length of each immunization;

(2) weighing 3-4 weeks after the last immunization, measuring the total length, and drawing a growth curve.

The results are shown in figures 1-4, where changes in body weight and length are most significant and intuitive for detecting the utility of the mstn nucleic acid vaccine. During the period from the first immunization to the second immunization, the weight and the body length of the epinephelus coioides in the three experimental groups have no obvious change compared with the control group; during the second immunization to the third immunization and the third interview to the sampling period, the body weight and body length growth/weight rate and the body weight and body weight rate of the epinephelus coioides of the three experimental groups generally show a trend of increasing along with the increase of the injection dose compared with the control group, through statistical analysis, the growth rate and the weight rate of the experimental group with the injection dose of 200 mu g and the control group have significant difference during the second injection to the third injection, and the weight gain rate of the experimental group with the injection dose of 100 mu g and the control group has significant difference; during the period from the third injection to the sampling, the growth rate and the weight gain rate of the experimental group injected with the dose of 100 mug were significantly different from those of the control group.

Research on influence of two-mstn autologous nucleic acid vaccine on epinephelus coioides muscle tissue

1. Fish body tissue sample Collection

After 3-4 weeks of culture after the third immunization, each group of experimental fish was anesthetized with eugenol, and muscle tissue sampling was performed, with the tissue mass about 5mm × 5mm × 2mm in size.

2. Sample fixation and embedding

(1) Immediately putting the cut tissue blocks into 4% paraformaldehyde for fixation for 24 h;

(2) dehydrating the tissue blocks by 75%, 85%, 95%, 100% and 100% ethanol solutions for 0.5h respectively;

(3) the absolute ethyl alcohol and the dimethylbenzene are mixed for 2 hours in equal amount, and the dimethylbenzene are respectively 0.5 hour;

(4) paraffin was melted by heating to 56 ℃ and the tissue blocks were embedded.

3. Slicing, spreading and baking slices

(1) Trimming the embedded paraffin block, and clamping the paraffin block on a clamping table of a slicer;

(2) fixing the blade on a cutter holder with the edge facing outwards;

(3) pushing the screw to enable the blade to be close to the paraffin block but not to exceed the knife edge, and adjusting the position and the angle between the blade and the paraffin block to be about 15 degrees;

(4) adjusting the thickness adjuster to cut strips of about 4-10 μm;

(5) shaking the rotating wheel, cutting the paraffin block into wax tapes, lifting the wax tapes with a writing brush, and continuously shaking the rotating wheel to cut into slices;

(6) breaking the wax belt when the length of the wax belt reaches 7-8cm, and flattening the wax belt in a 56 ℃ water bath;

(7) and (3) pasting the flattened wax tape on a glass slide, and drying in a thermostat at 45 ℃.

HE staining

(1) Dewaxing with xylene for 5min for the first time and 10min for the second time,

(2) absolute ethyl alcohol and dimethylbenzene are mixed according to the proportion of 1:1 for 5 min; absolute ethyl alcohol for 3 min; 95% alcohol for 3 min; 80% alcohol for 3 min; 70% alcohol for 3 min; 50% alcohol for 3 min;

(3) staining with hematoxylin dye for 3 min;

(4) washing off the dye for 10min with running water;

(5) then 0.5% hydrochloric acid ethanol is used for differentiation for 5s, and then the redundant dye is washed for 10 min;

(6) and (3) dehydrating: 95% alcohol for 2 min; 50% alcohol for 2 min; 70% alcohol for 2 min; 80% alcohol for 2 min; 95% alcohol for 2 min;

(7) staining the eosin solution for 20s, and washing the eosin solution twice in 95% alcohol after staining;

(8) and (3) dehydrating: 95% alcohol for 2 min; absolute ethyl alcohol for 2 min;

(9) transparent, sealing sheet: carrying out transparency on absolute ethyl alcohol and xylene according to the ratio of 1:1 for 2 min; 2min of dimethylbenzene; second xylene for 2 min;

(10) sealing with resin, and air drying.

5. Slice observation and calculation

The sections were observed and photographed under a microscope and the muscle fiber cross-sectional area was calculated by software.

As shown in FIGS. 5 to 8, the muscle of Epinephelus coioides was sectioned in paraffin and HE stained, and photographed under a microscope (a, b, c, d). The average relative cross-sectional area of the individual muscle fibers of the control group and the three experimental groups was calculated by performing picture processing through Adobe Photoshop CS6 software, as shown in fig. 9, the average relative cross-sectional area of the muscle fibers of the experimental group is larger than that of the control group in general, and the average relative cross-sectional area of the muscle fibers is increased along with the increase of the immune dose of the experimental group compared with the control group, and the significance (e) of the experimental groups with the injection dose of 100 mug and 200 mug compared with the control group is found through statistical analysis.

Research on influence of mstn autologous nucleic acid vaccine on mstn signal channel downstream of epinephelus coioides and related genes

1. Fish body tissue sample Collection

Each group was immunized 3 times with 2 weeks intervals, and after the third immunization, samples were taken after 3-4 weeks of culture, and muscle tissue samples were collected.

2. Total RNA extraction

(1) Sterilizing all scissors and tweezers with 75% ethanol, wrapping with tinfoil paper, sterilizing in autoclave, packaging RNA gun head with brand-new box, and pre-cooling at 4 deg.C with high-speed centrifuge.

(2) Taking out the sample from a refrigerator at-80 ℃, unfreezing the sample on ice, taking an RNase-Free 1.5mL centrifuge tube and adding 600 mu L

reagent (Invitrogen), then the sample was put therein, and the sample tissue piece was pulverized with a grinding rod, followed by addition of 400. mu.L

reagent (Invitrogen) was added to 1mL, mixed, and allowed to stand at room temperature for 5 min.

(3) Adding 200 μ L chloroform, shaking and mixing on a shaker, and standing at room temperature for 15 min.

(4) Centrifugation was carried out at 12000rpm at 4 ℃ for 15min, and the supernatant was aspirated into a new RNase-Free 1.5mL centrifuge tube, taking care not to aspirate a white precipitate.

(5) Adding 500 μ L isopropanol, mixing by turning upside down, and standing at room temperature for 10 min.

(6) Centrifuging at 12000rpm and 4 deg.C for 10min, discarding supernatant, adding 1mL 75% ethanol prepared with DEPC sterilized water, mixing, and standing on ice for 3-5 min.

(7) Removing supernatant at 12000rpm and 4 deg.C for 5min, sucking out excessive liquid, removing RNA precipitate, opening cover on a clean bench, and drying at room temperature for 3-5 min.

(8) The RNA precipitated was dissolved in about 50. mu.L of DEPC sterilized water according to the amount of RNA precipitated in the RNase-Free 1.5mL centrifuge tube.

(9) And (3) measuring the concentration of the dissolved RNA, and if the OD 260/280 value is more than 1.8 and less than 2.0, proving that the quality of the extracted RNA is qualified and carrying out subsequent experiments.

(10) Gel electrophoresis is used for further detecting the quality of the extracted RNA, the extracted RNA is placed in an imaging system for 20min under the voltage of 170V, and if the electrophoresis image is 3 strips, the 28S strip is brightest, the 18S strip is slightly dark, and the 5S strip is darkest, the quality of the extracted RNA is proved to be qualified, and subsequent experiments can be carried out;

(11) RNA was stored in a low temperature freezer at-80 ℃.

Synthesis of cDNA

Synthesis of cDNA Using reverse transcription ReverTra

qPCR RT Kit (TOYOBO) Kit, according to the instructions as follows:

(1) taking 1 mu g of RNA in a PCR tube, carrying out water bath denaturation at 65 ℃ for 5min, and carrying out ice bath for 5 min;

(2) the reverse transcription system was formulated as follows:

TABLE 5 RNA reverse transcription System

(3) Reaction procedure: 25min at 37 ℃; at 98 ℃ for 5 min; 4 ℃, infinity;

(4) the synthesized cDNA was stored at-80 ℃ until use.

4. Real-time fluorescent quantitative PCR

(1) RT-qPCR use

Green real polymerase PCR Master Mix (TOYOBO) kit, following the instructions, the following table:

TABLE 6 fluorescent quantitative PCR System

(2) Reaction procedure: according to the reaction System shown in the table above, adding each reagent into a 384-well plate in a dark place, mixing uniformly, centrifuging for 15-20s, and starting PCR reaction in a Thermo Fisher Quantum studio 5 Real-Time fluorescent quantitative PCR instrument, wherein the reaction program is set as the first step: at 95 ℃ for 10 min; the second step is that: 95 ℃ for 15 s; the third step: 15s at 55 ℃; the fourth step: 72 ℃ for 20s (40 cycles of the second to fourth steps).

5. Data processing and statistical analysis

Experimental data were analyzed for significance using one-way ANOVA analysis of variance using Graphpad prism 7.0 software. And significant differences are indicated by x when P < 0.05; differences were very significant when P <0.01, indicated by; differences were extremely significant when P <0.001, indicated by x.

Subcellular localization images were processed by Adobe Photoshop CS6 software. Myofibroblast cross-section images the relative cross-sectional area of individual myofibers was calculated by Adobe Photoshop CS6 software, the formula:

statistical analysis was then performed by Graphpad prism 7.0 software. Flow cytometry employed Flowjo software for image analysis.

And sampling the muscles of the experimental fish after the culture is finished, extracting tissue RNA and carrying out reverse transcription to prepare a cDNA template. The expression quantity of mstn signal paths such as smad3, myod, myog, p21 and mrf4 and related genes downstream of the mstn signal paths is detected by RT-qPCR. As shown in FIGS. 10-14, after immunization of mstn autologous nucleic acid vaccine, mRNA expression levels of smad3 and mrf4 in the three dose experimental groups were decreased compared to the control group, and statistical analysis showed that smad3 was not significantly different between the doses of 50. mu.g and 100. mu.g, that difference was significant between the doses of 200. mu.g, that no significant difference was present between the doses of 50. mu.g and 50. mu.g of mrf4, and that difference was significant between the doses of 100. mu.g and 200. mu.g; the mRNA expression levels of myod, myog and p21 all increased, but significant differences were observed at doses of 200. mu.g.

In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims

1. A construction method of mstn self-gene vaccine based on Epinephelus coioides is characterized by comprising the following steps:

2. The construction method of the epinephelus coioides mstn-based self-gene vaccine according to claim 1, wherein a recombinant plasmid and vector double enzyme digestion system of the exogenous gene comprises: 1.0 μ L of Nhe I, 1.0 μ L of Xho I, less than or equal to 2.0 μ g gene/plasmid, 2.0 μ L of 10 XBuffer, no more than 20.0 μ L of ddH₂0，20.0μL Total Volume。

3. The construction method of the Epinephelus coioides mstn-based self-gene vaccine as claimed in claim 1, wherein the method for recovering the fragments in S2 by using the DNA fragment purification kit comprises the following steps:

s202, arranging Spin columns in the kit on a Collection Tube;

s205 repeats operation S204;

4. The construction method of the Epinephelus coioides mstn-based self-gene vaccine as claimed in claim 1, wherein the reaction system of successive reactions in S3 is as follows: 6.0. mu.L of exogenous gene fragment, 6.0. mu.L of vector fragment, 1.0. mu.L of LT4 DNA ligase, 1.0. mu.L of 10 XT 4 DNA Ligation Buffer, and 10.0. mu.L of Total Volume.

5. The construction method of the Epinephelus coioides mstn-based self-gene vaccine as claimed in claim 1, wherein the S4 transformation process comprises the following steps:

s405, uniformly dripping 100 mu L of liquid onto an LB flat plate containing resistance, coating and dispersing the liquid by using a coating rod, wherein the coating rod needs to be sterilized at high temperature before use and can be used when the temperature is lowered to room temperature, and the whole process is operated in a super clean bench;

S4083-4 h, taking the culture bacterial liquid for PCR verification;

s409, performing nucleic acid gel electrophoresis on the reaction product, and selecting a bacterial liquid with a positive electrophoresis result for testing.

6. The Epinephelus coioides mstn-based fish of claim 5The construction method of the somatic gene vaccine is characterized in that the reaction system in S408 comprises the following steps: 5.0. mu.L of PCR mix, 3.5. mu.L of Primer F/R, 1.0. mu.L of bacterial solution, 0.5. mu.L of ddH₂O, 11.0 μ L Total Volume, reaction program: 3min at 94 ℃; 35 cycles: 30s at 94 ℃; 1min at 55 ℃; 72 ℃ for 10min, 4 ℃ infinite.

7. The construction method of the Epinephelus coioides mstn-based self-gene vaccine as claimed in claim 1, wherein the mass culture and purification steps of the S5 plasmid comprise:

s507, placing the mixture in ice water for ice bath for 10min, filtering the mixture on 8 layers of gauze, and taking all clear liquid;

s510 washing the machine again as in step S508 above;

s511, the sieve pores are changed into UFP-300-C-2U, the parameters are set as Mixer 40 and Feed50, the solution obtained in S510 is purified continuously, the solution is purified to half of the original amount, and the obtained plasmid solution is stored.

8. The construction method of the Epinephelus coioides mstn-based self-gene vaccine as claimed in claim 1, wherein the step of plasmid concentration in S6 comprises:

9. The mstn self-gene vaccine constructed based on the construction method of any one of claims 1-8 is used for immunization in epinephelus coioides.