AU2020103352A4 - Porcine Neuromedin B polypeptide as well as preparation method and application of polyclonal antibody thereof - Google Patents
Porcine Neuromedin B polypeptide as well as preparation method and application of polyclonal antibody thereof Download PDFInfo
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- 101800001639 Neuromedin-B Proteins 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 102100038819 Neuromedin-B Human genes 0.000 claims abstract description 62
- 238000000034 method Methods 0.000 claims abstract description 32
- 230000003053 immunization Effects 0.000 claims abstract description 29
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 25
- 229920001184 polypeptide Polymers 0.000 claims abstract description 24
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 239000000427 antigen Substances 0.000 claims abstract description 22
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 230000008878 coupling Effects 0.000 claims abstract description 14
- 238000010168 coupling process Methods 0.000 claims abstract description 14
- 238000005859 coupling reaction Methods 0.000 claims abstract description 14
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- 238000011587 new zealand white rabbit Methods 0.000 claims abstract description 8
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- YPFNACALNKVZNK-MFNIMNRCSA-N (2s)-2-[(2-aminoacetyl)amino]-n-[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3r)-1-[[2-[[(2s)-1-[[(2s)-1-[[(2s)-1-amino-4-methylsulfanyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-hydroxy-1- Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CN)[C@@H](C)O)C1=CC=CC=C1 YPFNACALNKVZNK-MFNIMNRCSA-N 0.000 description 40
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- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 description 1
- 101001039696 Homo sapiens Lysophospholipid acyltransferase 7 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
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- 230000036760 body temperature Effects 0.000 description 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
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- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
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- 229960002175 thyroglobulin Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
- C07K7/086—Bombesin; Related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57572—Gastrin releasing peptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/5758—Gastrin releasing peptide
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/549—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic with antigen or antibody entrapped within the carrier
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9406—Neurotransmitters
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the fields of biochemistry and molecular immunology, and
relates to a porcine Neuromedin B polypeptide and a preparation method and application of a
polyclonal antibody thereof. The preparation method comprises the following steps of:
coupling a C-terminal modified polypeptide of the porcine Neuromedin B polypeptide with
carrier protein keyhole limpet hemocyanin (KLH) to obtain NMB modified polypeptide-KLH
coupled protein; emulsifying the NMB modified polypeptide-KLH coupled protein and
immunizing New Zealand white rabbits; collecting and separating serum containing a rabbit
anti-porcine NMB modified polypeptide antibody; and purifying the prepared NMB polyclonal
antibody by adopting a Protein G spin column method. The polypeptide antigen and the
polyclonal antibody have the characteristics of being simple in preparation, low in cost and
high in titer, and can be specifically combined with NMB protein in pig tissues.
-1/6
5 IC 15 2 20 3 4 ! 5 50 55 50 W6 7D 75 ED 85 90 D5 100 105 110 115 120
0- aai lyrofa bt-le-il
DSurfam Prbbft t- Em~in
Figure1I
Description
-1/6
lyrofa 5 IC 15 2 20 3 4! 5 50 55 50 W6 7D 75 ED 85 90 D5 100 105 110 115 120
- aai bt-le-il
DSurfam Prbbft t- Em~in
Figure1I
Porcine Neuromedin B polypeptide as well as preparation method and
application of polyclonal antibody thereof
The invention belongs to the fields of biochemistry and molecular immunology,
and relates to a porcine Neuromedin B polypeptide as well as a preparation method
and an application of an antibody thereof. The prepared antibody is mainly used for
detecting natural protein antigens. The invention particularly relates to a preparation
method of a rabbit polyclonal antibody for resisting the porcine Neuromedin B
polypeptide.
Neuromedin B (NMB), a Neuromedin that was isolated and purified from
porcine spinal cords in 1983, belongs to the family of mammalian Bombesin
(BN)-like peptides. In vivo, NMB has a variety of biological effects, including
stimulating smooth muscle contraction, regulating gastrointestinal motility, ingestion
and body temperature, regulating hormone secretion, mediating stress and fear
responses, affecting behavior and memory, promoting cell growth, regulating
reproduction, and the like, through combination with receptors (NMBR or BB1) on
the surface of cell membranes.
Since the discovery of NMB, expression and distribution of NMB and its
receptors in human, rat, mouse and other animals have been studied by Chinese and
foreign scholars. It is found that NMB and its receptors are widely expressed in the
central nervous system and peripheral tissues and organs. Pig is the major economic
livestock and one of the main sources of animal protein in China and also serves as an
important model animal. It is an important responsibility for animal husbandry and veterinary work to ensure the health of pigs and improve their productivity and reproductive ability. It also provides an important reference value for further research on human diseases. By analyzing the porcine NMB sequence, we found that porcine
NMB includes signal peptides of 24 amino acids, conserved sequences NMB-32 and
NMB-10 (GNLWATGHFM), which are the same as those of human, rat and mouse. In
addition, homology alignment results show that porcine NMB nucleotide and amino
acid sequences are highly homological with other mammals. We found through the
fluorescence quantitative PCR that NMB mRNA was also widely expressed in the
central nervous system and various peripheral tissues and organs of pigs, indicating
that NMB may play a role as a neurotransmitter or a neuro-medium in the central or
peripheral tissues and organs of pigs. However, due to the lack of the porcine NMB
protein antibody detection in the market and the complex process, high cost and long
time consumption for preparing the porcine NMB monoclonal antibody, the research
of the NMB in the pig body is mainly centralized the gene level, and the functional
research of the NMB in the pig is seriously restricted. Therefore, the preparation of
the antibody for the porcine NMB protein is particularly important. It not only can be
used for researching the physiological function of the GRP in a pig body, but can also
satisfy the demand for the antibody in the market.
In order to solve the technical problems, the invention provides a porcine
Neuromedin B polypeptide as well as a preparation method and application of a
polyclonal antibody thereof, wherein the polypeptide antigen and the polyclonal
antibody have the characteristics of being simple in preparation, low in cost and high
in titer, and can be specifically combined with NMB protein in pig tissues.
In order to achieve the above object, the present invention is realized by the
following technical scheme:
According to the full-length amino acid sequence of the porcine NMB protein such as the sequence list SEQ-1, a porcine NMB polypeptide antigen such as the sequence list SEQ-2 is selected.
The invention provides a porcine Neuromedin B polypeptide, and the sequence
of the porcine Neuromedin B polypeptide is shown in a sequence list SEQ-2.
The invention provides a porcine Neuromedin B polypeptide, and the sequence
of the porcine Neuromedin B polypeptide is shown in a sequence list SEQ-3.
SEQ-1, molecular type and characteristics: the full-length amino acid sequence
of the NMB protein with length of 121 aa;
The sequence is as follows:
MetThrLeuArgAlaArgGlyAlaArgLeuLeuGlyGlyLeuLeuPhePheThrLeuLeuAla
AlaGlyAlaAlaProLeuSerTrpAspLeuProGluProArgSerArgAlaGlyLysIleArgValHisPro
ArgGlyAsnLeuTrpAlaThrGlyHisPheMETGlyLysLysSerLeuGluProProAsnProSerLeu
LeuGlyThrThrHisHislleSerLeuArgAspGlnArgLeuGlnLeuSerHisAspLeuLeuArgleLe
uLeuGlnLysLysAlaLeuGlyLeuSerLeuSerGlyProAlaSerHisThrProTyrArgArgLeuLeu
ValGlnThrLeuGlu
SEQ-2, molecular type and characteristics: NMB polypeptide antigen with
length of 20 aa
The sequence is as follows:
AspLeuProGluProArgSerArgAlaGlyLysIleArgValHisProArgGlyAsnLeu
SEQ-3, molecular type and characteristics: NMB modified polypeptide,
C-terminal added with a Cys, with a length of 21 aa
The sequence is as follows:
AspLeuProGluProArgSerArgAlaGlyLysIleArgValHisProArgGlyAsnLeuCys
The invention also provides a preparation method of a polyclonal antibody of
the porcine Neuromedin B polypeptide, which comprises the following steps: coupling the C-terminal modified polypeptide of the porcine Neuromedin B polypeptide with carrier protein keyhole limpet hemocyanin (KLH) to obtain NMB modified polypeptide-KLH coupling protein; emulsifying NMB modified polypeptide-KLH coupling protein and immunizing
New Zealand white rabbits;
collecting and separating serum containing rabbit anti-porcine NMB modified
polypeptide antibody; and
purifying the prepared NMB polyclonal antibody was purified by Protein G spin
column method.
Furthermore, the C-terminal modified polypeptide of the porcine Neuromedin B
polypeptide is coupled with the carrier protein keyhole limpet hemocyanin KLH
through a coupling agent Sulfo-SMCC.
Furthermore, the immunization method comprises the following steps of:
injecting emulsified antigen into an immunized New Zealand white rabbit by adopting
a back subcutaneous multi-point injection method; carrying out first immunization,
dissolving 1 mg of complete antigen in 1 mL of a PBS solution, and fully emulsifying
the solution with an equal volume of Freund's complete adjuvant; carrying out
reinforcement immunization after the first immunization for 2 weeks; carrying out
reinforcement immunization for 3 times; collecting antiserum after the fourth
immunization, anaesthetizing the rabbit, taking a large amount of blood by adopting
an abdominal aorta bleeding method, collecting the blood, performing oblique
standing for about 1 hour at 37 °C, then transferring the blood to a refrigerator at 4 °C,
performing standing for about 12 hours, fully separating out the antiserum,
centrifuging and separating the antiserum, and performing packaging and storing in an
environment of -80 °C.
Further, the method for detecting the titer of the antiserum comprises the
following steps of: diluting the NMB polypeptide antigen into 1-10 g/mL by using a
coating liquid, adding 100 L of the diluted antigen solution each ELISA plate well, putting the ELISA plate in a wet box, performing coating at a temperature of 4 °C for overnight, firstly incubating the ELISA plate in a 37 °C box for 0.5 h; discarding the coating solution, adding a washing liquid into the coated ELISA plate, performing drying with gauze cloth or filter paper, and washing for 3 times, with 3-5 min for each time; adding 250 L of blocking buffer into each well of the ELISA plate, putting the
ELISA plate in the wet box, and performing incubation for 2 h at 37 °C and washing
the plate in the same way mentioned above; adding the antibody diluent to a blank
control, adding non-immunized serum to a negative control, adding antiserum dilutes
according to dilution ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400,
1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, 1:1638400 and 1:3276800 to an experimental group, and then putting the ELISA plate in the wet box
at 37 °C for incubation of 1-2 h, and washing the plate in the same way as mentioned
above; adding 100 L of a horseradish peroxidase labeled goat anti-rabbit secondary
antibody with a dilution ratio of 1:3000 to each well of the ELISA plate, putting the
ELISA plate in the wet box for incubation of 1-2 h, and washing the plate in the same
way as mentioned above; directly adding 100 L TMB-urea hydrogen peroxide
substrate solution to each well of the ELISA plate, performing incubation at room
temperature or 37 °C for 5-30 min; adding 100 L of 2 M sulfuric acid stop solution
to each well of the ELISA plate and ending the reaction; placing the ELISA plate in an
ELISA plate reader and measuring the absorbance at 450 nm; calculating the antibody
titer when the ratio with the negative control serum is greater than 2.1.
Further, antibody purification was carried out by: sucking 10 mL of binding
buffer with a syringe, injecting into the column drop by drop, and preventing air from
entering the column at an injection rate of 1 mL/min; sucking 0.5 mL of antiserum
and 1.5 mL of binding buffer, and adding them to the column; washing the column
with 10 mL of binding buffer; adding 200 L of 1 M Tris-HCl to the EP tube from
which the protein was collected; eluting the column with 3 mL of elute buffer; and
collecting the purified NMB antibody.
Further, the purified NMB polyclonal antibody was subjected to immunoblot detection of NMB protein in porcine tissues.
The invention also provides a polyclonal antibody of the porcine Neuromedin B
polypeptide, which is prepared by adopting any one of the methods.
The invention also provides the application of the polyclonal antibody of the
porcine Neuromedin B polypeptide in the aspect of detecting the NMB protein level
of pig tissues. The invention has the beneficial effects that:
1. The method comprises: analyzing the hydrophilicity, the surface accessibility,
the antigen index, the homology and the like of the NMB protein sequence on the
basis of the porcine NMB gene sequence obtained in the earlier stage, and screening a
suitable peptide segment sequence as a target sequence for carrying out artificial
synthesis;
2. C-terminal modification is carried out on the synthesized peptide segment
sequence, and the peptide segment sequence is coupled with the carrier protein
keyhole limpet hemocyanin (KLH) to obtain the NMB modified polypeptide-KLH
coupling protein.
3. The method also comprises: immunizing New Zealand white rabbits with the
prepared polypeptide complex, and detecting the titer of the antiserum by an indirect
ELISA method, wherein the titer of the prepared antiserum is more than 1: 1638400,
and the titer of the purified antibody reaches over 1: 204800.
4. The NMB polyclonal antibody in antiserum was purified by Protein G spin
column method. The NMB polypeptide antibody prepared by the invention can be
specifically combined with the NMB protein in pig tissues, remedies the blank in the
field of porcine NMB protein detection and research, and lays a foundation for the
research of NMB functions in pigs.
Figure 1 is a diagram showing analysis of the hydrophilicity, surface accessibility, and antigen index of porcine NMB Protean sequences using bioinformatics software DNAstar (Protean);
Figure 2 is a diagram showing HPLC purity of a porcine NMB modified polypeptide with a purity of 95.89%;
Figure 3 is a mass spectrum detection result diagram of a porcine NMB modified polypeptide;
Figure 4 is a diagram showing antibody titers detected by the indirect ELISA method, wherein the antibody titers of the antisera were 1: 1638400, and the purified antibody titers were 1: 204800;
Figure 5 is an SDS-PAGE electrophoresis diagram of NMB polyclonal antibody purified by Protein G spin column method;
Figure 6 is a Western Blot detection result diagram for detecting the expression level of the NMB protein in the porcine pituitary by using the NMB polyclonal antibody, wherein the detection result has clear bands and good specificity;
The present invention is further illustrated below by way of specific examples. It should be understood that the examples are presented merely to illustrate the invention and are not intended to limit the scope of application of the invention. The experimental methods described in the following examples are conventional methods, if not specified, and the reagents and materials, if not specified, are commercially available.
Example 1 Sequence Analysis of Porcine NMB Protein and Design and Synthesis of NMB Polypeptide
As shown in porcine NMB gene sequence analysis: The ORF of porcine NMB gene was 366 bp, with code of 121 aa.
The hydrophilic property, surface accessibility and antigen index of porcine
NMB protein were analyzed by using a protean module in DNAstar software. The
sequence homology was analyzed by blastn in NCBI blast. A suitable sequence
DLPEPRSRAGKIRVHPRGNL was selected as antigen (30 aa-49 aa). In order to
realize coupling with the carrier protein, which can increase the immunogenicity of
the polypeptide, the selected polypeptide sequence is subjected to C-terminal
modification and added with cysteine (Cys), so that the finally synthesized
polypeptide sequence is DLPEPRSRAGKIRVHPRGNLC. The purity of the
synthesized polypeptide was 95.89% by HPLC, and the molecular weight of the
synthesized polypeptide was 2332.68 Da by mass spectrometry.
Example 2 Coupling of the C-Terminal Modified Peptide Fragment of Porcine
NMB with Carrier Protein KLH
There are a variety of carrier proteins for coupling with polypeptides, among
which keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin
(OVA) and bovine thyroglobulin (THY) are most commonly used. In view of the
relatively high antigenicity, KLH is the most commonly used polypeptide coupling
vector. Therefore, KLH is selected as a carrier protein, and a coupling agent
Sulfo-SMCC is used for coupling a C-terminal modified peptide segment of 5.0 mg of
porcine NMB with 5.0 mg of carrier protein KLH. In this way, an NMB modified
polypeptide-KLH coupling protein, namely a complete antigen, is obtained.
Example 3 Immunization of Experimental Animals and Preparation of
Antiserum
Male New Zealand white rabbits were selected as immunizing animals. Blood
samples were collected from the peripheral auricular vein of rabbits before the first
immunization (before the blood samples were fed in the morning), and were used as
control serum for subsequent ELISA detection.
For the first immunization, 1 mg of complete antigen was dissolved in 1 mL of
PBS solution and fully emulsified with an equal volume of Freund's complete adjuvant. The emulsified antigen was injected into the immunized New Zealand white rabbits by a back subcutaneous multi-point injection method. In two weeks after the first immunization, booster immunization was performed once. The experimental animals were immunized with 1 mg of complete antigen dissolved in 1 mL PBS solution and emulsified with the same volume of Freund's incomplete adjuvant. After one week of booster immunization, the rabbit auricular vein was sampled, the titers of the antibodies were detected by ELISA, and the dose required for the third booster immunization and immunization and the number of booster immunization were confirmed. The invention carries out boosting immunization for three times before and after the immunization.
The step comprises: collecting antiserum after the fourth immunization,
anaesthetizing the rabbit, taking a large amount of blood by adopting an abdominal
aorta bleeding method, collecting the blood, performing oblique standing for about 1
hour at 37 °C, then transferring the blood to a refrigerator at 4 °C, performing standing
for about 12 hours, fully separating out the antiserum, centrifuging and separating the
antiserum, and performing packaging and storing in an environment of -80 °C.
Example 4 Detection of Antiserum Titers by Indirect ELISA
The method comprises: diluting the NMB polypeptide antigen into 1-10 g/mL
by using a coating liquid, adding 100 L of the diluted antigen solution each ELISA
plate well, putting the ELISA plate in a wet box, performing coating at a temperature
of 4 °C for overnight, firstly incubating the ELISA plate in a 37 °C box for 0.5 h;
discarding the coating solution, adding a washing liquid into the coated ELISA plate
(adding 200 L till it was full), performing drying with gauze cloth or filter paper, and
performing washing for 3 times, with 3-5 minutes for each time; adding 250 L of
blocking buffer into each well of the ELISA plate, putting the ELISA plate in the wet
box, and performing incubation for 2 h at 37 °C and washing the plate in the same
way mentioned above; adding the antibody diluent to a blank control, adding
non-immunized serum to a negative control, adding antiserum dilutes according to
dilution ratios of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800,
1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200, 1:1638400 and 1:3276800 to an experimental group, and then putting the ELISA plate in the wet box
at 37 °C for incubation of 1-2 h, and washing the plate in the same way as mentioned
above; adding 100 L of a horseradish peroxidase labeled goat anti-rabbit secondary
antibody with a dilution ratio of 1:3000 to each well of the ELISA plate, putting the
ELISA plate in the wet box for incubation of 1-2 h, and washing the plate in the same
way as mentioned above; directly adding 100 L TMB-urea hydrogen peroxide
substrate solution to each well of the ELISA plate, performing incubation at room
temperature or 37 °C for 5-30 min; adding 100 L of 2 M sulfuric acid stop solution
to each well of the ELISA plate and ending the reaction; placing the ELISA plate in an
ELISA plate reader and measuring the absorbance at 450 nm; calculating the antibody
titer when the ratio with the negative control serum is greater than 2.1. The results
showed that the titer of antiserum was 1: 1638400 and that of purified antiserum was
1:204800.
Example 5 Antibody Purification
The prepared polyclonal antibody was purified by Protein G spin column
method by the following steps: antibody purification was carried out by sucking 10
mL of binding buffer with a syringe, injecting into the column drop by drop, and
preventing air from entering the column at an injection rate of 1 mL/min; sucking 0.5
mL of antiserum and 1.5 mL of binding buffer, and adding them to the column;
washing the column with 10 mL of binding buffer; adding 200 L of 1 M Tris-HCl
(pH = 9.0) to the EP tube from which the protein was collected; eluting the column
with 3 mL of elute buffer; and collecting the purified NMB antibody. The purified
antibody was stored in a refrigerator at -80 °C. The purity of the purified antibody was
determined by SDS-PAGE electrophoresis.
Example 6 Immunoblot Detection of NMB Protein in Pig Tissues
SDS-PAGE gel was prepared according to the standard method. 10 L of
porcine tissue lysate with a protein concentration of 5 mg/mL was added into the loading wells of a vertical electrophoresis tank. Then SDS-PAGE gel electrophoresis and 60 V-electrophoresis were carried out for 30 min. The electrophoresis was then stopped when the voltage was adjusted to 100 V until bromophenolblue was about 1.5 cm away from the bottom of the gel. After completion of SDS-PAGE electrophoresis, membrane transfer was carried out by a semi-dry method, and the protein was transferred onto an NC membrane. Sealing with 5% skimmed milk powder was carried out at a room temperature for 2 h. Washing with TBST was carried out for 4 times, wherein each time lasted for 10 min. The primary antibody (prepared NMB antibody, diluted at 1: 500) was added and overnight incubation at 4 °C was carried out. Washing with TBST was carried out for 5 times; the HRP labeled goat anti-rabbit secondary antibody was added, incubation was carried out at a room temperature for 2 h, and washing with TBST was carried out for 4 times; and the ECL luminescent solution was mixed for reacting with NC membrane for 1 min, and fluorescent luminescent images were collected. The experimental results of this example show that the expression level of NMB is higher in the porcine pituitary.
Claims (1)
- Claims1. A porcine Neuromedin B polypeptide, wherein the sequence is shown inSEQ-2.2. A porcine Neuromedin B polypeptide, wherein the sequence is shown inSEQ-3.3. A method for preparing a polyclonal antibody of the porcine Neuromedin Bpolypeptide, comprising the following steps:coupling the C-terminal modified polypeptide of the porcine Neuromedin Bpolypeptide according to claim 2 with carrier protein keyhole limpet hemocyanin(KLH) to obtain NMB modified polypeptide-KLH coupling protein;emulsifying NMB modified polypeptide-KLH coupling protein and immunizingNew Zealand white rabbits;collecting and separating serum containing rabbit anti-porcine NMB modifiedpolypeptide antibody; andpurifying the prepared NMB polyclonal antibody by a Protein G spin columnmethod.4. The method for preparing the polyclonal antibody of the porcine NeuromedinB polypeptide according to claim 3, wherein the C-terminal modified polypeptide ofthe porcine Neuromedin B polypeptide is coupled with the carrier protein keyholelimpet hemocyanin (KLH) through a coupling agent Sulfo-SMCC.5. The preparation method of the polyclonal antibody of the porcineNeuromedin B polypeptide according to claim 3, wherein the immunization methodcomprises: injecting an emulsified antigen into an immunized New Zealand whiterabbit by adopting a back subcutaneous multi-point injection method; carrying outfirst immunization, dissolving 1 mg of complete antigen in 1 mL of a PBS solution, and fully emulsifying the solution with an equal volume of Freund's complete adjuvant; carrying out reinforcement immunization after the first immunization for 2 weeks; carrying out reinforcement immunization for 3 times; collecting antiserum after the fourth immunization, anaesthetizing the rabbit, taking a large amount of blood by adopting an abdominal aorta bleeding method, collecting the blood, performing oblique standing for about 1 hour at 37 °C, then transferring the blood to a refrigerator at 4 °C, performing standing for about 12 hours, fully separating out the antiserum, centrifuging and separating the antiserum, and packaging and storing in an environment of -80 °C.6. The method for preparing the polyclonal antibody of the porcine NeuromedinB polypeptide according to claim 3, wherein the method for detecting the titer of theantiserum comprises: diluting the NMB polypeptide antigen into 1-10 pg/mL by usinga coating liquid, adding 100 pL of the diluted antigen solution each ELISA plate well,putting the ELISA plate in a wet box, performing coating at a temperature of 4 °C forovernight, firstly incubating the ELISA plate in a 37 °C box for 0.5 h; discarding thecoating solution, adding a washing liquid into the coated ELISA plate, performingdrying with gauze cloth or filter paper, and performing washing for 3 times, with 3-5minutes for each time; adding 250 pL of blocking buffer into each well of the ELISAplate, putting the ELISA plate in the wet box, and performing incubation for 2 h at37 °C and washing the plate in the same way mentioned above; adding the antibodydiluent to a blank control, adding non-immunized serum to a negative control, addingantiserum dilutes according to dilution ratios of 1:100, 1:200, 1:400, 1:800, 1:1600,1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600, 1:819200,1:1638400 and 1:3276800 to an experimental group, and then putting the ELISA platein the wet box at 37 °C for incubation of 1-2 h, and washing the plate in the same wayas mentioned above; adding 100 pL of a horseradish peroxidase labeled goatanti-rabbit secondary antibody with a dilution ratio of 1:3000 to each well of theELISA plate, putting the ELISA plate in the wet box for incubation of 1-2 h, andwashing the plate in the same way as mentioned above; directly adding 100 pLTMB-urea hydrogen peroxide substrate solution to each well of the ELISA plate,performing incubation at a room temperature or 37 °C for 5-30 min; adding 100 PL of2 M sulfuric acid stop solution to each well of the ELISA plate and ending thereaction; placing the ELISA plate in an ELISA plate reader and measuring theabsorbance at 450 nm; calculating the antibody titer when the ratio with the negativecontrol serum is greater than 2.1.7. The method for preparing the polyclonal antibody of the porcine NeuromedinB polypeptide according to claim 3, wherein the method for purifying the antibodycomprises: sucking 10 mL of binding buffer with a syringe, injecting it into thecolumn drop by drop, and preventing air from entering the column at an injection rateof 1 mL/min; sucking 0.5 mL of antiserum and 1.5 mL of binding buffer, and addingthem to the column; washing the column with 10 mL of binding buffer; adding 200 PLof 1 M Tris-HCl to the EP tube from which the protein was collected; eluting thecolumn with 3 mL of elute buffer; and collecting the purified NMB antibody.8. The method for preparing the polyclonal antibody of the porcine NeuromedinB polypeptide according to claim 3, wherein the purified NMB polyclonal antibody issubjected to immunoblot detection of NMB protein in porcine tissues.9. The polyclonal antibody of the porcine Neuromedin B polypeptide, whereinthe polyclonal antibody is prepared by the method according to any one of claims 3 to8.10. Application of the polyclonal antibody of the porcine Neuromedin Bpolypeptide according to claim 9 in detecting the level of NMB protein in porcinetissues.-1/6-Figure 1-2/6- Nov 2020mV72013.233' 630540450 202010335236027018012.886' 12.723' 12.138' 11.470' 900-90 2 4 6 8 10 12 14 16 18 20 22 24minFigure 2-3/6-Figure 3-4/6- Nov 2020AntiserumPurified serum Value 2020103352Dilution factorFigure 4-5/6-Figure 5-6/6- Nov 2020Pituitary 2020103352Figure 6
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