CN109942705A - A kind of MSTN nano antibody, construction method and its application - Google Patents
A kind of MSTN nano antibody, construction method and its application Download PDFInfo
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- CN109942705A CN109942705A CN201910299045.6A CN201910299045A CN109942705A CN 109942705 A CN109942705 A CN 109942705A CN 201910299045 A CN201910299045 A CN 201910299045A CN 109942705 A CN109942705 A CN 109942705A
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Abstract
The invention belongs to biomedicine technical field, a kind of MSTN nano antibody, construction method and its application are disclosed, the nano antibody library of sheep MSTN gene expression product is constructed；It is obtained by display technique of bacteriophage for MSTN gene expression product specific nano antibody；Construct bivalent sheep MSTN nano antibody.Effect of the MSTN nano antibody provided by the invention on mouse, which is presented as, increases animal muscle content, drops lower animal fat content.When weight gain is main with muscle weight gain, sheep MSTN nano antibody is presented as that weight gain is more than untreated control group；Mouse shows nano antibody group muscle weight significantly more than control group, and muscle accounts for the ratio of whole body also significantly beyond control group.Sheep MSTN nano antibody can promote animal muscle to grow.
Description
Technical field
The invention belongs to biomedicine technical field more particularly to a kind of MSTN nano antibodies, construction method and its application.
Background technique
Currently, the prior art commonly used in the trade is such that
MSTN is the gene of regulation muscle growth in animal body.Primarily serve the effect for inhibiting muscle growth.MSTN missing
Animal can be promoted double-muscling shape occur.When sheep MSTN gene action is interfered, the growth of Sheep Muscle can be promoted,
Can more mutton preferably be provided for people of all nationalities.By antigen-antibody reaction principle, the work that antibody inhibits MSTN is prepared
It can inhibit inhibition of the MSTN gene expression product to muscle growth with approach, the Sheep Fattening time can be shortened and increase flesh
The yield of meat.Horse is first brave to wait amplification pig (MSTN) gene, and the crude extract of the recombination MSTN albumen of expression is for being immunized mouse
The influence to weight is studied, mouse weight is immunized than control weight apparent increase, experimental mice is than control group mice weight
10.5%, significant difference (P < 0.05).Sheep and mouse MSTN genetic homology are higher.The MSTN gene order of mammal is high
Degree is conservative, and mouse, pig, chicken homology are 100%, and there was only 3 nucleotide differences between ox and sheep.MSTN gene is in sheep heredity
It guards, makes a variation less always in evolution.The genetic diversity of goat MSTN gene is very low.Sheep MSTN gene promoter is in difference
Sheep variety between it is also relatively conservative.Highly conserved MSTN provides strong for production nano antibody promotion ovine growth
It ensures.
By sheep MSTN protein immunization mouse, verification the verifying results can also be played.Nano antibody enters in Mice Body, Bu Huiyin
Play the immune response of mouse.Therefore, mouse is an outstanding platform for verifying nano antibody function.By preparing anti-sheep MSTN
The nano antibody of albumen, antagonism MSTN albumen inhibit MSTN gene activation approach, play the role of promoting ovine growth.Nanometer
Antibody passes through after high-temperature process, and albuminous degeneration is decomposed into polypeptide and amino acid, is digested in alimentary canal as amino acid, then
It is absorbed by animal, the approach for being administered to animal is not belonging to transgenic animals scope.
Nano antibody has the difference of essence as a kind of new antibody and general antibody structure.Nano antibody can be controlled
Treat the disease that some conventional antibodies can not treat.Nano antibody has the advantages such as small in size, stability is high, binding force is strong.
The advantage of nano antibody first is that the change of antibody can be achieved the goal by editor's gene.It is anti-obtaining nanometer
After body sequence, it can be haved the function that by being modified sequence to antibody modification.Specific aim transformation is carried out to antibody,
Make antibody that there is targetedly characteristic.Immune response can be reduced, the function of antibody is modified, popularityization transformation modification can be carried out
Deng.Also the groups with special construction such as linker link albumin can be used, to make nano antibody have different features.
Nano antibody possesses advantageous advantage in the context of detection of antigen, and obtains in not synantigen context of detection proud
Achievement.
In conclusion problem of the existing technology is:
(1) in the prior art, promote muscle growth effect for raising sheep MSTN nano antibody and lack theories integration.
(2) prior art is the preparation for MSTN polyclonal antibody, and it is more to only have this team to carry out sheep MSTN
Clonal antibody promotes sheep increasing weight experiment.
(3) MSTN antibody is not applied to production by the prior art.
(4) the problems such as prior art produces MSTN antibody higher cost, and there are the heterologous of antibody.
Solve the meaning of above-mentioned technical problem:
Display technique of bacteriophage is a kind of technology by phage expression foreign protein.The principle that elutriation uses is solid phase
Carrier Panning methods are enriched with by the method for ELISA and detect antibody with high specificity.Firstly, the antigen coat that antibody will be directed to
On a solid carrier, antigen blockade is reduced into nonspecific combination with albumen.Then be incorporated on solid phase carrier, not by
The bacteriophage elution of elution.After the bacteriophage elution of specific binding, bacteriophage is collected, and is expanded.It obtains being enriched with it
Antibody afterwards, then multiple screening operation is carried out, the antibody of high specific can be obtained.After being sequenced, just wanted
Purpose antibody, aim sequence.It gathers around that instrument and equipment in need is common, principle is simple, technical requirements are not high, is suitble to production nanometer
The advantages that antibody.M13 single stranded phage is common helper phage (Helper Phage).Can for bacteriophage duplication and
Proliferation provides the substances such as required enzyme (Flori defect).M13K07 belongs to M13 series, has kalamycin resistance.
M13K07 cannot be used as cloning vector.M13K07 belongs to filobactivirus, when expanding in bacterial body, bacterium will not be killed
Extremely.The lytic phages such as T7, T4 need to be purified first in elutriation, to guarantee the purity of albumen.The breeding speed of M13
Spend it is slower, expand numerous bacteriophage be secreted into it is extracellular.By being centrifuged to culture, available recombination, with destination protein
Phage particle.But storage capacity is bigger than other bacteriophages.Facilitate purifying, and other secretion are not influenced by bacterium.It bites
The advantages of phage display, which is to establish aim sequence with destination protein, to be contacted.After constructing aim sequence library, Ke Yijin
Row repeatedly screening.Library can be expanded and be saved by clone.This ensure that even if only one specificity is anti-
Body also can be repeatedly enriched with and be increased the content of specific antibody.A possibility that screening ideal protein is greatly increased,
Facilitate screening operation.
In early period, clone sheep MSTN gene constructs the prokaryotic expression system of sheep MSTN gene, and carries out in laboratory
Cytotoxicity verifying；After MSTN antigen-immunized animal, polyclonal antibody is prepared, is verified on sheep, is had to sheep
Apparent growth promoting function；Two-humped camel is immunized in antigen, takes leucocyte to carry out reverse transcription, constructs the nanometer of sheep MSTN gene
Antibody library.
By preparing MSTN nano antibody, may be implemented to produce antibody by prokaryotic expression.Nano antibody (Nanobody,
Nb) by the variable region preparation of heavy chain antibody in clone's camel body, volume is nanoscale, has and completely combines antigenic capacity.
Nano antibody have stable in physicochemical property, Gao Teyi, Gao Qinhe, low toxicity, it is soluble high, be not easy to cause immune response, be easy to produce,
It is easy to the advantages such as transformation.For nano antibody compared with conventional antibodies, the ability with antigen binding can replace tradition there is no reducing
Antibody and antigen binding.Bivalent and multifunctional nano antibody hundred times higher than monovalent nano antibody binding ability are to thousands of times.
The nano antibody R&D cycle is short, and risk is small, less investment, and output is big.Nano antibody can be used prokaryotic expression system and be expressed,
It can largely be expressed.Drug price can be greatly reduced using nano antibody substitution mab treatment, reduced
Treatment cost and be expected to improve therapeutic effect.
Nano antibody can keep conformation constant in harsh environment.Nano antibody heat resistanceheat resistant is especially good, can be in room temperature
Lower storage one week or more.And superpower resistance to acid and alkali can make nano antibody preferably resist different environment, can also increase
The sphere of action of nano antibody.The small volume of single domain heavy chain antibody also makes it obtain lower immunogenicity.Keep animal long
Phase injection albumen is possibly realized.The advantage that nano antibody has conventional antibodies no, also obtains in the research field of nano antibody
Proud achievement.The small volume of single domain heavy chain antibody, but have the function of heavy chain antibody and antigen binding, without Fc
End, will not cause complementary reaction.Nano antibody and the binding site of antigen are also different with monoclonal antibody.Nano antibody can be more
Closely be integrated on antigen, and can be incorporated into traditional antigen binding less than place.Building is with virus and bacterium surface
Product is that the library of antigen needs to select representational strain, has the strain for the treatment of or detection value.Monoclonal antibody is due to being easy
Cause complementary reaction, and volume is relatively too big, it is not easy to enter organization internal.
Research finds that the MSTN of various animals is relatively conservative, the MSTN difference very little of mouse and sheep.Since mouse obtains
Convenient, experimental implementation is simple, and effect occurs very fast.Therefore sheep MSTN antibody is carried out to the preliminary identification of effect on mouse.
Because mouse trunk includes a large amount of bones and its hetero-organization, and in hind leg, muscle accounts for major part, also can be easily separated, and has and represents
Property.Therefore, only mouse hind leg is separated, carries out muscle growth comparison.There is a judgement with the effect to nano antibody.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of MSTN nano antibody, construction method and its applications.
The invention is realized in this way a kind of construction method of MSTN nano antibody uses display technique of bacteriophage, pass through
The antibody of the method elutriation high specificity of ELISA principle.Detection and the high clone of antigen binding power, send bacterium solution to sequencing.Made
With the library R1, storage capacity is 1.8 × 10 when library construction12pfu/mL。
Monovalent sheep MSTN nano antibody is configured to bivalent nano antibody, by flexible linker (-
GGGGSGGGGSGGGGS- aim sequence) is connected as bivalent gene, constructs prokaryotic expression pET-30 (A) prokaryotic expression system.
Antibody activity is detected using SDS-PAGE electrophoresis and ELISA.To monovalent sheep MSTN nano antibody and bivalent sheep MSTN
Nano antibody carries out great expression.
Kunming mice is bought, grouping injection PBS, BSA, MSTN, monovalent nano antibody, bivalent nano antibody observe mouse
Changes of weight, observation muscle morphology variation production slice carry out histological observation；It carries out primary repetition to test, is divided into unit price and receives
Rice antibody group, bivalent nano antibody group, control group, every group 10.
It specifically includes:
Step 1: the nano antibody library of building sheep MSTN gene expression product；
Step 2: it is obtained by display technique of bacteriophage for MSTN gene expression product specific nano antibody；
Step 3: building bivalent sheep MSTN nano antibody SEQ ID NO:3.
(nano antibody gene order: (KpnI)
GGTACCCAGCTGCAACTGGTTGAAAGCGGTGGTGGTAGCGTTCAGGCAGGCGGTAGCCTGAGCCTGAG
CTGTGCAGCAAG CGGTTATACCTATGTTAGCCGTTATATGGGTTGGTTTCGTCAGGCACCGGGTAATGAACGTGA
AGGTGTTGCAACCATTTATAC CGCAGGTATTAGCACCTATTATGATGCAAGCGTTAAAGGTCGTTTCACCATCAG
CAAAGATAATGCCAAAAATACCGTTTACCT GCAGATGAATAGCCTGAAACCGGAAGATACCGCAATGTATTATTG
TGCAGCCACCCATGATGATTATGGTGGTAGTGGTAGCC GTCTGAGTCCGGCAAGCTATGCATATTGGGGTCAGGG
CACCCAGGTTACCGTTAGCAGCGAACCGAAAACCCCGAAACCGC AGGGTCCGCGTGGTCTGGGTGGTGGTGGAAG
TGGTGGCGGTGGTTCAGGCGGTGGCGGTAGTCAACTGCAGCTGGTAGAA TCAGGTGGTGGCTCAGTTCAAGCCGG
TGGTAGCCTGTCACTGTCATGTGCAGCCTCAGGCTATACATATGTTTCACGCTACAT GGGCTGGTTCCGTCAAGC
CCCTGGCAACGAACGCGAAGGCGTGGCCACAATTTATACAGCTGGCATTTCAACATATTATGAC GCCTCAGTGAA
AGGTCGCTTTACGATTTCAAAAGACAATGCGAAAAACACGGTGTATCTGCAAATGAATTCACTGAAACCTGA GGA
CACAGCCATGTACTACTGTGCCGCAACACACGATGACTATGGCGGTAGCGGTTCACGTCTGTCACCGGCATCATAT
GCCT ACTGGGGACAGGGTACACAGGTGACAGTTAGTTCAGAACCTAAAACACCTAAACCTCAAGGCCCTCGTGGT
CTGCTCGAG (XhoI))。
Amino acid sequence:
GTQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGVATIYTAGISTYYDASVKG
RF TISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWGQGTQVTVSSEPKTPKPQG PRG
LGGGGSGGGGSGGGGSQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGV ATIYTAGISTY
YDASVKGRFTISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWG
QGTQVTVSSEPKTPKPQGPRGLLE。
Further, in step 2, specific nano antibody is specific to prepare are as follows:
(1) acquisition of target fragment, design of primers；
(2) nano antibody sequence amplification；
(3) bivalent sheep MSTN nano antibody sequence PCR product glue recycles；
(4) double digestion is carried out to pET-30A carrier；
(5) recovery product, pET-30A carrier are connected；
(6) inducing expression of albumen；
(7) SDS-PAGE is detected；
(8) ELISA verifies protein active；
(9) great expression of nano antibody collects albumen and is ready for subsequent experimental.
Further, in step 3, bivalent sheep MSTN nano antibody is constructed, it is raw that verifying sheep MSTN nano antibody promotees muscle
Long effect.
Another object of the present invention is to provide a kind of sheep MSTN nano antibodies.
Further, the acquisition of target fragment, in design of primers, specifically include:
Using MSTN nano antibody as purpose sequence, bivalent nano antibody is prepared；.
Primer are as follows: GAATTCATGCAGTTGCAGCTCGTGG,
GCGGCCGCAAGGCCTCGG。
Further, nano antibody sequence amplification includes: bivalent sheep MSTN nano antibody sequence PCR condition
After end of reaction, 4 DEG C of preservation products.
Further, bivalent sheep MSTN nano antibody sequence PCR product glue, which recycles, includes:
The gel for running through electrophoresis is placed in the UV lamp, and target fragment is cut；The glue cut is put into EP pipe.To EP
Soltion B is added in pipe, places the gel in 60 DEG C, constantly shakes, gel is waited to melt.Gel after thawing is added
In collecting pipe, 3 minutes, 7000r/min are stood, is centrifuged 3min.Collecting pipe is removed, is eluted using Wash Soluion,
Elution is twice.Centrifugation thoroughly removes Wash Soluion.DNA is eluted using Elution Buffer；Carry out gel electrophoresis inspection
Survey time receives product purity.
Further, pET-30A carrier is carried out in double digestion, double digestion is carried out to carrier using BamHI, XhoI；37℃
React 2h.
Further, by recovery product, pET-30A carrier connection in, 50 DEG C of reaction 1h of PCR instrument.
Another object of the present invention is to provide a kind of MSTN that the construction method using the MSTN nano antibody constructs
Nano antibody.
Another object of the present invention is to provide a kind of detection method of MSTN nano antibody, described MSTN nanometers anti-
The detection method of body includes:
1) preparation of competent cell
The DH5 α of preservation is subjected to plate setting-out culture, picking monoclonal colonies.By the bacterium colony of picking, 5mL LB training is added
It supports in base, is incubated overnight；It second day, is transferred in 50mL culture medium, culture to logarithmic growth phase；Logarithmic growth phase
DH5 α carries out ice bath；It is centrifuged using high speed low temperature centrifugal machine, collects precipitating；0.1 mol of 6mL low temperature is injected to precipitating
CaCl2Suspend precipitating；It dispenses, it is quick-frozen in liquid nitrogen, it is put into -80 DEG C of refrigerators and saves；
2) it is transformed into expression bacterium DE3
The product connected is added in EP pipe, water-bath is adjusted to 42 DEG C, EP pipe is put into rapidly, is during which constantly shaken
It is dynamic, reaction time 90s；EP pipe is taken out, 2min on ice is put in.Centrifugation takes and precipitates painting plate, 30 μ g/ml kanamycins of addition,
34 μ g/ml chloramphenicol；Second day, by monoclonal picking；
The inducing expression of albumen
Picking monoclonal is incubated overnight；It takes 100 μ L of culture solution to be added to 20mL to contain in the culture medium of Kana and chloramphenicol,
Inducing expression is to logarithmic growth phase；The IPTG of final concentration of 0.5mmol is added；It is dispensed into different triangular flasks；Setting is different
Condition: first bottle, using 15 DEG C, overnight shaking table culture；It second bottle, 25 DEG C, induces overnight；Third bottle, 37 DEG C, 220 r/
Min cultivates 4h；The group for being not added with IPTG is set as negative control；4000r/min collects bacterium, and 500 μ L are added into precipitating
PBS is blown and beaten repeatedly；Ultrasonication is carried out, supernatant and precipitating are collected；The precipitating being collected into is molten using the progress of solubilization of inclusion bodies liquid
Solution；Protein loading buffer is added in sample, boils 10min；
3) SDS-PAGE is detected:
12% separation gel is prepared, separation gel is mixed, is added in gel maker, by the bubble of generation using using minute hand to choose
It out, cannot there are bubbles in glue.Ethyl alcohol is added above separation gel using pipettor；After 20min, ethyl alcohol is poured out；It waits
After ethyl alcohol volatilization is clean, 5% concentration glue of preparation is added, and is inserted into comb；Gel maker is put in testing stand and stands 2h；By comb
It is removed from glue.Glue is put into electrophoresis tank, 1 × electrophoresis liquid is added.Drive the bubble among gap out of；By bivalent sheep MSTN
For nano antibody sample pipetting volume into concentration glue, albumen Marker is added in 8 microlitres of every hole；Adjusting voltage is 80 V, and 30min is carried out
Gel electrophoresis is concentrated；Separation gel 120V, 60min.After 3-4h, when bromophenol blue being waited to reach glue bottom, stop electrophoresis；Glue is separated down
Come, is placed in deionized water and is cleaned.It immerses in dyeing liquor, in dyeing 35min on constant-temperature table.Prepare fresh decoloration
Liquid, it is clear to band in repeatedly decolourizing on constant-temperature table, when glue decoloration is clean, stop decoloration.It is cleaned using deionized water；It puts
It is placed in Bio-RAD agglutination imager, corrects the position of glue, adjust pattern color and contrast；
4) ELISA verifies protein active
MSTN albumen is diluted using coating buffer, is coated with elisa plate, the hole 200ng/, 4 degree are incubated overnight.PBST is washed 3 times.
Using the 100 microlitres of every holes 5%PBSM (PBS+ skimmed milk power), 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.By nano antibody
100,1000,10000,100000,1000000,10000000,100000000 times of gradient dilution, PBS is added in control group, is added
100 microlitres of every holes, 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.The secondary antibody 100 that 10000 times of diluted anti-alpacas are added is micro-
Every hole is risen, 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.100 microlitres of developing solution are added, 37 degrees Celsius, is incubated for 15 minutes,
50 microlitres of terminate liquid are added, microplate reader reads OD value.
In conclusion advantages of the present invention and good effect are as follows:
The present invention provides the nano antibody library of building sheep MSTN gene expression product.It is obtained by display technique of bacteriophage
To for MSTN gene expression product specific nano antibody.Bivalent sheep MSTN nano antibody is constructed, further verifies and mentions
High sheep MSTN nano antibody promotees muscle growth effect.Its function of promoting muscle growth is verified in Mice Body.
Promote mouse growth verifying: mouse is grouped, BSA albumen, MSTN albumen, MSTN nanometers of monovalent sheep is anti-
Body, bivalent sheep MSTN nano antibody inject mouse, and the mouse for injecting PBS is verified as a control group.The weight of animals variation is observed,
After experiment terminates, musculature production slice is taken, meat fiber situation of change is observed.As the result is shown: bivalent nano antibody
Group is compared with PBS control group and BSA control group, and muscle is significantly increased with bone.Meat fiber quantity PBS control group is more monovalent to be received
Rice antibody group is few, the significant difference of p=0.042 < 0.05；PBS control group is few compared with bivalent nano antibody group, and p=0.015 < 0.05 is poor
It is different significant.Monovalent nano antibody group and the bivalent nano antibody group difference of p=0.209 > 0.05 is not significant.After bivalent nano antibody group
Limb weight is most heavy.Bivalent nano antibody is than control group weight, the significant difference of p=0.03 < 0.05, bivalent nano antibody ratio MSTN
Group weight, the significant difference of p=0.027 < 0.05, for bivalent nano antibody than monovalent nano antibody weight, the difference of p=0.008 < 0.01 is extremely aobvious
It writes.Second of mouse test results: bivalent nano antibody group hind leg is significantly greater than monovalent nano antibody group, monovalent nano antibody group
It is big compared with control group.Control group, monovalent nano antibody, the final total weight of bivalent nano antibody do not have otherness.Leg weighing discovery
Difference is extremely significant between bivalent nano antibody and monovalent nano antibody, p=0.004 < 0.01, bivalent nano antibody and control group it
Between difference it is extremely significant, p=0.000 < 0.01.Conclusion: nano antibody group mouse muscle volume significantly increases, and muscle accounts for whole body specific gravity
It dramatically increases, and the weight gain of bivalent nano antibody group is more than monovalent nano antibody group, statistical analysis otherness is significant；Bivalent sheep
MSTN nano antibody can increase the quantity of meat fiber.Bivalent sheep MSTN nano antibody has the function for increasing muscle weight
Can, and increasing muscle weight is more than monovalent group；When weight gain is main with muscle weight gain, sheep MSTN nano antibody is presented as increasing
It is again more than the control group of PBS；When weight gain is main with lipopexia, sheep MSTN nano antibody is presented as increase muscle weight
And increase weight, the differences such as gaining effect and fat weight gain are unobvious in certain time.
The present invention provides the nano antibody library of building sheep MSTN gene expression product.It is obtained by display technique of bacteriophage
To for MSTN gene expression product specific nano antibody.Bivalent sheep MSTN nano antibody is constructed, further verifies and mentions
High sheep MSTN nano antibody promotees muscle growth effect.
Effect of the MSTN nano antibody on mouse, which is presented as, increases animal muscle content, drops lower animal fat content.When
Weight gain with muscle weight gain be it is main when, sheep MSTN nano antibody be presented as weight gain be more than untreated control group；When weight gain is with rouge
When fat accumulation is main, sheep MSTN nano antibody, which is presented as, to be increased muscle weight and increases weight, in a short time gaining effect and fat product
Tired gaining effect does not have too many differences.Mouse shows nano antibody group muscle weight significantly more than control group, and muscle accounts for whole body
Ratio is also significantly beyond control group.Bivalent nano antibody group animal, meat fiber quantity increases in muscle section unit of display area
Add.Sheep MSTN nano antibody can promote animal muscle to grow.
The present invention can link two nano antibodies by adding amino acid between two nano antibodies, play increase and make
With the purpose of effect.It will be attached for the different nano antibodies of same antigen by linker, antigen-antibody can be increased
In conjunction with efficiency.The amino acid sequence for linking two albumen is referred to as Linker.Outstanding linker can be such that fusion protein plays
Maximum effect, and itself is unaffected.The length of linker also affects the conformation and function of albumen.It selects too long
Linker is easy to be selected too short linker by enzyme hydrolysis then and will affect the performance of protein function.Common linker be (-
GGGGSGGGGSGGGGS-).The molecular weight of glycine (glycine, Gly, G) is smaller, flexibility with higher.Serine
(serine, Ser, S) hydrophily with higher.For suppressing virus replication, bivalent nano antibody validity is at least unit price
60 times of nano antibody.
Detailed description of the invention
Fig. 1 is MSTN nano antibody and its application method flow chart provided in an embodiment of the present invention.
Fig. 2 is bivalent nano antibody gene amplification product schematic diagram provided in an embodiment of the present invention.
Fig. 3 is the restriction enzyme digestion and electrophoresis figure of pET-30A provided in an embodiment of the present invention.
Fig. 4 is the SDS analysis result schematic diagram of various concentration IPTG provided in an embodiment of the present invention, different temperatures.
Fig. 5 be various concentration IPTG provided in an embodiment of the present invention, different temperatures Western blot analysis result show
It is intended to.
Fig. 6 is elisa assay schematic diagram provided in an embodiment of the present invention.
Fig. 7 is SDS-PAGE verification result schematic diagram provided in an embodiment of the present invention.
Fig. 8 is mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Fig. 9 is mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Figure 10 is musculature slice schematic diagram provided in an embodiment of the present invention.
Figure 11 is mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Figure 12 is mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Figure 13 is mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In the prior art, promote muscle growth effect for raising sheep MSTN nano antibody and lack theories integration.
It elaborates with reference to the accompanying drawing to application principle of the invention；
As shown in Figure 1, MSTN nano antibody provided in an embodiment of the present invention and its application method are as follows:
S101: the nano antibody library of building sheep MSTN gene expression product；
S102: it is obtained by display technique of bacteriophage for MSTN gene expression product specific nano antibody；
S103: building bivalent sheep MSTN nano antibody.
In step S103, bivalent sheep MSTN nano antibody SEQ ID NO:3.
(nano antibody gene order: (KpnI)
GGTACCCAGCTGCAACTGGTTGAAAGCGGTGGTGGTAGCGTTCAGGCAGGCGGTAGCCTGAGCCTGAG
CTGTGCAGCAAG CGGTTATACCTATGTTAGCCGTTATATGGGTTGGTTTCGTCAGGCACCGGGTAATGAACGTGA
AGGTGTTGCAACCATTTATAC CGCAGGTATTAGCACCTATTATGATGCAAGCGTTAAAGGTCGTTTCACCATCAG
CAAAGATAATGCCAAAAATACCGTTTACCT GCAGATGAATAGCCTGAAACCGGAAGATACCGCAATGTATTATTG
TGCAGCCACCCATGATGATTATGGTGGTAGTGGTAGCC GTCTGAGTCCGGCAAGCTATGCATATTGGGGTCAGGG
CACCCAGGTTACCGTTAGCAGCGAACCGAAAACCCCGAAACCGC AGGGTCCGCGTGGTCTGGGTGGTGGTGGAAG
TGGTGGCGGTGGTTCAGGCGGTGGCGGTAGTCAACTGCAGCTGGTAGAA TCAGGTGGTGGCTCAGTTCAAGCCGG
TGGTAGCCTGTCACTGTCATGTGCAGCCTCAGGCTATACATATGTTTCACGCTACAT GGGCTGGTTCCGTCAAGC
CCCTGGCAACGAACGCGAAGGCGTGGCCACAATTTATACAGCTGGCATTTCAACATATTATGAC GCCTCAGTGAA
AGGTCGCTTTACGATTTCAAAAGACAATGCGAAAAACACGGTGTATCTGCAAATGAATTCACTGAAACCTGA GGA
CACAGCCATGTACTACTGTGCCGCAACACACGATGACTATGGCGGTAGCGGTTCACGTCTGTCACCGGCATCATAT
GCCT ACTGGGGACAGGGTACACAGGTGACAGTTAGTTCAGAACCTAAAACACCTAAACCTCAAGGCCCTCGTGGT
CTGCTCGAG (XhoI))。
Amino acid sequence: SEQ ID NO:4
GTQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGVATIYTAGISTYYDASVKG
RF TISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWGQGTQVTVSSEPKTPKPQG PRG
LGGGGSGGGGSGGGGSQLQLVESGGGSVQAGGSLSLSCAASGYTYVSRYMGWFRQAPGNEREGV ATIYTAGISTY
YDASVKGRFTISKDNAKNTVYLQMNSLKPEDTAMYYCAATHDDYGGSGSRLSPASYAYWG
QGTQVTVSSEPKTPKPQGPRGLLE。
Specific nano antibody provided in an embodiment of the present invention, it is specific to prepare are as follows:
(1) acquisition of target fragment, design of primers；
(2) nano antibody sequence amplification；
(3) bivalent sheep MSTN nano antibody sequence PCR product glue recycles；
(4) double digestion is carried out to pET-30A carrier；
(5) recovery product, pET-30A carrier are connected；
(6) inducing expression of albumen；
(7) SDS-PAGE is detected；
(8) WB verifies protein active；
(9) great expression of nano antibody collects albumen and is ready for subsequent experimental.
In step S103, building bivalent sheep MSTN nano antibody provided in an embodiment of the present invention, verifying sheep MSTN is received
Meter Kang Ti promotees muscle growth effect.
It is provided in an embodiment of the present invention to be increased weight using sheep MSTN nano antibody applied to animal muscle, specific steps are as follows:
(1) ELISA specific detection antigen-antibody combines；
(2) effect of the verifying bivalent sheep MSTN nano antibody on mouse；
(3) bivalent sheep MSTN nano antibody repeats experimental verification on mouse.
It is provided in an embodiment of the present invention to be increased weight using sheep MSTN nano antibody applied to mouse muscle.
It is provided in an embodiment of the present invention to apply animal using sheep MSTN nano antibody, increase Lean mass, reduces animal
Fat content.
Application principle of the invention is further described below with reference to specific experiment；
1, material
(1) bacterial strain, carrier
Rosetta (DE3) bacterial strain is saved by zoonosis research laboratory.PET-30a expression vector is bought from health
Century biotech firm.
(2) reagent
1 main agents of table
(3) instrument
2 key instrument of table
2, method
(1) acquisition of target fragment, design of primers
Laboratory has obtained a nano antibody high with MSTN antigen-binding specificity early period, and carries out meticulous born of the same parents' poison
Property experiment and mouse growth promotion experiment.Promote the mouse muscle speed of growth obvious.Therefore, as a purpose by the nano antibody
Sequence prepares bivalent nano antibody.By aim sequence Song Huanuo Time Technology Co., Ltd, centre uses flexibility linker
(- GGGGSGGGGSGGGGS-) connection.
3 design of primers of table
(2) nano antibody sequence amplification
1) PCR system
4 pcr amplification reaction system of table
(primer concentration dilution: 400 μ L ddH are added in 1OD2O)
2) bivalent sheep MSTN nano antibody sequence PCR condition
5 PCR reaction condition of table
After end of reaction, 4 DEG C of preservation products.
(3) bivalent sheep MSTN nano antibody sequence PCR product glue recycles
The gel for running through electrophoresis is placed in the UV lamp, and target fragment is cut.Notice that the gel volume cut should not be too
Greatly.The glue cut is put into EP pipe.Soltion B is added into EP pipe, places the gel in 60 DEG C, constantly shakes, etc.
Melt to gel.Gel after thawing is added in collecting pipe, stands 3 minutes, 7000r/min, is centrifuged 3min.Collecting pipe is taken
Under, it is eluted using Wash Soluion, elution is twice.Centrifugation thoroughly removes Wash Soluion.Use Elution
Buffer elutes DNA.Carry out detected through gel electrophoresis recovery product purity.
(4) double digestion is carried out to pET-30A carrier
Double digestion is carried out to carrier using BamHI, XhoI.
6 digestion system of table
37 DEG C of reaction 2h.
(5) digestion products glue recycles
Reclaimer operation step with it is upper identical.
(6) recovery product, pET-30A carrier are connected
7 linked system of table
50 DEG C of reaction 1h of PCR instrument are set.
1) preparation of competent cell:
The DH5 α of preservation is subjected to plate setting-out culture, picking monoclonal colonies.By the bacterium colony of picking, 5mL LB training is added
It supports in base, is incubated overnight.It second day, is transferred in 50mL culture medium, culture to logarithmic growth phase.Logarithmic growth phase
DH5 α carries out ice bath.It is centrifuged using high speed low temperature centrifugal machine, collects precipitating.0.1 mol of 6mL low temperature is injected to precipitating
CaCl2, which suspends, to be precipitated.It dispenses, it is quick-frozen in liquid nitrogen, it is put into -80 DEG C of refrigerators and saves.
2) it is transformed into expression bacterium DE3:
The product connected is added in EP pipe, water-bath is adjusted to 42 DEG C, EP pipe is put into rapidly, is during which constantly shaken
It is dynamic, reaction time 90s.EP pipe is taken out, 2min on ice is put in.Centrifugation takes and precipitates painting plate, 30 μ g/ml kanamycins of addition,
34 μ g/ml chloramphenicol.Second day, by monoclonal picking, it is sent to the sequencing of Shanghai Sheng Gong biotechnology Co., Ltd.
(8) inducing expression of albumen:
By sequencing, correctly clone carries out protecting bacterium work, and picking monoclonal is incubated overnight.100 μ L of culture solution is taken to be added to
20mL contains in the culture medium of Kana and chloramphenicol, inducing expression to logarithmic growth phase.It is added final concentration of 0.5mmol's
IPTG.It is dispensed into different triangular flasks.Different conditions is set: first bottle, using 15 DEG C, staying overnight shaking table culture；Second bottle,
It 25 DEG C, induces overnight；Third bottle, 37 DEG C, 220r/min cultivates 4h；The group for being not added with IPTG is set as negative control.4000r/
Min collects bacterium, and 500 μ L PBS are added into precipitating and blow and beat repeatedly.Ultrasonication is carried out, supernatant and precipitating are collected.It will collect
To precipitating dissolved using solubilization of inclusion bodies liquid.Protein loading buffer is added in sample, boils 10min.
(9) SDS-PAGE is detected:
Verify bivalent sheep MSTN nano antibody inducing expression.Instrument needed for glue is installed, distilled water is added and tests
Card whether leak adhesive.12% separation gel is prepared, separation gel is mixed, is added in gel maker, the bubble of generation is used into minute hand
Choose, it cannot there are bubbles in glue.Ethyl alcohol is added above separation gel using pipettor, guarantees to flatten above separation gel.
After 20min, ethyl alcohol is poured out.After waiting ethyl alcohol volatilization clean, 5% concentration glue of preparation is added, and is inserted into comb.By gel maker
It is put in testing stand and stands 2h.Slow and neat removes comb from glue.Glue is put into electrophoresis tank, 1 × electrophoresis liquid is added.
Drive the bubble among gap out of.By bivalent sheep MSTN nano antibody sample pipetting volume into concentration glue, egg is added in 8 microlitres of every hole
White Marker.Adjusting voltage is 80 V, and 30min carries out concentration gel electrophoresis.Separation gel 120V, 60min.After 3-4h, bromine phenol is waited
When indigo plant reaches glue bottom, stop electrophoresis.Glue is separated, is placed in deionized water and is cleaned.It immerses in dyeing liquor, Yu Heng
35min is dyed on warm shaking table.Fresh destainer is prepared, it is clear to band in repeatedly decolourizing on constant-temperature table, when glue decoloration is clean
When, stop decoloration.It is cleaned using deionized water.It is placed in Bio-RAD agglutination imager, corrects the position of glue, adjust pattern
Color and contrast carry out preservation of taking pictures.
(10) ELISA verifies protein active:
MSTN albumen is diluted using coating buffer, is coated with elisa plate, the hole 200ng/, 4 degree are incubated overnight.PBST is washed 3 times.
Using the 100 microlitres of every holes 5%PBSM (PBS+ skimmed milk power), 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.By two nanometers
100,1000,10000,100000,1000000,10000000,100000000 times of antibody gradient dilution, PBS is added in control group,
100 microlitres of every holes are added, 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.The secondary antibody of 10000 times of diluted anti-alpacas is added
100 microlitres of every holes, 37 degrees Celsius are incubated for 1 hour.PBST is washed 3 times.100 microlitres of developing solution are added, 37 degrees Celsius, is incubated for 15
Minute, 50 microlitres of terminate liquid are added, microplate reader reads OD value.
3, the great expression of nano antibody:
By monovalent sheep MSTN nano antibody and bivalent sheep MSTN nano antibody according to middle MSTN antigen operating procedure into
Row great expression and purifying.It collects albumen and is ready for subsequent experimental.
4, result:
(1) bivalent sheep MSTN nano antibody
PCR product is subjected to agarose gel electrophoresis.Purpose band is clear as the result is shown, and size meets.
As shown in Fig. 2, bivalent nano antibody gene amplification product schematic diagram provided in an embodiment of the present invention.
Note: M.DNA Marker DL3000；The PCR product of 1:Nanobody gene
M.DNA Marker DL3000；1:PCR production of Nanobody gene
(2) recycling of pET-30A carrier double enzyme digestion product
Digestion products run agarose gel electrophoresis figure as shown in Fig. 2, purpose band size is 5000bp.Verifying is such as Fig. 3 institute
Show:
As shown in figure 3, the restriction enzyme digestion and electrophoresis figure of pET-30A provided in an embodiment of the present invention.
Note: M1.pET-30A digestion products；M2.pET-30A digestion products；SM331.DNA Marker DL6000；
M1.pET-30A Digestion with BamHI+XhoI；M2.pET-30A Digestion with BamHI+
XhoI； SM331.DNA Marker DL6000；
(3) the protein induced expression of bivalent sheep MSTN nano antibody expression vector
SDS-PAGE shows albumen successful expression, and albumen is inclusion body expression, and when albumen is at 27 DEG C, expression contents are most
It is high.The best induced concentration of IPTG is 1mmoL.
As shown in figure 4, the SDS of various concentration IPTG provided in an embodiment of the present invention, different temperatures analyzes result schematic diagram.
Note: M:Protein marker；1: before luring；2:0.5mM IPTG, 15 DEG C of supernatants；3:0.5mM IPTG, 15 DEG C heavy
It forms sediment；4:0.5mM IPTG, 27 DEG C of supernatants；5:0.5mM IPTG, 27 DEG C of precipitatings；6:0.5mM IPTG, 37 DEG C of supernatants；7:0.5 mM
IPTG, 37 DEG C of precipitatings；8:1.0mM IPTG, 15 DEG C of supernatants；9:1.0mM IPTG, 15 DEG C of precipitatings；10:1.0mM IPTG, 27 DEG C
Supernatant；11:1.0mM IPTG, 27 DEG C of precipitatings；12:1.0mM IPTG, 37 DEG C of supernatants；13:1.0mM IPTG, 37 DEG C of precipitatings.
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) present invention is further retouched below with reference to the ELISA verifying of bivalent sheep MSTN nano antibody expression albumen
It states.
As shown in fig. 6, elisa assay is shown, albumen has biological activity.
According to SDS-PAGE and ELISA as a result, bivalent sheep MSTN nano antibody constructs successfully, by inducible protein success
Expression.
(5) below with reference to the great expression of nano antibody, the invention will be further described.
Copurification unit price sheep MSTN albumen 6 times collects monovalent sheep MSTN protein 70 mg.Purify bivalent sheep MSTN
Albumen 7 times, collect protein 70 mg.After protein renaturation finishes, except the endotoxin of deproteinized, it to be used for zoopery.
It is further described below with reference to 2 pairs of application principles of the invention of experiment.
Experiment 2；
1.1 material
1.1.1 experimental animal
Affiliated hospital, Xinjiang Medicine University Experimental Animal Center buys a monthly age no-special pathogen kunming mice 75.Stone
He Zi150Tuan company Yang Chang, buy 2015 sheep Yearling lamb 9.
1.1.2 experiment reagent
With experiment three.
1.1.3 laboratory apparatus
8 key instrument of table
1.2 method
1.2.1 Western Blot specific detection antigen-antibody combines
Using sheep bivalent MSTN nano antibody as antigen, transferring film is carried out.Sheep MSTN antigen is that primary antibody is incubated for, rabbit-anti
People's MSTN monoclonal antibody is secondary antibody incubation, and goat-anti rabbit HRP labelled antibody is three anti-incubations.
1.2.2 compliance test result of the bivalent sheep MSTN nano antibody on mouse
1.2.2.1 the preparation of experiment mice
Buy a monthly age kunming mice, laboratory observation two days later, weigh.By minimum two of weight, with body
Highest 10 removals of weight.Remaining 25 kunming mices, five groups, every group five are divided by weight from low to high.Then, five in group
Mouse is randomly divided into five groups of PBS, BSA, MSTN, Univalent, Bivalent.Feeding condition: sufficient drinking-water, when guarantee
There is mouse grain at quarter.Unlimited feeding guarantees the growth of mouse.
Weighing in mouse every two days is primary, and injection is primary.Medial Thigh Skin intramuscular injection.
PBS group, per injection PBS30 μ L；
BSA group, per injection BSA30 μ L (60 μ g)；
MSTN group, 30 μ L (60 μ g) of per injection unit price sheep MSTN nano antibody；
Univalent group, 30 μ L (60 μ g) of per injection unit price sheep MSTN nano antibody；
Bivalent group, 30 μ L (60 μ g) of per injection bivalent sheep MSTN nano antibody.
1.2.2.2 muscle profile variation is dissected and observed
Mouse after weighing is put to death.Mouse is dissected, takes muscle between two hind leg of mouse and two hind legs and bone simultaneously
It takes pictures preservation.The tissue that will be removed immerses in 4% formalin and 4% paraformaldehyde solution, in case subsequent experimental.It will
Extra tissue is dispensed into polybag, and -80 DEG C of refrigerators save.
1.2.2.3 production sections observation musculature variation
(1) it draws materials: after mouse is butchered, mouse being handled rapidly.Using scalpel, right hind stock four-head is separated
Flesh.Tissue selection size is suitable, should not be too big, is suitble to production slice.Cleaned using physiological saline, wash away bloodstain and
Other pollutants.New 4% formalin of preparing, which is stored in, with the ratio of 1:20 fixes 48h.
(2) it removes fixer: tissue block being wrapped up using gauze, is placed in container, rinse 25h using clear water.
(3) it modifies: tissue block being placed in station, is modified using scalpel.Every mouse makes two parts, and portion is flesh
Fibre transections, portion are that muscle fibre is longitudinal sectional.Guarantee that notch is smooth, and guarantees vertical with section.
(4) it is dehydrated: carrying out dehydrating operations using ethyl alcohol, tissue is placed among the alcohol of various concentration, moisture is displaced.
It is first placed in 30% alcohol, is dehydrated 3h；Later, it is placed in 50% alcoholic solution, is dehydrated 3h；It is placed in 70% alcoholic solution
12h；It is placed in 80% alcohol 1h；It is placed in 90% alcohol 1h；It is placed in 95% alcohol 1h；It is placed in 100% alcohol 15min；It is placed in
100% alcohol 15min, the alcohol more renewed；It is placed in 100% alcohol 15min, the alcohol more renewed.The volume and tissue of alcohol
The ratio between volume be 49:1.
(5) transparent: tissue being first placed in 20min in the solution that dimethylbenzene is mixed with ethyl alcohol 1:1.Tissue is taken out, is placed in
20min in dimethylbenzene.
(6) it is impregnated with: first the impurity in wax being removed, is guaranteed free from foreign meter in the wax used.Then, tissue is placed in thawing
Soft wax in 0.5h；It takes out tissue and is placed in new soft wax 0.5h；Tissue is taken out, 0.5h in the hard wax of thawing is placed in；Tissue is taken out,
It is placed in 0.5h in new hard wax.
(7) it embeds: getting out mold, hard wax is melted, the tissue being impregnated with taking-up is placed among hard wax.Wait thawing
Hard wax is cooling.When embedding, the face of the section of tissue block and wax stone is consistent.
(8) moulding: taking embedded wax stone, is repaired wax stone neatly using scalpel.Tissue is set to protrude from wax stone.And
Periphery is regular, preferably square.When repairing, keep wax stone integrality and tissue it is complete.Avoid incised tissue.
(9) it is sliced: paying attention to grain of meat when slice, it is crosscutting longitudinal sectional to be parallel to meat fiber perpendicular to meat fiber
Direction.
(10) it cleans glass slide: glass slide being placed in cleaning solution and is cleaned, then cleaned using clear water.Later using distillation
Water cleaning, steeps in alcoholic solution.
(11) open up piece and bonding die: the tissue after taking slice is placed in 37 DEG C of warm water.After flattening completely, load glass is used
Piece is viscous out by tissue.Histotomy position is adjusted, 3h in 38 DEG C of baking ovens is put in.
1.2.2.4 HE is dyed
(1) it takes histotomy to be dipped in 16min in dimethylbenzene, then takes out and be placed in 16min in new dimethylbenzene.
(2) dimethylbenzene and dehydrated alcohol mixed liquor are prepared, 3min is impregnated.Then 6min is respectively impregnated with dehydrated alcohol respectively.
It is placed in 80% alcohol, 6min.Taking-up is placed in 5min in distilled water.
(3) hematoxylin 6min is used, is reacted fully.Then make 5s wash with distilled water.Be put into 1% acidic alcohol it is molten
4s in liquid.It is cleaned using distilled water.
(4) Yihong liquid for using 0.5% infiltrates 2min, cleaning.
(5) it is put into 5s in 80%, 95%, is then dipped in 6min in dehydrated alcohol.
(6) it takes out, impregnates 7min using carboxylol.It is respectively placed in new dimethylbenzene and impregnates three times, every time
2min.Resin, covered are added dropwise on glass slide.After drying, it is stored at shady and cool drying.
1.2.3 bivalent sheep MSTN nano antibody repeats experimental verification on mouse.
1.2.3.1 zoopery:
Buy a monthly age, weight is in kunming mice 40 of 15 grams.After Xinjiang Medicine University's purchase, fed in laboratory
It supports three days, observation mouse growth variation.The kunming mice of health is picked out, subsequent experimental is carried out.It is divided into 3 groups: PBS group,
Univalent group, Bivalent group, carry out experimental verification by every group 10.Every other day carry out injection and experiment of weighing.PBS
Every 30 μ L PBS of injection of group are as a control group；MSTN nanometers of 30 μ L (60mg) of Univalent group per injection monovalent sheep is anti-
Body；Every group of injection of Bivalent group 30 μ L (60mg) bivalent sheep MSTN nano antibody.First weigh, it is rear to inject.It observes and records
Mouse state.
1.2.3.2 anatomic observation muscle volume variation:
By mouse break neck put to death, separate two hind legs, preservation of taking pictures.Two hind legs are weighed.
1.2.3.3 production sections observation musculature variation
It takes mouse leg long adductor muscle (adductor longus muscle), production meat fiber slice.
2 results:
2.1 sheep bivalent MSTN nano antibody ELISA specific detection antigen-antibodies are used in combination；
As shown in fig. 6, elisa assay schematic diagram provided in an embodiment of the present invention.
2.2 nano antibody first time mouse compliance test results
2.2.1 SDS detects albumen
SDS verifying is carried out to monovalent nano antibody, bivalent nano antibody and MSTN albumen before injection, shows expression
Albumen is correct, does not decompose, and can carry out zoopery.SDS detects protein band.
As shown in fig. 7, SDS-PAGE verification result schematic diagram provided in an embodiment of the present invention.
Note: M:Protein marker；1,2,3: monovalent sheep MSTN nano antibody；4,5: MSTN nanometers of bivalent sheep is anti-
Body；6:MSTN albumen
M:Protein marker；1,2,3:the univalent nanobody；4,5:the bivalent
nanobody；6:MSTN protein
2.2.2 mouse hind leg morphological observation:
Growth promotion experiment is carried out in Mice Body for the first time, from 2015.12.21-2015.01.03 totally 15 days, injects albumen
6 times.Mouse is handled, result is observed.
As shown in figure 8, mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Note: PBS: the control group of PBS is injected；BSA: the control group of albumin is injected；MSTN injects sheep MSTN protein groups；
Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent: injection bivalent sheep MSTN nano antibody group.
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.
Isolated hind leg is measured, discovery bivalent nano antibody group muscle volume is greater than control group and monovalent nanometer
Antibody group.And monovalent nano antibody group is greater than PBS control group.To keep control obvious, right hind is separated, carry out different groups it
Between compare.
As shown in figure 9, mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Note: PBS: the control group of PBS is injected；BSA: the control group of albumin is injected；MSTN injects sheep MSTN protein groups；
Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent: injection bivalent sheep MSTN nano antibody group.
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 results show that bivalent nano antibody group and PBS control group and BSA control group compare, muscle is significantly increased with bone.
2.2.3 Histomorphological:
As shown in Figure 10, musculature provided in an embodiment of the present invention is sliced schematic diagram.
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
In different histotomies, the region of same area is selected, muscle fibre quantity between more different groups.PBS pairs
According to group, p=0.042 < 0.05 significant difference few compared with monovalent nano antibody group；PBS control group is few compared with bivalent nano antibody group, p=
0.015 < 0.05 significant difference.Monovalent nano antibody group and the bivalent nano antibody group difference of p=0.209 > 0.05 is not significant.
2.2.4 mouse weight is analyzed
9 mouse weight of table
Note: PBS: the control group of PBS is injected；BSA: the control group of albumin is injected；MSTN injects sheep MSTN protein groups；
Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent: injection bivalent sheep MSTN nano antibody group.
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.
Wherein BSA control group ratio MSTN control group weight, the significant difference of p=0.023 < 0.05, BSA group are more anti-than monovalent nanometer
The significant difference of weight p=0.047 < 0.05.Bivalent nano antibody is compared with MSTN group weight, the significant difference of p=0.045 < 0.05.Total weight
Situation of change is that bivalent nano antibody group and BSA control group growth rate are most fast, and weight gain is most.MSTN group weight gain is minimum.
The weight gain of bivalent nano antibody group is apparently higher than PBS group, but difference is not significant.
10 mouse hind leg weight of table
Note: PBS: the control group of PBS is injected；BSA: the control group of albumin is injected；MSTN injects sheep MSTN protein groups；
Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent: injection bivalent sheep MSTN nano antibody group.
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.
Bivalent nano antibody group hind leg weight is most heavy.Bivalent nano antibody is than control group weight, the difference of p=0.03 < 0.05
Significantly, bivalent nano antibody ratio MSTN group weight, the significant difference of p=0.027 < 0.05, bivalent nano antibody is than monovalent nano antibody
Weight, p=0.008 < 0.01 difference are extremely significant.
By two groups of data of comparison it can be found that bivalent nano antibody group does not have in total weight with other groups (removing MSTN group)
There is apparent difference, but again upper obvious with other differences in hind leg, and is one group most heavy in each group.Illustrate, bivalent is received
The ratio that the muscle of rice antibody group accounts for body is the largest, and Lean mass is most one group in vivo.
2.3 nano antibodies repeat to test in Mice Body
2.3.1 muscle morphology is observed
Experiment uses 30 kunming mices, from 2016.1.26-2016.2.22 days, carries out 30 days altogether.PBS group every injection
30 μ L PBS are as a control group；The monovalent sheep MSTN nano antibody of 30 μ L (60mg) of Univalent group per injection；Bivalent
Every group of injection 30 μ L (60mg) bivalent sheep MSTN nano antibody of group.
As shown in figure 11, mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Fig.2-6 Mouse hindlimb muscle morphology diagram
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
It is observed that bivalent nano antibody group, muscle is full.Right hind is separated, is intuitively compared.And claimed
Weight.
As shown in figure 12, mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
To wherein it amplify two groups of pictures, more intuitive comparison.Bivalent nano antibody group hind leg is significantly greater than monovalent nano antibody
Group, monovalent nano antibody group are big compared with control group.
As shown in figure 13, mouse hind leg muscle form schematic diagram provided in an embodiment of the present invention.
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
2.3.2 changes of weight
11 mouse weight of table
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
After experiment, mouse is handled.Control group, monovalent nano antibody, the final total weight of bivalent nano antibody do not have
Otherness.In terms of promoting weight gain, without apparent difference (table 11).
12 mouse hind leg weight of table
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
Control group, monovalent nano antibody, two hind leg of bivalent nano antibody weight statistics do not have otherness.But in weight,
Bivalent nano antibody group is higher than monovalent nano antibody group, and monovalent nano antibody group is higher than control group.When dissected discovery, has bright
Significant difference is different.Therefore leg is separated, is individually weighed (table 12).
13 mouse unilateral hindlimb weight of table
Note: PBS: the control group of PBS is injected；Univalent: monovalent sheep MSTN nano antibody group is injected；Bivalent:
Inject bivalent sheep MSTN nano antibody group.
PBS:PBS injected control group；Univalent:Injection Unit sheep MSTN
Nanobodies； Bivalent:sheep MSTN injection bivalent Nanobody.
Difference is extremely significant between leg weighing discovery bivalent nano antibody and monovalent nano antibody, and p=0.004 < 0.01 is double
Difference is extremely significant between valence nano antibody and control group, p=0.000 < 0.01 (table 13).
In table 11, bivalent nano antibody group does not have apparent otherness with other two groups, but the bivalent nanometer in table 13
Two groups of othernesses of antibody group and other are extremely significant.In total weight, bivalent nano antibody group is minimum, but in leg muscle
In weight, bivalent nano antibody is highest.
The intracorporal Lean mass of bivalent nano antibody group and ratio are highest in an experiment.Repetition experiment on mouse
It further confirms that nano antibody mainly has the function of promoting muscle growth, while promoting animal muscle growth, can promote
Into Bone Development for Animals, the accumulation of fat is reduced.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
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<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> 34
<211> 301
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 34
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 (9)
1. a kind of construction method of MSTN nano antibody, which is characterized in that the construction method MSTN of the MSTN nano antibody
Nano antibody and its application method include:
Step 1: the nano antibody library of building sheep MSTN gene expression product；
Step 2: it is obtained by display technique of bacteriophage for MSTN gene expression product specific nano antibody；
Step 3: building bivalent sheep MSTN nano antibody SEQ ID NO:3.
2. the construction method of MSTN nano antibody as described in claim 1, which is characterized in that in the step 2, specificity
The preparation method of nano antibody includes:
(1) acquisition of target fragment, design of primers；
(2) nano antibody sequence amplification；
(3) bivalent sheep MSTN nano antibody sequence PCR product glue recycles；
(4) double digestion is carried out to pET-30A carrier；
(5) recovery product, pET-30A carrier are connected；
(6) inducing expression of albumen；
(7) SDS-PAGE is detected；
(8) ELISA verifies protein active；
(9) great expression of nano antibody collects albumen and is ready for subsequent experimental.
3. the construction method of MSTN nano antibody as claimed in claim 2, which is characterized in that the acquisition of target fragment, primer
In design, specifically include:
Using MSTN nano antibody as purpose sequence, bivalent nano antibody is prepared；
Primer are as follows: upstream primer 5'-GAATTCATGCAGTTGCAGCTCGTGG-3', downstream primer 5'-
GCGGCCGCAAGGCCTCGG-3'。
4. the construction method of MSTN nano antibody as claimed in claim 2, which is characterized in that nano antibody sequence amplification packet
It includes: bivalent sheep MSTN nano antibody sequence PCR condition
After end of reaction, 4 DEG C of preservation products.
5. the construction method of MSTN nano antibody as claimed in claim 2, which is characterized in that bivalent sheep MSTN nano antibody
Sequence PCR product glue recycles
The gel for running through electrophoresis is placed in the UV lamp, and target fragment is cut；The glue cut is put into EP pipe；Into EP pipe
Soltion B is added, places the gel in 60 DEG C, constantly shakes, gel is waited to melt；Gel after thawing is added and is collected
Guan Zhong stands 3 minutes, 7000r/min, is centrifuged 3min；Collecting pipe is removed, is eluted using Wash Soluion, is eluted
Twice；Centrifugation thoroughly removes Wash Soluion.DNA is eluted using Elution Buffer；Detected through gel electrophoresis is carried out to return
Receive product purity.
6. the construction method of MSTN nano antibody as claimed in claim 2, which is characterized in that carried out to pET-30A carrier double
In digestion, double digestion is carried out to carrier using BamHI, XhoI；37 DEG C of reaction 2h.
7. the construction method of MSTN nano antibody as claimed in claim 2, which is characterized in that
By recovery product, pET-30A carrier connection in, 50 DEG C of reaction 1h of PCR instrument.
8. a kind of MSTN nano antibody that the construction method using MSTN nano antibody described in claim 1 constructs.
9. a kind of detection method of MSTN nano antibody as described in claim 1, which is characterized in that the MSTN nano antibody
Detection method includes:
1) preparation of competent cell
The DH5 α of preservation is subjected to plate setting-out culture, picking monoclonal colonies.By the bacterium colony of picking, 5mL LB culture medium is added
In, it is incubated overnight；It second day, is transferred in 50mL culture medium, culture to logarithmic growth phase；The DH5 α of logarithmic growth phase
Carry out ice bath；It is centrifuged using high speed low temperature centrifugal machine, collects precipitating；It is outstanding to precipitating injection 6mL low temperature 0.1mol CaCl2
It drifts along shallow lake；It dispenses, it is quick-frozen in liquid nitrogen, it is put into -80 DEG C of refrigerators and saves；
2) it is transformed into expression bacterium DE3:
The product connected is added in EP pipe, water-bath is adjusted to 42 DEG C, EP pipe is put into rapidly, is during which constantly shaken,
Reaction time 90s；EP pipe is taken out, 2min on ice is put in；Centrifugation takes precipitating to apply plate, and 30 μ g/ml kanamycins, 34 μ are added
G/ml chloramphenicol；Second day, by monoclonal picking；
The inducing expression of albumen:
Picking monoclonal is incubated overnight；It takes 100 μ L of culture solution to be added to 20mL to contain in the culture medium of Kana and chloramphenicol, induce
It expresses to logarithmic growth phase；The IPTG of final concentration of 0.5mmol is added；It is dispensed into different triangular flasks；Different items is set
Part: first bottle, using 15 DEG C, shaking table culture is stayed overnight；It second bottle, 25 DEG C, induces overnight；Third bottle, 37 DEG C, 220r/min culture
4h；The group for being not added with IPTG is set as negative control；4000r/min collects bacterium, and 500 μ L PBS are added into precipitating and blow repeatedly
It beats；Ultrasonication is carried out, supernatant and precipitating are collected；The precipitating being collected into is dissolved using solubilization of inclusion bodies liquid；By sample
Protein loading buffer is added, boils 10min；
3) SDS-PAGE is detected:
12% separation gel is prepared, separation gel is mixed, is added in gel maker, by the bubble of generation using using minute hand to choose, no
It can there are bubbles in glue；Ethyl alcohol is added above separation gel using pipettor；After 20min, ethyl alcohol is poured out；Ethyl alcohol is waited to wave
After hair shaft is net, 5% concentration glue of preparation is added, and is inserted into comb；Gel maker is put in testing stand and stands 2h；By comb from glue
It removes；Glue is put into electrophoresis tank, 1 × electrophoresis liquid is added；Drive the bubble among gap out of；By bivalent sheep MSTN nano antibody
For sample pipetting volume into concentration glue, albumen Marker is added in 8 microlitres of every hole；Adjusting voltage is 80V, and 30min carries out concentration glue electricity
Swimming；Separation gel 120V, 60min；After 3-4h, when bromophenol blue being waited to reach glue bottom, stop electrophoresis；Glue is separated, is placed in
It is cleaned in deionized water；It immerses in dyeing liquor, in dyeing 35min on constant-temperature table；Fresh destainer is prepared, Yu Hengwen shakes
Repeatedly decoloration is clear to band on bed, when glue decoloration is clean, stops decoloration；It is cleaned using deionized water；It is placed in Bio-RAD
It is aggregated in imager, corrects the position of glue, adjust pattern color and contrast；
4) ELISA verifies protein active: diluting MSTN albumen using coating buffer, is coated with elisa plate, the hole 200ng/, 4, which spend night, incubates
It educates；PBST is washed 3 times；Using 5%PBSM100 microlitres of every hole, 37 degrees Celsius are incubated for 1 hour；PBST is washed 3 times；Nanometer is resisted
100,1000,10000,100000,1000000,10000000,100000000 times of body gradient dilution, PBS is added in control group, adds
Enter 100 microlitres of every holes, 37 degrees Celsius are incubated for 1 hour；PBST is washed 3 times；The secondary antibody 100 of 10000 times of diluted anti-alpacas is added
Microlitre every hole, 37 degrees Celsius are incubated for 1 hour, and PBST is washed 3 times；100 microlitres of developing solution are added, 37 degrees Celsius, is incubated for 15 minutes,
50 microlitres of terminate liquid are added, microplate reader reads OD value.
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| CN110760517A (en) * | 2019-10-09 | 2020-02-07 | 天津大学 | Antagonistic PD-1 camel antibody analogue AP gene, protein and application |
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