CN109942705B

CN109942705B - MSTN nano antibody, construction method and application thereof

Info

Publication number: CN109942705B
Application number: CN201910299045.6A
Authority: CN
Inventors: 陈创夫; 盛金良; 吴鹏; 李尤简; 郭吉星
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2019-04-15
Filing date: 2019-04-15
Publication date: 2022-10-18
Anticipated expiration: 2039-04-15
Also published as: CN109942705A

Abstract

The invention belongs to the technical field of biological medicines, and discloses an MSTN (myostatin) nano antibody, a construction method and application thereof, wherein a nano antibody library of a sheep MSTN gene expression product is constructed; obtaining a specific nano antibody aiming at an MSTN gene expression product by a phage display technology; and constructing the bivalent sheep MSTN nano antibody. The MSTN nano antibody provided by the invention has the effects of increasing the muscle content of animals and reducing the fat content of the animals on mice. When the weight gain is mainly muscle weight gain, the sheep MSTN nano antibody is shown to gain more weight than the untreated control group; the mice show that the muscle weight of the nano-antibody group is obviously higher than that of the control group, and the proportion of the muscle in the whole body is also obviously higher than that of the control group. The sheep MSTN nano antibody can promote the growth of animal muscle.

Description

MSTN nano antibody, construction method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an MSTN nano antibody, a construction method and application thereof.

Background

Currently, the current state of the art commonly used in the industry is such that:

MSTN is a gene that regulates muscle growth in animals. Mainly plays a role in inhibiting the growth of muscles. The lack of MSTN can promote the development of the double-muscle trait in the animal. When the action of the sheep MSTN gene is interfered, the growth of sheep muscle can be promoted, and more mutton can be better provided for people of all families. According to the principle of antigen-antibody reaction, the action way of preparing the antibody for inhibiting the MSTN can inhibit the inhibition of the MSTN gene expression product on the muscle growth, can shorten the sheep fattening time and increase the muscle yield. The porcine (MSTN) gene is amplified by Malayong and the like, and a crude extract of the expressed recombinant MSTN protein is used for immunizing mice to study the influence on the body weight, the body weight of the immunized mice is obviously increased compared with that of a control group, and the difference of the body weight of the experimental group mice is more than 10.5 percent of that of the control group mice (P is less than 0.05). Sheep and mouse MSTN gene have high homology. The MSTN gene sequence of the mammal is highly conserved, the homology of mouse, pig and chicken is 100%, and the difference between cattle and sheep is only 3 nucleotides. The MSTN gene is always conserved in sheep genetic evolution, and has less variation. The genetic diversity of the goat MSTN gene is very low. The sheep MSTN gene promoter is also relatively conserved between different sheep species. The highly conserved MSTN provides a powerful guarantee for producing the nano antibody to promote the growth of sheep.

The sheep MSTN protein is used for immunizing mice, and the verification effect can also be achieved. The nano antibody can enter the body of a mouse, and can not cause the immune reaction of the mouse. Therefore, mice are an excellent platform for verifying the function of nanobodies. The sheep MSTN protein is antagonized by preparing the nano antibody for resisting the sheep MSTN protein, and the MSTN gene activation pathway is inhibited, so that the sheep growth is promoted. After the nano antibody is subjected to high-temperature treatment, protein is denatured and decomposed into polypeptide and amino acid, the polypeptide and the amino acid are digested into the amino acid in the digestive tract, and then the amino acid is absorbed by animals, and the way of injecting the nano antibody into the animals does not belong to the category of transgenic animals.

The nanometer antibody as a new antibody is fundamentally different from the general antibody structure. The nano-antibody can treat diseases which can not be treated by the traditional antibody. The nano antibody has the advantages of small volume, high stability, strong binding force and the like.

One of the advantages of nanobodies is that the alteration of the antibody can be achieved by editing the gene. After obtaining the nano antibody sequence, the sequence can be modified to achieve the effect of modifying the antibody. The antibody is subjected to targeted modification, so that the antibody has targeted characteristics. Can reduce immune reaction, modify the function of antibody, and perform humanized modification. The linker can also be used for linking groups with special structures such as albumin and the like, so that the nano antibody has different characteristics. The nano antibody has unique advantages in the aspect of antigen detection, and achieves outstanding performances in the aspect of different antigen detection.

In summary, the problems of the prior art are as follows:

(1) In the prior art, theoretical support is lacked for improving the muscle growth promoting effect of the sheep MSTN nano antibody.

(2) In the prior art, the MSTN polyclonal antibody is prepared, and only the team performs an experiment for promoting sheep weight gain by using the sheep MSTN polyclonal antibody.

(3) The MSTN antibodies are not applied to production in the prior art.

(4) The MSTN antibody produced by the prior art has high cost and the problems of heterogeneity of the antibody and the like.

The significance of solving the technical problems is as follows:

the phage display technology is a technology for expressing foreign proteins by phage. The panning adopts the principle of a solid phase carrier panning method, and enriches and detects high specificity antibodies by an ELISA method. First, antigens against antibodies are coated on a solid support, and the antigens are blocked with proteins to reduce non-specific binding. The phage bound to the solid support, which have not been eluted, are then eluted. After elution of specifically bound phage, the phage are collected and amplified. Obtaining the enriched antibody, and then carrying out a plurality of screening operations to obtain the antibody with high specificity. After sequencing, the desired target antibody and target sequence are obtained. The method has the advantages of common required instruments and equipment, simple principle, low technical requirement, suitability for producing the nano antibody and the like. M13 single-stranded Phage is a commonly used Helper Phage (Helper Phage). Can provide substances such as required enzyme (Flori defect) for the replication and proliferation of the phage. M13K07 belongs to the M13 series and has kanamycin resistance. M13K07 cannot be used as a cloning vector. M13K07 belongs to filamentous bacteriophage, and does not kill bacteria when amplified in vivo. Lytic bacteriophages such as T7 and T4 need to be purified first during panning to ensure the purity of the protein. M13 has a slow propagation rate, and the propagated phage is secreted to the outside of the cell. Recombinant phage particles with the protein of interest can be obtained by centrifugation of the culture. But the library capacity was larger than other phages. Convenient purification and is not affected by other secretion of bacteria. Phage display has the advantage of linking the sequence of interest to the protein of interest. After construction of the library of sequences of interest, multiple screens may be performed. The library can be amplified and stored by cloning. This ensures that even a single specific antibody will be enriched multiple times and the content of specific antibody will be increased. Greatly increasing the possibility of screening ideal protein and facilitating the screening work.

Cloning a sheep MSTN gene in the early stage of a laboratory, constructing a prokaryotic expression system of the sheep MSTN gene, and carrying out cytotoxicity verification; after an animal is immunized by the MSTN antigen, a polyclonal antibody is prepared, and the growth promoting effect on sheep is obvious when the polyclonal antibody is verified on sheep; and (3) immunizing the bactrian camel by the antigen, and performing reverse transcription on the leucocyte to construct a nano antibody library of the sheep MSTN gene.

The antibody can be produced through prokaryotic expression by preparing the MSTN nano antibody. The nano antibody (Nanobody, nb) is prepared by cloning the variable region of a heavy chain antibody in a camel body, has the volume of nano level and has complete antigen combining capability. The nano antibody has the advantages of stable physicochemical property, high specificity, high affinity, low toxicity, high solubility, difficult immune reaction, easy production, easy modification and the like. Compared with the traditional antibody, the nano antibody has no reduction of the capacity of combining with the antigen and can replace the traditional antibody to combine with the antigen. Bivalent and multifunctional nanobodies have hundreds to thousands of times higher binding capacity than monovalent nanobodies. The nano antibody has short research and development period, small risk, less investment and large output. The nano antibody can be expressed by using a prokaryotic expression system, and can be expressed in a large quantity. The nano antibody is used for replacing the monoclonal antibody for treatment, so that the price of the medicine can be greatly reduced, the treatment cost is reduced, and the treatment effect is expected to be improved.

Nanobodies may remain conformationally unchanged in harsh environments. The nano antibody has particularly good heat resistance and can be stored at room temperature for more than one week. The super-strong acid and alkali resistance can enable the nano antibody to better resist different environments, and can also increase the action range of the nano antibody. The small size of the single domain heavy chain antibody also makes it less immunogenic. Makes it possible for animals to inject protein for a long time. The nano antibody has the advantages that the traditional antibody does not have, and the nano antibody also achieves the performance of a person in the research field of the nano antibody. The single domain heavy chain antibody is small in size, but has the function of binding the heavy chain antibody to an antigen, does not have an Fc end, and does not cause a complement reaction. The binding site of nanobodies to antigens is also different from that of monoclonal antibodies. Nanobodies can bind more tightly to antigens and can bind to places where traditional antigens do not bind. The construction of libraries using viral and bacterial surface products as antigens requires the selection of representative strains, strains with therapeutic or detection value. Monoclonal antibodies are not readily accessible to the interior of the tissue due to their susceptibility to complement reaction and their relatively large size.

The study found that the MSTNs of the various animals were relatively conserved and that the MSTNs of mice and sheep differed very little. Because the mouse is convenient to obtain, the experiment operation is simple, and the effect is quicker. Therefore, preliminary validation of the effect of the sheep MSTN antibody on mice was performed. Since the carcass of a mouse contains a large amount of bone and other tissues, the muscles in the hind limb are the majority and are also easily isolated, as is typical. Therefore, only the hind limbs of the mice were isolated and muscle growth comparisons were performed. To have a decision on the effect of nanobodies.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an MSTN nano antibody, a construction method and application thereof.

The construction method of the MSTN nano antibody is realized by using phage display technology and panning the antibody with strong specificity by using an ELISA principle method. Detecting the clone with high binding force with antigen, and sequencing the bacterial liquid. The R1 library used was constructed at a library volume of 1.8X 10 ¹² pfu/mL。

The MSTN nano antibody of the monovalent sheep is constructed into a bivalent nano antibody, a target sequence is connected into a bivalent gene through a flexible linker (-GGGGSGGGGSGGS-), and a prokaryotic expression pET-30 (A) prokaryotic expression system is constructed. Antibody activity was detected using SDS-PAGE electrophoresis and ELISA. And (3) carrying out mass expression on the univalent sheep MSTN nano antibody and the bivalent sheep MSTN nano antibody.

Purchasing Kunming mice, injecting PBS, BSA, MSTN, monovalent nanometer antibody and bivalent nanometer antibody in groups, observing the weight change of the mice, observing the morphological change of muscles, manufacturing slices and carrying out histological observation; and performing repeated experiments, wherein the repeated experiments are divided into a monovalent nanometer antibody group, a bivalent nanometer antibody group and a control group, and each group comprises 10 antibodies.

The method specifically comprises the following steps:

the method comprises the following steps: constructing a nano antibody library of a sheep MSTN gene expression product;

step two: obtaining a specific nano antibody aiming at an MSTN gene expression product by a phage display technology;

step three: constructing bivalent sheep MSTN nano antibody SEQ ID NO:3.

(nanometer antibody gene sequence (KpnI)

GGTACCCAGCTGCAACTGGTTGAAAGCGGTGGTGGTAGCGTTCAGGCAGGCGGTAGCCTGAGCCTGAGCTGTGCAGCAAG CGGTTATACCTATGTTAGCCGTTATATGGGTTGGTTTCGTCAGGCACCGGGTAATGAACGTGAAGGTGTTGCAACCATTTATAC CGCAGGTATTAGCACCTATTATGATGCAAGCGTTAAAGGTCGTTTCACCATCAGCAAAGATAATGCCAAAAATACCGTTTACCT GCAGATGAATAGCCTGAAACCGGAAGATACCGCAATGTATTATTGTGCAGCCACCCATGATGATTATGGTGGTAGTGGTAGCC GTCTGAGTCCGGCAAGCTATGCATATTGGGGTCAGGGCACCCAGGTTACCGTTAGCAGCGAACCGAAAACCCCGAAACCGC AGGGTCCGCGTGGTCTGGGTGGTGGTGGAAGTGGTGGCGGTGGTTCAGGCGGTGGCGGTAGTCAACTGCAGCTGGTAGAA TCAGGTGGTGGCTCAGTTCAAGCCGGTGGTAGCCTGTCACTGTCATGTGCAGCCTCAGGCTATACATATGTTTCACGCTACAT GGGCTGGTTCCGTCAAGCCCCTGGCAACGAACGCGAAGGCGTGGCCACAATTTATACAGCTGGCATTTCAACATATTATGAC GCCTCAGTGAAAGGTCGCTTTACGATTTCAAAAGACAATGCGAAAAACACGGTGTATCTGCAAATGAATTCACTGAAACCTGA GGACACAGCCATGTACTACTGTGCCGCAACACACGATGACTATGGCGGTAGCGGTTCACGTCTGTCACCGGCATCATATGCCT ACTGGGGACAGGGTACACAGGTGACAGTTAGTTCAGAACCTAAAACACCTAAACCTCAAGGCCCTCGTGGTCTGCTCGAG (XhoI))。

Amino acid sequence:

GTQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGVATIYTAGISTYYDASVKGRF TISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWGQGTQVTVSSEPKTPKPQG PRGLGGGGSGGGGSGGGGSQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGV ATIYTAGISTYYDASVKGRFTISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWG QGTQVTVSSEPKTPKPQGPRGLLE。

further, in the second step, the specific nano antibody is specifically prepared by:

(1) Obtaining a target fragment and designing a primer;

(2) Amplifying a nano antibody sequence;

(3) Recovering PCR product gel of a bivalent sheep MSTN nano antibody sequence;

(4) Carrying out double enzyme digestion on the pET-30A vector;

(5) Connecting the recovered product with a pET-30A vector;

(6) Induced expression of the protein;

(7) SDS-PAGE detection;

(8) The protein activity is verified by ELISA;

(9) And (3) carrying out mass expression on the nano antibody, and collecting protein to prepare for subsequent experiments.

Further, in the third step, a bivalent sheep MSTN nano antibody is constructed, and the muscle growth promoting effect of the sheep MSTN nano antibody is verified.

The invention also aims to provide the sheep MSTN nano-antibody.

Further, the obtaining of the target fragment and the design of the primer specifically include:

preparing a bivalent nano antibody by taking the MSTN nano antibody as a target sequence; .

The primers are as follows: GAATTCATGCAGTTGCAGCTCGTGG,

GCGGCCGCAAGGCCTCGG。

Further, nanobody sequence amplification comprises: PCR condition of bivalent sheep MSTN nano antibody sequence

After the reaction was complete, the product was stored at 4 ℃.

Further, the recovery of PCR product gel of the bivalent sheep MSTN nano antibody sequence comprises the following steps:

placing the gel subjected to electrophoresis under an ultraviolet lamp, and cutting off the target fragment; the cut gel was placed in an EP tube. Add solution B to the EP tube, place the gel at 60 ℃ with constant shaking, and wait for the gel to melt. Adding the melted gel into a collecting pipe, standing for 3 minutes at 7000r/min, and centrifuging for 3min. The collection tube was removed and eluted twice using Wash solution. Wash Soluion was removed thoroughly by centrifugation. Eluting the DNA by using Elution Buffer; and carrying out gel electrophoresis detection to recover the product purity.

Further, carrying out double enzyme digestion on the pET-30A vector by using BamHI and XhoI; react for 2h at 37 ℃.

Further, the recovered product was ligated with pET-30A vector and reacted for 1 hour at 50 ℃ in a PCR apparatus.

The invention also aims to provide the MSTN nano antibody constructed by the construction method of the MSTN nano antibody.

Another objective of the present invention is to provide a method for detecting the MSTN nanobody, which comprises:

1) Preparation of competent cells

The preserved DH5 alpha is subjected to plate line drawing culture, and a monoclonal colony is picked. Adding the picked colonies into 5mL of LB culture medium, and carrying out overnight culture; the next day, transferring the cells into a 50mL culture medium, and culturing the cells to a logarithmic phase; taking DH5 alpha in logarithmic growth phase to carry out ice bath; centrifuging by using a high-speed low-temperature centrifuge, and collecting precipitates; 6mL of low-temperature 0.1 mol CaCl was injected into the precipitate ₂ Suspension precipitation; the mixture is subpackaged and packaged into a small package,quickly freezing in liquid nitrogen, and storing in a refrigerator at-80 deg.C;

2) Transformed into expression bacterium DE3

Adding the connected product into an EP tube, adjusting a water bath kettle to 42 ℃, quickly putting the EP tube into the water bath kettle, continuously shaking the EP tube during the process, and reacting for 90s; the EP tube was removed and placed on ice for 2min. Centrifuging, collecting precipitate, plating, and adding 30 μ g/ml kanamycin and 34 μ g/ml chloramphenicol; the next day, single clones were picked;

inducible expression of proteins

Picking a monoclonal for overnight culture; adding 100 mu L of culture solution into 20mL of culture medium containing Kana and chloramphenicol, and inducing expression to logarithmic phase; adding IPTG with the final concentration of 0.5 mmol; subpackaging into different triangular flasks; different conditions are set: first bottle, using 15 deg.C, overnight shake culture; second bottle, 25 ℃, overnight induction; culturing in a third bottle at 37 deg.C for 4h at 220 r/min; the group without IPTG addition was set as negative control; collecting bacteria at 4000r/min, adding 500 μ L PBS into the precipitate, and repeatedly beating; carrying out ultrasonic crushing, and collecting supernatant and precipitate; dissolving the collected precipitate with an inclusion body dissolving solution; adding the sample into a protein loading buffer, and boiling for 10min;

3) SDS-PAGE detection:

preparing 12% separation glue, mixing the separation glue uniformly, adding into a glue maker, and picking out generated bubbles by using a long needle without leaving bubbles in the glue. Adding ethanol above the separation gel by using a liquid shifter; after 20min, pouring out the ethanol; after the ethanol is completely volatilized, preparing 5% concentrated glue, adding the concentrated glue, and inserting a comb; placing the glue maker on a test bed and standing for 2 hours; the comb was removed from the gel. The gel was placed in an electrophoresis tank and 1 Xelectrophoresis solution was added. Removing air bubbles in the gap; adding a bivalent sheep MSTN nano antibody sample into concentrated gel, wherein each hole is 8 microliters, and adding a protein Marker; regulating voltage to 80V, and performing gel concentration electrophoresis for 30min; the gel was isolated at 120V,60min. After 3-4h, stopping electrophoresis when bromophenol blue reaches the bottom of the gel; the glue is separated and placed in deionized water for cleaning. Immersing in staining solution, and staining for 35min on constant temperature shaking table. Preparing fresh decolorizing solution, decolorizing for many times on a constant temperature shaking table until strips are clear, and stopping decolorizing when the gel is completely decolorized. Cleaning with deionized water; placing in a Bio-RAD agglutination imager, correcting the position of the glue, and adjusting the color and contrast of the pattern;

4) ELISA for protein Activity

MSTN protein was diluted with coating solution, ELISA plates coated, 200 ng/well, 4 degrees overnight incubation. PBST washing 3 times. 5% PBSM (PBS + skim milk powder) 100 microliters per well was used, incubated at 37 degrees Celsius for 1 hour. PBST washing 3 times. The nanobody was diluted by 100, 1000, 10000, 100000, 1000000, 10000000, 100000000 times in a gradient manner, PBS was added to the control group, 100 μ l per well was added, and incubation was performed at 37 ℃ for 1 hour. PBST washing 3 times. 10000 times of diluted secondary antibody against alpaca, 100 microliters per well, was added and incubated at 37 ℃ for 1 hour. PBST was washed 3 times. Adding 100 microliters of developing solution, incubating at 37 ℃ for 15 minutes, adding 50 microliters of stop solution, and reading the OD value by a microplate reader.

In summary, the advantages and positive effects of the invention are:

the invention provides a nano antibody library for constructing a sheep MSTN gene expression product. Obtaining the specific nano antibody aiming at the MSTN gene expression product by a phage display technology. And (3) constructing a bivalent sheep MSTN nano antibody, and further verifying and improving the muscle growth promoting effect of the sheep MSTN nano antibody. The function of promoting muscle growth was verified in mice.

And (3) mouse growth promotion verification: the mice are divided into groups, BSA (bovine serum albumin), MSTN (MSTN protein), monovalent sheep MSTN (myostatin) nano antibody and bivalent sheep MSTN nano antibody are injected into the mice, and the mice injected with PBS (phosphate buffer solution) are used as a control group for verification. And (4) observing the weight change of the animal, taking muscle tissues to prepare slices after the experiment is finished, and observing the change condition of muscle fibers. The results show that: muscle and bone were significantly increased in the diabody group compared to the PBS control group and the BSA control group. The number of muscle fibers in the PBS control group is less than that in the monovalent nanometer antibody group, and the difference of p = 0.042-straw 0.05 is obvious; the PBS control group was less than the diabody group, and p =0.015<0.05 was significantly different. The monovalent nanobody group and the bivalent nanobody group p =0.209 >. The bivalent nanobody group hind limb weight was the heaviest. Bivalent nanobodies were heavier than the control group, with p =0.03<0.05 differences being significant, bivalent nanobodies were heavier than the MSTN group, p =0.027<0.05 differences being significant, bivalent nanobodies were heavier than monovalent nanobodies, and p =0.008<0.01 differences being very significant. Results of the second mouse experiment: the hind limb of the bivalent nano antibody group is obviously larger than that of the monovalent nano antibody group, and the monovalent nano antibody group is larger than that of the control group. The final total weight of the control group, the monovalent nanometer antibody and the bivalent nanometer antibody has no difference. Leg weighing revealed that the difference between bivalent nanobody and monovalent nanobody was very significant, p =0.004 was-0.01, and the difference between bivalent nanobody and control group was very significant, p =0.000 was-0.01. And (4) conclusion: the muscle volume of the mice in the nano antibody group is obviously increased, the proportion of the muscles in the whole body is obviously increased, the weight gain of the bivalent nano antibody group is more than that of the univalent nano antibody group, and the statistical analysis difference is obvious; the bivalent sheep MSTN nanobody can increase the number of muscle fibers. The bivalent sheep MSTN nano antibody has the function of increasing the muscle weight, and the muscle weight is increased over a monovalent group; when the weight gain is mainly muscle weight gain, the sheep MSTN nano antibody is embodied as a control group with the weight gain exceeding PBS; when the weight gain is mainly fat accumulation, the sheep MSTN nano antibody is embodied as increasing muscle weight and increasing weight, and the difference between the weight gain effect and the fat weight gain is not obvious within a certain time.

The invention provides a nano antibody library for constructing a sheep MSTN gene expression product. Obtaining the specific nano antibody aiming at the MSTN gene expression product by a phage display technology. And (3) constructing a bivalent sheep MSTN nano antibody, and further verifying and improving the muscle growth promoting effect of the sheep MSTN nano antibody.

The MSTN nano antibody has the effects of increasing the muscle content of animals and reducing the fat content of the animals on mice. When the weight gain is mainly muscle weight gain, the sheep MSTN nano antibody is shown to gain more weight than the untreated control group; when the weight gain is mainly fat accumulation, the sheep MSTN nano antibody is reflected by increasing muscle weight and gaining weight, and the weight gain effect and the fat accumulation weight gain effect in a short period are not greatly different. The mice show that the muscle weight of the nano-antibody group is obviously higher than that of the control group, and the proportion of the muscle in the whole body is also obviously higher than that of the control group. In the animals of the bivalent nanobody group, muscle sections showed an increase in the number of muscle fibers per unit area. The sheep MSTN nano antibody can promote the growth of animal muscle.

According to the invention, the two nano antibodies can be linked by adding the amino acid between the two nano antibodies, so that the purpose of increasing the action effect is achieved. Different nano antibodies aiming at the same antigen are connected through a linker, so that the antigen-antibody combination efficiency can be increased. The amino acid sequence linking two proteins is called Linker. The excellent linker can enable the fusion protein to play the most role without influencing the fusion protein. The length of the linker also affects the conformation and function of the protein. The excessively long linker is easily hydrolyzed by enzyme, and the excessively short linker influences the function of the protein. A commonly used linker is (-GGGGSGGGGSGGS-). Glycine (Gly, G) has a small molecular weight and high flexibility. Serine (Ser, S) has a high hydrophilicity. Bivalent nanobodies are at least 60 times more effective than monovalent nanobodies for inhibiting viral replication.

Drawings

FIG. 1 is a flow chart of an MSTN nanobody and an application method thereof provided by an embodiment of the present invention.

FIG. 2 is a schematic diagram of a bivalent nanobody gene amplification product provided in the embodiment of the present invention.

FIG. 3 shows the restriction electrophoresis of pET-30A provided by the present invention.

FIG. 4 is a graph showing the results of SDS analysis of different concentrations of IPTG and different temperatures provided by an embodiment of the present invention.

FIG. 5 is a schematic diagram of the results of Western blot analysis at different temperatures and different concentrations of IPTG provided by the embodiment of the invention.

FIG. 6 is a schematic diagram of an ELISA assay provided in an embodiment of the invention.

FIG. 7 is a diagram showing the results of SDS-PAGE verification provided in the example of the present invention.

FIG. 8 is a schematic diagram of the morphology of the muscle of the hind limb of the mouse according to the embodiment of the present invention.

FIG. 9 is a schematic diagram of the morphology of the muscle of the hind limb of the mouse according to the embodiment of the present invention.

Fig. 10 is a schematic diagram of a muscle tissue slice according to an embodiment of the present invention.

FIG. 11 is a schematic diagram of the morphology of the muscle of the hind limb of a mouse according to an embodiment of the present invention.

FIG. 12 is a schematic diagram of the morphology of the muscle of the hind limb of the mouse according to the embodiment of the present invention.

FIG. 13 is a schematic diagram of the morphology of the muscle of the hind limb of a mouse according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

In the prior art, theoretical support for improving the muscle growth promoting effect of the sheep MSTN nano antibody is lacked.

The application principle of the present invention will be described in detail with reference to the accompanying drawings;

as shown in fig. 1, the MSTN nanobody and the application method thereof provided by the embodiment of the present invention are:

s101: constructing a nano antibody library of a sheep MSTN gene expression product;

s102: obtaining a specific nano antibody aiming at an MSTN gene expression product by a phage display technology;

s103: and constructing the bivalent sheep MSTN nano antibody.

In step S103, the bivalent sheep MSTN nano antibody SEQ ID NO:3.

(nanometer antibody gene sequence (KpnI)

Amino acid sequence: the amino acid sequence of SEQ ID NO:4

The specific nano antibody provided by the embodiment of the invention is specifically prepared by the following steps:

(1) Obtaining a target fragment and designing a primer;

(2) Amplifying a nano antibody sequence;

(3) Recovering PCR product gel of a bivalent sheep MSTN nano antibody sequence;

(4) Carrying out double enzyme digestion on the pET-30A vector;

(5) Connecting the recovered product with a pET-30A vector;

(6) Induced expression of the protein;

(7) SDS-PAGE detection;

(8) WB to verify protein activity;

In step S103, the bivalent sheep MSTN nano antibody is constructed, and the muscle growth promoting effect of the sheep MSTN nano antibody is verified.

The sheep MSTN nano antibody applied to animal muscle weight gain provided by the embodiment of the invention comprises the following specific steps:

(1) Detecting antigen-antibody binding specifically by ELISA;

(2) Verifying the effect of the bivalent sheep MSTN nano antibody on a mouse;

(3) The bivalent sheep MSTN nano antibody is verified on a mouse by repeated experiments.

The sheep MSTN nano antibody provided by the embodiment of the invention is applied to mouse muscle weight gain.

The sheep MSTN nano antibody is applied to animals, so that the muscle content is increased, and the fat content of the animals is reduced.

The application principle of the invention is further explained by combining specific experiments;

1. material

(1) Bacterial strain and carrier

The Rosetta (DE 3) strain was maintained by a zoonotic research laboratory. pET-30a expression vector was purchased from Yukang, a century organism.

(2) Reagent

TABLE 1 Main reagents

(3) Instrument for measuring the position of a moving object

TABLE 2 Main instruments

2. Method of producing a composite material

(1) Obtaining of target fragment and design of primer

A nano antibody with high binding specificity with MSTN antigen is obtained in the early stage of a laboratory, and a cytotoxicity test and a mouse growth-promoting test are carried out. The growth speed of the muscle of the mouse is obviously promoted. Therefore, the nanobody is used as a target sequence to prepare a bivalent nanobody. The target sequence is sent to Huanuo time science and technology Limited liability company, and is connected by using flexible linker (-GGGGSGGGGSGGS-).

TABLE 3 primer design

(2) Nano antibody sequence amplification

1) PCR system

TABLE 4 PCR amplification reaction System

(dilution of primer concentration: 1OD addition of 400. Mu.L ddH ₂ O)

2) PCR condition of bivalent sheep MSTN nano antibody sequence

TABLE 5 PCR reaction conditions

After the reaction was complete, the product was stored at 4 ℃.

(3) Recovery of PCR product gel of bivalent sheep MSTN nano antibody sequence

The gel after running the electrophoresis is placed under an ultraviolet lamp, and the target fragment is cut off. Note that the cut gel volume is not too large. The cut gel was placed in an EP tube. Add solution B to the EP tube, place the gel at 60 deg.C with constant shaking, and wait for the gel to melt. Adding the melted gel into a collecting pipe, standing for 3 minutes at 7000r/min, and centrifuging for 3min. The collection tube was removed and eluted twice using a Wash solution. Wash Soluion was removed thoroughly by centrifugation. The DNA was eluted using an Elution Buffer. And carrying out gel electrophoresis detection to recover the product purity.

(4) Double digestion of pET-30A vector

The vector was double digested with BamHI and XhoI.

TABLE 6 enzyme digestion System

React for 2h at 37 ℃.

(5) Recovery of enzyme digestion product gel

The recovery procedure was the same as above.

(6) The recovered product and the pET-30A vector were ligated

TABLE 7 connection System

The PCR instrument was set to react for 1h at 50 ℃.

1) Preparation of competent cells:

the preserved DH5 alpha is subjected to plate line drawing culture, and a monoclonal colony is picked. The colonies picked were added to 5mL of LB medium and cultured overnight. The next day, the cells were transferred to 50mL of medium and cultured to logarithmic growth phase. DH 5. Alpha. Was taken in the logarithmic growth phase and ice-cooled. And (4) centrifuging by using a high-speed low-temperature centrifuge, and collecting precipitates. 6mL of low temperature 0.1 mol CaCl2 suspension precipitate was injected into the precipitate. Subpackaging, quick freezing in liquid nitrogen, and storing in-80 deg.C refrigerator.

2) Transformation into an expression bacterium DE3:

the joined product was added to an EP tube, the water bath was adjusted to 42 ℃ and the EP tube was quickly placed while shaking constantly, the reaction time being 90s. The EP tube was removed and placed on ice for 2min. The pellet was plated by centrifugation, and 30. Mu.g/ml kanamycin and 34. Mu.g/ml chloramphenicol were added. The next day, single clones were picked and sent to Shanghai Biotechnology, LLC for sequencing.

(8) Induced expression of the protein:

and (4) carrying out bacterium preservation on the clone with the correct sequencing, and picking out the monoclonal for overnight culture. 100. Mu.L of the culture broth was added to 20mL of a culture medium containing Kana and chloramphenicol, and expression was induced to logarithmic growth phase. IPTG was added to a final concentration of 0.5 mmol. Subpackaging into different triangular flasks. Different conditions were set: first bottle, using 15 deg.C, overnight shake culture; second bottle, 25 ℃, overnight induction; culturing in a third bottle at 37 ℃ for 4h at 220 r/min; the group to which no IPTG was added was set as a negative control. The bacteria were collected at 4000r/min and 500. Mu.L of PBS was added to the pellet and the pipetting was repeated. And (4) carrying out ultrasonic disruption, and collecting supernatant and precipitate. The collected precipitate was solubilized using an inclusion body solubilization solution. The sample was added to the protein loading buffer and boiled for 10min.

(9) SDS-PAGE detection:

verifying the induction expression of the bivalent sheep MSTN nano antibody. And (5) after the instruments required for glue making are installed, adding distilled water to verify whether glue leaks or not. Preparing 12% separation glue, mixing the separation glue uniformly, adding the separation glue into a glue maker, and picking out generated bubbles by using a long needle without leaving bubbles in the glue. And adding ethanol above the separation gel by using a pipettor to ensure that the upper part of the separation gel is flattened. After 20min, the ethanol was decanted. After the ethanol was completely volatilized, 5% concentrated gum was prepared and a comb was inserted. And (5) placing the glue maker on a test bed for standing for 2h. The comb was slowly and neatly removed from the gel. The gel was placed in an electrophoresis tank and 1 Xelectrophoresis solution was added. Removing the air bubbles in the gap. And (3) adding a bivalent sheep MSTN nano antibody sample into the concentrated gel, wherein each hole is 8 microliters, and adding a protein Marker. The voltage is adjusted to 80V, and the gel concentration electrophoresis is carried out for 30min. Isolate gel 120V,60min. And after 3-4h, stopping electrophoresis when bromophenol blue reaches the bottom of the gel. The glue is separated and placed in deionized water for cleaning. Immersing in staining solution, and staining for 35min on constant temperature shaking table. Preparing fresh decolorizing solution, decolorizing for many times on a constant temperature shaking table until strips are clear, and stopping decolorizing when the gel is completely decolorized. And cleaning with deionized water. And (4) placing the sample in a Bio-RAD agglutination imager, correcting the position of the glue, adjusting the pattern color and contrast, and photographing for storage.

(10) Protein activity was verified by ELISA:

MSTN protein was diluted with coating solution, ELISA plates coated, 200 ng/well, 4 degrees overnight incubation. PBST was washed 3 times. 5% PBSM (PBS + skim milk powder) 100. Mu.l per well was used and incubated at 37 ℃ for 1 hour. PBST was washed 3 times. Two nanobodies were diluted by 100, 1000, 10000, 100000, 1000000, 10000000, 100000000 times in gradient, PBS was added to the control group, 100 μ l per well was added, and incubation was performed at 37 ℃ for 1 hour. PBST was washed 3 times. 10000 times of diluted secondary antibody against alpaca, 100 microliters per well, was added and incubated at 37 ℃ for 1 hour. PBST washing 3 times. Adding 100 microliters of developing solution, incubating at 37 ℃ for 15 minutes, adding 50 microliters of stop solution, and reading the OD value by a microplate reader.

3. Large scale expression of nanobodies:

and carrying out mass expression and purification on the bivalent sheep MSTN nano antibody and the bivalent sheep MSTN nano antibody according to the MSTN antigen operation steps. The protein was collected ready for subsequent experiments.

4. As a result:

(1) Bivalent sheep MSTN nano antibody

The PCR product was subjected to agarose gel electrophoresis. The result shows that the target stripe is clear and the size is consistent.

As shown in fig. 2, a schematic diagram of a bivalent nanobody gene amplification product provided in the embodiment of the present invention.

Note: M.DNA Marker DL3000;1

M.DNA Marker DL3000；1:PCR production of Nanobody gene

(2) Recovery of pET-30A vector double enzyme digestion product

The agarose gel electrophoresis pattern of the digestion product is shown in FIG. 2, and the size of the target band is 5000bp. The verification is shown in fig. 3:

as shown in FIG. 3, the restriction electrophoresis diagram of pET-30A provided in the examples of the present invention.

Note: m1.PET-30A enzyme digestion product; m2.PET-30A enzyme digestion product; dna Marker DL6000;

M1.pET-30A Digestion with BamHI+XhoI；M2.pET-30A Digestion with BamHI+XhoI； SM331.DNA Marker DL6000；

(3) Bivalent sheep MSTN nano antibody expression vector protein induction expression

SDS-PAGE shows that the protein is successfully expressed, the protein is expressed by inclusion bodies, and the expression content is highest when the protein is at 27 ℃. The optimum induction concentration of IPTG was 1mmoL.

As shown in fig. 4, the SDS analysis results provided by the embodiment of the present invention are schematically shown for different concentrations of IPTG and different temperatures.

Note: m: protein marker;1: before induction; 2:0.5mM IPTG, supernatant at 15 ℃;3:0.5mM IPTG, precipitation at 15 ℃;4:0.5mM IPTG, supernatant at 27 ℃;5:0.5mM IPTG, precipitation at 27 ℃;6:0.5mM IPTG, supernatant at 37 ℃;7:0.5 mM IPTG, precipitation at 37 ℃;8:1.0mM IPTG, supernatant at 15 ℃;9:1.0mM IPTG, precipitation at 15 ℃;10:1.0mM IPTG, supernatant at 27 ℃;11:1.0mM IPTG, precipitation at 27 ℃;12:1.0mM IPTG, supernatant at 37 ℃;13:1.0mM IPTG, precipitation at 37 ℃.

M:Protein Marker；1.DE3 without IPTG induction；2.Supernatant of DE3 with15℃,0.5mM IPTG；3.Pallet of DE3 with15℃,0.5mM IPTG；4.Supernatant of DE3 with27℃,0.5mM IPTG；5. Pallet of DE3 with27℃,0.5mM IPTG；6.Supernatant of DE3 with37℃,0.5mM IPTG；7.Pallet of DE3 with37℃,0.5mM IPTG；8.Supernatant of DE3 with15℃,1mM IPTG；9.Pallet of DE3 with 15℃,1mM IPTG；10.Supernatant of DE3 with27℃,1mM IPTG；11.Pallet of DE3 with27℃,1mM IPTG；12.Supernatant of DE3 with37℃,1mM IPTG；13.Pallet of DE3 with37℃,1mM IPTG；

(4) The invention is further described below in connection with the ELISA validation of the bivalent sheep MSTN nanobody expression protein.

As shown in fig. 6, ELISA analysis showed that the protein had biological activity.

According to the results of SDS-PAGE and ELISA, the bivalent sheep MSTN nano antibody is successfully constructed and successfully expressed by induced protein.

(5) The invention is further described below in connection with the expression of nanobodies in large numbers.

And co-purifying the monovalent sheep MSTN protein for 6 times, and collecting 70mg of the monovalent sheep MSTN protein. The bivalent sheep MSTN protein is purified 7 times, and 70mg of the protein is collected. After the protein renaturation is finished, the endotoxin of the protein is removed and the protein is used for animal experiments.

The principle of the invention is further described below in connection with experiment 2.

Experiment 2;

1.1 materials

1.1.1 Experimental animals

75 one-month-old Kunming mice without specific pathogen were purchased from the laboratory animal center, affiliated Hospital, xinjiang medical university. In a 150-group sheep farm, 9 lambs in the year 2015 are purchased.

1.1.2 Experimental reagents

The same as experiment three.

1.1.3 Experimental instruments

TABLE 8 Main instruments

1.2 methods

1.2.1 Specific detection of antigen-antibody binding by Western Blot

And (3) taking the sheep bivalent MSTN nano antibody as an antigen to carry out membrane transfer. The sheep MSTN antigen is used as a primary antibody for incubation, the rabbit anti-human MSTN monoclonal antibody is used as a secondary antibody for incubation, and the sheep anti-rabbit HRP labeled antibody is used as a tertiary antibody for incubation.

Effect verification of 1.2.2 bivalent sheep MSTN nano antibody on mice

1.2.2.1 preparation of laboratory mice

One month old Kunming mice were purchased and weighed after two days of laboratory observation. The two with the lowest body weight were removed, together with the 10 with the highest body weight. The remaining 25 Kunming mice were divided into five groups of five mice each based on body weight from low to high. Then, five mice in the group were randomly divided into five groups of PBS, BSA, MSTN, univalent, and Bivalent. Feeding conditions are as follows: sufficient drinking water ensures that rats and mice have food all the time. The feeding is not limited, and the growth of the mice is ensured.

Mice were weighed every two days and injected once. Intramuscular injection into the thigh.

PBS group, 30 μ L PBS per injection;

BSA group, 30 μ L (60 μ g) BSA per injection;

MSTN group, each injection monovalent sheep MSTN nanobody 30 u L (60 u g);

univalent group, 30 μ L (60 μ g) of monovalent sheep MSTN nanobody per injection;

bivalent group, 30 μ L (60 μ g) of Bivalent sheep MSTN nanobody per injection.

1.2.2.2 anatomical observations of muscle shape changes

The weighed mice were sacrificed. Dissecting the mouse, taking two hind limbs of the mouse and the muscle and the bone between the two hind limbs, and photographing for storage. The removed tissue was immersed in 4% formaldehyde solution and 4% paraformaldehyde solution for subsequent experiments. Packaging the redundant tissue into plastic bags, and storing in a refrigerator at-80 deg.C.

1.2.2.3 preparation of sections to observe changes in muscle tissue

(1) Material taking: after the mice were slaughtered, the mice were quickly treated. The right hind thigh quadriceps was isolated using a scalpel. The tissue is suitable for being selected, is not too large, and is suitable for being made into slices. And (4) washing with physiological saline to remove bloodstains and other pollutants. The method comprises the following steps of 1: the proportion of 20 is preserved in newly prepared 4 percent formaldehyde solution for fixation for 48 hours.

(2) Removing the stationary liquid: wrapping the tissue block with gauze, placing in a container, and washing with clear water for 25h.

(3) Trimming: the tissue mass was placed in the operating table and trimmed using a scalpel. Two copies were made per mouse, one for the muscle fiber transection and one for the muscle fiber rip. Ensure the cut to be smooth and vertical to the tangent plane.

(4) And (3) dehydrating: the dehydration was performed using ethanol, and the tissue was placed in alcohols of different concentrations to displace the water. Placing in 30% alcohol, dehydrating for 3 hr; then placing the mixture into 50% alcohol solution, and dehydrating for 3 hours; placing in 70% alcohol solution for 12h; placing in 80% alcohol for 1h; placing in 90% alcohol for 1h; placing in 95% alcohol for 1h; placing in 100% ethanol for 15min; placing in 100% alcohol for 15min, and replacing with new alcohol; placing in 100% alcohol for 15min, and replacing with new alcohol. The ratio of the volume of alcohol to the volume of tissue was 49:1.

(5) And (3) transparency: the tissue was first placed in a mixture of xylene and ethanol 1:1 for 20min in the mixed solution. The tissue was removed and placed in xylene for 20min.

(6) Soaking: firstly, removing impurities in the wax to ensure that the used wax does not contain impurities. Then, the tissue was placed in melted soft wax for 0.5h; taking out the tissue and placing in new soft wax for 0.5h; taking out the tissue, and placing in melted hard wax for 0.5h; the tissue was removed and placed in fresh hard wax for 0.5h.

(7) Embedding: the mold is prepared, the hard wax is melted, and the soaked tissue is removed and placed in the hard wax. Waiting for the melted hard wax to cool. During embedding, the cut surface of the tissue block is kept consistent with the surface of the wax block.

(8) Shaping: and taking the embedded wax block, and trimming the wax block by using a scalpel. The tissue protrudes from the wax block. And the periphery is regular, and is preferably square. The integrity of the wax block and the integrity of the tissue are maintained during repair. Avoiding cutting the tissue.

(9) Slicing: when the muscle is sliced, the muscle texture is observed, the transverse direction is perpendicular to the muscle fibers, and the longitudinal direction is parallel to the muscle fiber direction.

(10) Cleaning the glass slide: the slide glass is washed in a washing solution and then washed with clear water. Then, the mixture was washed with distilled water and soaked in an alcohol solution.

(11) Unfolding and adhering the sheet: the sliced tissue was taken and placed in warm water at 37 ℃. After being completely flattened, the tissue was taped out using a glass slide. The position of the tissue section is adjusted and the tissue section is placed in a 38 ℃ oven for 3h.

1.2.2.4 HE staining

(1) The tissue slices were immersed in xylene for 16min, and then taken out and placed in fresh xylene for 16min.

(2) Preparing mixed solution of xylene and absolute ethyl alcohol, and soaking for 3min. Then soaking with anhydrous ethanol for 6min respectively. Placing in 80% alcohol for 6min. Taking out and placing in distilled water for 5min.

(3) Hematoxylin was used for 6min to allow for complete reaction. Then washed with distilled water for 5s. Adding into 1% ethanol hydrochloride solution for 4s. The washing was performed using distilled water.

(4) Soaking in 0.5% eosin solution for 2min, and cleaning.

(5) Placing into 80%, 95% solution for 5s, and soaking in anhydrous ethanol for 6min.

(6) Taking out, and soaking in xylene carbolic acid for 7min. Soaking in new xylene for three times, each for 2min. The resin was dropped onto the slide and the cover slip was covered. Air-drying, and storing in a cool and dry place.

1.2.3 bivalent sheep MSTN nano-antibody is verified on a mouse by repeated experiments.

1.2.3.1 animal experiments:

40 Kunming mice, one month old and 15 grams in weight, were purchased. Mice were observed for growth changes after being purchased from Xinjiang medical university and fed in the laboratory for three days. Healthy Kunming mice were selected for subsequent experiments. The method comprises the following steps of (1) dividing into 3 groups: experimental validation was performed on PBS group, univalent group, bivalent group, 10 per group. Injections and weighing experiments were performed every other day. Each PBS group was injected with 30 μ L PBS as a control group; univalent group injected 30 μ L (60 mg) of monovalent sheep MSTN nanobody per injection; bivalent groups were injected with 30 μ L (60 mg) of Bivalent sheep MSTN nanobodies per group. Weighing first, then injecting. Mouse status was observed and recorded.

1.2.3.2 anatomical observations of muscle volume changes:

the mice were sacrificed by cervical dislocation, and the two hind limbs were separated and stored by photographing. Both hind limbs were weighed.

1.2.3.3 preparation of sections for observing changes in muscle tissue

Muscle fiber sections were prepared from mouse leg adductor longus muscles.

2, results:

2.1 sheep bivalent MSTN nano antibody ELISA specificity detection antigen antibody combination;

as shown in fig. 6, the ELISA assay provided by the embodiments of the present invention is schematically illustrated.

2.2 first mouse Effect verification of Nano antibody

2.2.1 SDS detection of proteins

And performing SDS verification on the monovalent nanometer antibody, the bivalent nanometer antibody and the MSTN protein before injection, wherein the expressed protein is correct and is not decomposed, and animal experiments can be performed. SDS detects protein bands.

As shown in FIG. 7, a schematic diagram of the SDS-PAGE result provided by the embodiment of the invention.

Note: m: protein marker;1,2,3: a monovalent sheep MSTN nanobody; 4,5: bivalent sheep MSTN nanobody; 6: MSTN proteins

M:Protein marker；1,2,3:the univalent nanobody；4,5:the bivalent nanobody；6:MSTN protein

2.2.2 morphological observation of mouse hind limbs:

the growth promotion experiment was performed in mice for the first time, and the protein was injected 6 times for 15 days from 2015.12.21-2015.01.03. Mice were treated and the results observed.

As shown in fig. 8, the embodiment of the present invention provides a schematic diagram of the morphology of the muscle of the hind limb of the mouse.

Note: PBS: a control group injected with PBS; BSA: control group injected with albumin; injecting sheep MSTN protein group; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent injection of Bivalent sheep MSTN Nanobody groups.

PBS:PBS injected control group；BSA:injection of albumin in the control group；MSTN: injection sheep MSTN protein；Univalent:Injection Unit sheep MSTN Nanobodies；Bivalent: sheep MSTN injection bivalent Nanobody.

The muscle volume of the bivalent nanobody group is larger than that of the control group and the monovalent nanobody group when the separated hind limbs are measured. And the monovalent nanobody group is larger than the PBS control group. For the control to be obvious, the right hind limb was isolated and compared between the different groups.

As shown in fig. 9, the muscle morphology of the hind limb of the mouse provided by the embodiment of the present invention is schematically shown.

Note: PBS: a control group injected with PBS; BSA: control group injected with albumin; MSTN injection sheep MSTN proteome; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent injection of Bivalent sheep MSTN Nanobody groups.

The results show that muscle and bone are significantly increased in the bivalent nanobody group compared with the PBS control group and the BSA control group.

2.2.3 histomorphological observations:

as shown in fig. 10, a muscle tissue slice is provided according to an embodiment of the present invention.

Note: PBS: a control group injected with PBS; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent injection of Bivalent sheep MSTN Nanobody groups.

PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.

In different tissue sections, regions of the same area were selected and the number of muscle fibers was compared between different groups. The PBS control group has less than the monovalent nanometer antibody group, and the difference of p =0.042 & ltn & gt 0.05 is obvious; the PBS control group was less than the diabody group, with p =0.015<0.05 differences being significant. The monovalent nanobody group and the bivalent nanobody group p =0.209 >.

2.2.4 mouse body weight analysis

TABLE 9 mouse body weights

Note: PBS: a control group injected with PBS; BSA: a control group injected with albumin; MSTN injection sheep MSTN proteome; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent injection of Bivalent sheep MSTN Nanobody groups.

Wherein the BSA control group is significantly different from the MSTN control group by p =0.023 and woven fabric by 0.05, and the BSA group is significantly different from the monovalent nanometer antibody by p =0.047 and woven fabric by 0.05. The bivalent nanobodies were heavier than the MSTN group, and p =0.045<0.05 were significantly different. The change condition of the total body weight is that the bivalent nanometer antibody group and the BSA control group have the fastest growth speed and the largest weight gain. The MSTN group had the least weight gain. The weight gain of the bivalent nano antibody group is obviously higher than that of the PBS group, but the difference is not obvious.

TABLE 10 mouse hind limb weight

Note: PBS: a control group injected with PBS; BSA: control group injected with albumin; MSTN injection sheep MSTN proteome; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent is an injection group of Bivalent sheep MSTN nanobodies.

The bivalent nanobody group hind limb weight was the heaviest. Bivalent nanobodies were heavier than the control group, with p =0.03<0.05 differences being significant, bivalent nanobodies were heavier than the MSTN group, p =0.027<0.05 differences being significant, bivalent nanobodies were heavier than monovalent nanobodies, and p =0.008<0.01 differences being very significant.

By comparing the two sets of data, it can be seen that the diabody group has no significant difference from the other groups (except the MSTN group) in the total body weight, but has significant difference from the other groups in the hindlimb weight, and is the heaviest group among the groups. It is shown that the muscle-to-body ratio of the diabody group is the largest, and the muscle content in vivo is the largest group.

2.3 repeated experiments of Nanobodies in mice

2.3.1 Observation of muscle morphology

The experiment was performed using 30 Kunming mice, starting from 2016.1.26-2016.2.22 for 30 days. Each PBS group was injected with 30 μ L PBS as a control group; univalent group injected 30 μ L (60 mg) of monovalent sheep MSTN nanobody per injection; bivalent groups were injected with 30 μ L (60 mg) of Bivalent sheep MSTN nanobodies per group.

As shown in fig. 11, the embodiment of the present invention provides a schematic diagram of the morphology of the muscle of the hind limb of the mouse.

Fig.2-6 Mouse hindlimb muscle morphology diagram

A bivalent nanobody group, full muscle, can be observed. The right hind limb was separated and visually compared. And weighed.

As shown in fig. 12, the embodiment of the present invention provides a schematic diagram of the morphology of the muscle of the hind limb of the mouse.

Note: PBS: a control group injected with PBS; univalent: injecting a monovalent sheep MSTN nano antibody group; bivalent is an injection group of Bivalent sheep MSTN nanobodies.

Two groups of pictures are amplified, so that the pictures are more visually compared. The hind limb of the bivalent nano antibody group is obviously larger than that of the monovalent nano antibody group, and the monovalent nano antibody group is larger than that of the control group.

As shown in fig. 13, the muscle morphology of the hind limb of the mouse provided by the embodiment of the present invention is schematically shown.

2.3.2 weight changes

TABLE 11 mouse body weights

After the experiment was completed, the mice were treated. The final total body weight of the control group, the monovalent nano antibody and the bivalent nano antibody has no difference. There was no significant difference in promoting weight gain (table 11).

TABLE 12 mouse hind limb weight

There was no difference in the hindlimb weight statistics of the control, monovalent nanobody, and bivalent nanobody. However, the bivalent nanobody group is higher than the monovalent nanobody group by weight, and the monovalent nanobody group is higher than the control group. When dissected, the difference is obvious. The legs were therefore separated and weighed separately (table 12).

TABLE 13 mouse unilateral hind limb weight

Leg weighing revealed that the difference between bivalent nanobodies and monovalent nanobodies was very significant, p =0.004 straw-0.01, and that the difference between bivalent nanobodies and control group was very significant, p =0.000 straw-0.01 (table 13).

In table 11, the bivalent nanobody group did not significantly differ from the other two groups, but the bivalent nanobody group was significantly different from the other two groups in table 13. In total body weight, the diabody group was the lowest, but in leg muscle weight, the diabody was the highest.

Muscle content and proportion in the bivalent nanobody group were the highest in the experiment. Repeated experiments on mice further prove that the nano antibody mainly has the function of promoting muscle growth, can promote the development of animal skeletons while promoting the muscle growth of animals, and reduces the accumulation of fat.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> river university

<120> MSTN nano antibody, construction method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaattcatgc agttgcagct cgtgg 25

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcggccgcaa ggcctcgg 18

<210> 3

<211> 903

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggtacccagc tgcaactggt tgaaagcggt ggtggtagcg ttcaggcagg cggtagcctg 60

agcctgagct gtgcagcaag cggttatacc tatgttagcc gttatatggg ttggtttcgt 120

caggcaccgg gtaatgaacg tgaaggtgtt gcaaccattt ataccgcagg tattagcacc 180

tattatgatg caagcgttaa aggtcgtttc accatcagca aagataatgc caaaaatacc 240

gtttacctgc agatgaatag cctgaaaccg gaagataccg caatgtatta ttgtgcagcc 300

acccatgatg attatggtgg tagtggtagc cgtctgagtc cggcaagcta tgcatattgg 360

ggtcagggca cccaggttac cgttagcagc gaaccgaaaa ccccgaaacc gcagggtccg 420

cgtggtctgg gtggtggtgg aagtggtggc ggtggttcag gcggtggcgg tagtcaactg 480

cagctggtag aatcaggtgg tggctcagtt caagccggtg gtagcctgtc actgtcatgt 540

gcagcctcag gctatacata tgtttcacgc tacatgggct ggttccgtca agcccctggc 600

aacgaacgcg aaggcgtggc cacaatttat acagctggca tttcaacata ttatgacgcc 660

tcagtgaaag gtcgctttac gatttcaaaa gacaatgcga aaaacacggt gtatctgcaa 720

atgaattcac tgaaacctga ggacacagcc atgtactact gtgccgcaac acacgatgac 780

tatggcggta gcggttcacg tctgtcaccg gcatcatatg cctactgggg acagggtaca 840

caggtgacag ttagttcaga acctaaaaca cctaaacctc aaggccctcg tggtctgctc 900

gag 903

<210> 4

<211> 301

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Thr Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala

1 5 10 15

Gly Gly Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Val

20 25 30

Ser Arg Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu

35 40 45

Gly Val Ala Thr Ile Tyr Thr Ala Gly Ile Ser Thr Tyr Tyr Asp Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Ala Ala Thr His Asp Asp Tyr Gly Gly Ser Gly Ser Arg Leu

100 105 110

Ser Pro Ala Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Gly Pro Arg Gly Leu Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Leu

145 150 155 160

Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu

165 170 175

Ser Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Val Ser Arg Tyr Met

180 185 190

Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Gly Val Ala Thr

195 200 205

Ile Tyr Thr Ala Gly Ile Ser Thr Tyr Tyr Asp Ala Ser Val Lys Gly

210 215 220

Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

225 230 235 240

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala

245 250 255

Thr His Asp Asp Tyr Gly Gly Ser Gly Ser Arg Leu Ser Pro Ala Ser

260 265 270

Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro

275 280 285

Lys Thr Pro Lys Pro Gln Gly Pro Arg Gly Leu Leu Glu

290 295 300

Claims

1. The MSTN nano antibody is characterized in that the nucleotide sequence of the MSTN nano antibody is shown as SEQ ID NO:3, respectively.

2. The detection method of the MSTN nanobody of claim 1, which comprises:

1) Preparation of competent cells

Performing plate line drawing culture on the stored DH5 alpha, and selecting a monoclonal colony; adding the picked colonies into 5mL of LB culture medium, and carrying out overnight culture; the next day, transferring the cells into a 50mL culture medium, and culturing the cells to a logarithmic phase; taking DH5 alpha in logarithmic phase to carry out ice bath; centrifuging by using a high-speed low-temperature centrifuge, and collecting precipitates; injecting 6mL into the sediment, and suspending and precipitating the CaCl2 with low temperature of 0.1 mol; subpackaging, quick freezing in liquid nitrogen, and storing in-80 deg.C refrigerator;

2) Transformation into an expression bacterium DE3:

adding the connected product into an EP tube, adjusting a water bath kettle to 42 ℃, quickly putting the EP tube into the EP tube, continuously shaking the EP tube during the process, and reacting for 90s; taking out the EP tube, and placing on ice for 2 min; centrifuging, taking a sediment coating plate, and adding 30 microgram/ml kanamycin and 34 microgram/ml chloramphenicol; the next day, single clones were picked;

induced expression of the protein:

selecting a monoclonal for overnight culture; adding 100 mu L of culture solution into a 20mL culture medium containing Kana and chloramphenicol, and performing induced expression to logarithmic phase; adding IPTG with the final concentration of 0.5 mmol; subpackaging into different triangular flasks; different conditions are set: first bottle, using 15 deg.C, overnight shake culture; second bottle, 25 ℃, overnight induction; culturing 4h in a third bottle at 37 ℃ and 220 r/min; the group without IPTG addition was set as negative control; 4000 Collecting bacteria at r/min, and adding 500 mu L PBS into the precipitate for repeated blow beating; carrying out ultrasonic crushing, and collecting supernatant and precipitate; dissolving the collected precipitate with an inclusion body dissolving solution; adding the sample into a protein loading buffer, and boiling for 10min;

3) SDS-PAGE detection:

preparing 12% separation glue, uniformly mixing the separation glue, adding the separation glue into a glue maker, and picking out generated bubbles by using a long needle, wherein the bubbles cannot be left in the glue; adding ethanol above the separation gel by using a pipettor; 20 After min, pouring out the ethanol; after the ethanol is completely volatilized, preparing 5% concentrated glue, adding the concentrated glue, and inserting a comb; placing the gel maker on a test bed and standing for 2h; taking the comb off the glue; putting the gel into an electrophoresis tank, and adding 1X electrophoresis solution; removing air bubbles in the gap; adding a bivalent sheep MSTN nano antibody sample into concentrated gel, wherein each hole is 8 microliters, and adding a protein Marker; regulating voltage to 80V, and performing gel concentration electrophoresis for 30 min; separating gel at 120V for 60 min;3-4 h, and stopping electrophoresis when bromophenol blue reaches the bottom of the gel; separating the glue, and placing the glue in deionized water for cleaning; soaking in staining solution, and dyeing on constant temperature shaking table for 35 min; preparing a fresh decoloring solution, decoloring for many times on a constant-temperature shaking table until strips are clear, and stopping decoloring when the glue is completely decolored; washing with deionized water; placing in a Bio-RAD agglutination imager, correcting the position of the glue, and adjusting the color and contrast of the pattern;

4) The ELISA verified protein activity: using coating solution to dilute MSTN protein, coating ELISA plate, 200 ng/hole, and incubating overnight at 4 ℃; PBST wash 3 times; incubation with 5% PBSM100. Mu.l per well at 37 ℃ for 1 hour; PBST wash 3 times; diluting the nano antibody by 100, 1000, 10000, 100000, 1000000, 10000000 and 100000000 times in a gradient manner, adding PBS into a control group, adding 100 microliters of PBS into each hole, and incubating for 1 hour at 37 ℃; PBST wash 3 times; adding 10000 times diluted secondary antibody against alpaca 100 microliter per hole, incubating at 37 ℃ for 1 hour, and washing for 3 times by PBST; adding 100 microliters of developing solution, incubating at 37 ℃ for 15 minutes, adding 50 microliters of stop solution, and reading the OD value by using an enzyme-labeling instrument.