CN111704667A - Bovine and goat milk casein monoclonal antibody, detection kit and application - Google Patents
Bovine and goat milk casein monoclonal antibody, detection kit and application Download PDFInfo
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Abstract
The invention provides a bovine and goat milk casein monoclonal antibody, a detection kit and application thereof, wherein the light chain variable region of the bovine and goat milk casein monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. The monoclonal antibody is prepared and screened by a hybridoma technology, can be combined with cow milk casein and goat milk casein but not with camel cheese protein, has the highest affinity when being combined with the cow milk casein and the goat milk casein, can be used for simultaneously detecting trace amounts of cow milk and goat milk doped in the camel milk, and has the advantages of simple and convenient detection operation and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a bovine and sheep milk casein monoclonal antibody, a detection kit and application.
Background
The camel milk has high nutritive value, does not contain allergen, does not cause anaphylactic reaction such as vomiting and diarrhea, is three times as rich in vitamin c as milk, has ten times of iron content as high as milk, contains 903 mg of calcium per 100 g of camel milk, and has high calcium content. The camel milk also contains a large amount of unsaturated fatty acid, iron and vitamin B which are required by a human body, and researches show that the camel milk contains insulin-like small molecular substances and has a certain treatment effect on type 2 diabetes. Because of the advantages of camel milk, there are some illegal merchants who add milk or goat milk to camel milk, or even use cow milk or goat milk to impersonate camel milk.
At present, the main methods for detecting the adulteration of cow milk and goat milk in camel milk include a non-immunological method and an immunological method. Non-immunological methods include gas chromatography, liquid chromatography, capillary electrophoresis and other techniques, which usually require professional personnel to perform the procedures, and have complicated operations and long detection time. In the immunological method, milk or goat milk adulteration in the camel milk is detected by a milk casein monoclonal antibody or a goat milk casein monoclonal antibody, but the prior art can only detect the goat milk adulteration or the goat milk adulteration at one time, and two detection test paper products are needed if the milk or goat milk adulteration in the camel milk needs to be detected, so that the operation is complicated.
Disclosure of Invention
The invention solves the technical problem of providing a casein monoclonal antibody of milk and goat milk, a detection kit and application thereof, wherein the monoclonal antibody can simultaneously identify the milk and the goat milk, the detection kit prepared by the monoclonal antibody can show positive when the milk or the goat milk or both of the milk and the goat milk are doped in the camel milk, and the doping of the milk and the goat milk in the camel milk can be rapidly detected at one time.
In order to solve the above problems, one aspect of the present invention provides a bovine and sheep casein monoclonal antibody, wherein the light chain variable region of the bovine and sheep casein monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. The monoclonal antibody is prepared and screened by a hybridoma technology, can be combined with cow milk casein and goat milk casein but not with camel cheese protein, has the highest affinity when being combined with the cow milk casein and the goat milk casein, can be used for simultaneously detecting trace amounts of cow milk and goat milk doped in the camel milk, and has the advantages of simple and convenient detection operation and high sensitivity.
The nucleotide sequence of SEQ ID NO: 1 has the following sequence structure (109):
DIELPQSPAILSASLGERVTMTRSPGTRLRASYLHWYQQKPGSSPKLWIHCPTDMPTRLPARFSGSGSGTSYSLTISSMEAEDAATYYCDKDYGTASAFGAGLSSRSNN。
the nucleotide sequence of SEQ ID NO: 2 (120) as follows:
QVQLQESGPGLVKPSQSLSLTCTVTGYSITSHNVRNWIRQFPGNKLEWMGHVGYCRGPTYSLSLKSRISITRDTSQNQFFLQLNSVTTEDTATYYCARSTMITTRRVGYWGQGTTVTVSS。
preferably, the bovine and sheep casein monoclonal antibody is an IgG antibody.
In another aspect of the present invention, there is provided a nucleotide sequence encoding the heavy chain variable region and the light chain variable region of the bovine and sheep casein monoclonal antibody.
Preferably, the nucleotide sequence of the variable region of the light chain of the bovine and sheep casein monoclonal antibody is shown as SEQ ID NO: 3, the nucleotide sequence of the heavy chain variable region of the bovine and sheep milk casein monoclonal antibody is shown as SEQ ID NO: 4, respectively.
The nucleotide sequence of SEQ ID NO: the sequence structure of 3 is as follows (327):
gacattgagctcccccagtctccagcaatcttgtctgcctctctaggggaacgggtcaccatgacccgctctcccggcacacgtttacgtgccagttacttgcactggtaccagcagaagccaggatcctcccccaaactctggattcattgcccaaccgacatgcctactcgactcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgcgacaaggattatggtaccgcatccgcgttcggagcgggactaagctcgagatcaaacaat。
the nucleotide sequence of SEQ ID NO: the sequence structure of 4 is as follows (360):
caggtccaactgcaggagtcaggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcaccagtcataatgtccggaactggatccggcagtttccaggaaacaaactggagtggatgggccacgtaggctactgtcgtggccctacctacagcctatctctcaaaagtcgaatctctatcactcgagacacatcccagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagatctactatgattaccacaagaagggtcggctactggggccaagggaccacggtcaccgtctcctca。
in a further aspect of the invention there is provided a vector comprising a nucleotide sequence as described above.
In a further aspect the invention provides a host cell comprising a nucleotide sequence as described above.
The invention further provides a milk and sheep milk casein detection kit, which comprises the milk and sheep milk casein monoclonal antibody.
In another aspect, the invention provides a kit for detecting milk or goat milk adulteration in camel milk, which comprises: a test strip and a microporous strip;
the test strip comprises a back plate and a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged on the back plate; the nitrocellulose is provided with a detection line and a quality control line, the detection line is coated with a mixture of milk casein and goat milk casein, the quality control line is coated with goat anti-mouse IgG, the detection line is arranged near one side of the sample pad, and the quality control line is arranged near one side of the water absorption pad;
the microporous battens are filled with the bovine and sheep milk casein monoclonal antibodies marked by the colloidal gold, and the bovine and sheep milk casein monoclonal antibodies are the bovine and sheep milk casein monoclonal antibodies.
The detection kit utilizes the principle of competitive inhibition immunochromatography. The principle is as follows: the milk casein and the goat milk casein in the sample liquid are combined with the specific bovine and goat milk casein monoclonal antibodies marked by the colloidal gold in the flowing process, and the combination of the antibodies and the milk casein or the goat milk casein on the nitrocellulose membrane detection line (T) is inhibited. If the content of the casein in the milk and the casein in the goat milk in the sample liquid is larger than the detection limit, the detection line does not develop color, and the result is positive; otherwise, the detection line shows red, and the result is negative.
Preferably, the mass ratio of the cow milk casein to the sheep milk casein in the mixture of the cow milk casein and the sheep milk casein is 1: 1.
The invention also provides the application of the casein monoclonal antibody in the detection of milk or goat milk adulteration in camel milk.
Compared with the prior art, the invention has the following beneficial effects:
the monoclonal antibody of the casein of the cow and the sheep is immune to casein of cow and coated by casein of the sheep, is prepared by a hybridoma technology, is screened out, can be combined with the casein of cow and the casein of sheep but not with the casein of camel, and is the monoclonal antibody with the highest affinity combined with the casein of cow and the casein of sheep, can be used for simultaneously detecting trace amounts of cow milk and goat milk doped in camel milk, and has the advantages of simple and convenient detection operation and high sensitivity. The kit for detecting the adulteration of the milk or the goat milk in the camel milk utilizes the principle of competitive inhibition immunochromatography, the milk casein and the goat milk casein in the sample liquid are combined with the specific bovine and goat milk casein monoclonal antibodies marked by the colloidal gold in the flowing process, and the combination of the antibodies and the bovine casein or the goat milk casein on the nitrocellulose membrane detection line is inhibited, so that the detection line is not colored when the content of the milk casein or the goat milk casein in the sample liquid is greater than the detection limit, and the detection result is positive; otherwise, the detection line is red, and the result is negative; the kit can rapidly detect the adulteration of the milk and the goat milk in the camel milk through one-time operation, and has simple and convenient operation and high detection sensitivity.
Drawings
FIG. 1 is a plasmid diagram of NabaiFC-Entry1 eukaryotic expression vector: wherein Kozak is enhancer, MusFC is Fc fragment of mouse IgG; PolyA is an mRNA 3' end regulating sequence, AmpR is an ampicillin resistance gene, and CMV is a promoter;
FIG. 2 is a diagram of a NabaiFC-Entry1-VH recombinant plasmid;
FIG. 3 is a diagram of a NabaiFC-Entry1-VH recombinant plasmid;
FIG. 4 is a diagram showing the identification of bovine and ovine casein monoclonal antibodies prepared in example 3 of the present invention;
FIG. 5 is a schematic view of the structure of a test strip in example 4 of the present invention;
FIG. 6 shows the results of the determination of pure camel milk, camel milk containing 0.2% cow milk, camel milk containing 0.2% goat milk, camel milk powder containing 0.2% cow milk powder, and camel milk powder containing 0.2% goat milk powder using the kit in example 5 of the present invention.
Wherein: 1-a back plate; 2-sample pad; 3-nitrocellulose membrane; 4-absorbent pad; 5-detection line; 6-quality control line.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 preparation of bovine and ovine Casein monoclonal antibodies
1. Preparation of casein antigen of cow and sheep milk
Centrifuging at low temperature and high speed (12000 rpm,15min,4 deg.C) to remove fat and impurity precipitate in milk; the obtained supernatant was dialyzed overnight in 0.01M PBS (pH = 7.2) buffer, and was milk casein. The same method is adopted to extract the sheep cheese protein.
2. Cow and sheep milk casein antigen immune mouse
The purified milk casein is used as immunogen to immunize Balb/c female mice of 5-6 weeks old respectively. The first immunization was carried out using 100. mu.L of Freund's complete adjuvant emulsified with 100. mu.L of phosphate buffer solution (0.01M PBSpH7.4) containing 60. mu.g of bovine and ovine casein, measured at 10. mu.g/mouse, and each subsequent booster immunization using Freund's incomplete adjuvant emulsified with phosphate buffer solution of bovine casein (0.01M PBS pH7.4), at a dose consistent with that of the first immunization. Two weeks after the initial immunization, the booster immunization is carried out once every 14 days for 3 times in total, when the antibody titer is not increased any more, the last immunization is carried out, the intraperitoneal injection is directly carried out by using 0.01M of milk casein and phosphate buffer solution with pH of 7.4 without adding an immunization adjuvant at the last time, and the dosage is the same as the initial immunization.
3. Screening of bovine and sheep milk casein monoclonal antibody
And (3) taking blood from the tail part and simultaneously detecting the titer of the serum on the goat milk casein coated plate, wherein the result is shown in table 1, taking a mouse with the highest titer on the goat milk casein coated plate, and taking spleen cells of the mouse to perform cell fusion with myeloma cells SP2/0 according to a ratio of 10:1 under the aseptic condition.
And (3) screening by adopting bovine and sheep milk casein coated enzyme-labeled plates respectively, and screening hybridoma cells by a limiting dilution method to obtain 3 monoclonal antibodies aiming at identifying the milk casein and the sheep milk casein respectively. As in table 2 below.
Balb/C mice were sensitized by intraperitoneal injection with Freund's incomplete adjuvant at a dose of 0.5 mL/mouse one week before administration. The hybridoma cell suspension with the cell concentration of 1.2 ten thousand/mL is injected into the abdominal cavity of the mouse, and the dosage is 0.5 mL/mouse. And 7-10 days after the hybridoma cells are inoculated, ascites is collected and repeatedly collected for a plurality of times, and the supernatant is collected by centrifuging the collected ascites at 5000rpm/min each time. Storing in a refrigerator at-20 deg.C.
TABLE 1
TABLE 2
4. Purification and determination of casein monoclonal antibody of cow and sheep
Purifying the N antigen monoclonal ascites antibody by adopting an octanoic acid-ammonium sulfate precipitation method. The specific method comprises the following steps: ascites 1 mL was taken and the name of ascites was recorded. Adding 3 mL acetic acid-sodium acetate solution (0.06M, pH 4.0), mixing for 5min, adding 10 μ L n-octanoic acid, stirring for 15min, filtering absorbent cotton, and centrifuging at 12000rpm for 15 min. The supernatant was taken, and an equal volume of saturated ammonium sulfate (i.e., 50% ammonium sulfate final concentration) was added, and the mixture was allowed to stand at 4 ℃ for 3 hours and centrifuged at 12000rpm for 15 min. The supernatant was discarded, and 1.8 mL of PBS (0.01M, pH7.2) was added to dissolve the precipitate, and 1/2 volumes of saturated ammonium sulfate were added thereto, and the mixture was allowed to stand at 4 ℃ for 3 hours and centrifuged at 12000rpm for 15 min. The supernatant was discarded, and 0.45 mL of PBS (0.01M, pH7.2) was added to dissolve the precipitate. Dialyzing in 0.01M PBS (pH7.4), collecting after three times of liquid change, measuring the concentration, adjusting the concentration to 1.00 mg/mL, subpackaging, and storing at-20 ℃ for later use. The monoclonal antibody titers after purification are given in table 3 (indirect method), table 4 (blocking method).
TABLE 3
TABLE 4
Note: the antibody concentration is 1mg/mL, the antibody is diluted by 10000 times with camel milk, the sample adding is 50 muL, the adding amount of goat milk and cow milk is 5 thousandths, and the sample adding is 50 muL.
Example 2 sequencing of bovine and ovine Casein monoclonal antibodies
Measuring titer according to the purified monoclonal antibody, selecting 5B10-1G3 as a candidate monoclonal antibody, extracting mRNA from 5B10-1G3 hybridoma cells, extracting the mRNA by using an RNase Mini KiT extraction KiT of QIAGEN company, and freezing the extracted nucleic acid at-80 ℃ for later use according to the KiT instructions. Reverse transcription PCR amplification was performed using SuperScriptTM amplification System for First Strand cDNA Synthesis kit from ThermoFisher, Inc.
The method comprises the following specific steps: adding 1 mug of total RNA into a 0.2 mL microcentrifuge tube, and supplementing a proper amount of DEPC H2And O makes the total volume reach 11 mu L. Adding 10 uM Oligo (dT) 12-181 muL into the tube, gently mixing uniformly, and centrifuging; heating at 70 deg.C for 10min, immediately inserting the microcentrifuge tube into ice bath for at least 1 min; taking a 0.2 mL PCR tube, and sequentially adding the following reagents: 2 muL of first strand cDNA; 2 muL of upstream primer (10 pM); 2 muL of downstream primer (10 pM); dNTP (2mM) 4 muL; 10x PCR buffer5 μ L; taq enzyme (2 u/muL) 1 muL. Mixing and centrifuging. Incubating at 42 deg.C for 2-5 min; adding Superscript II 1 muL, and incubating in water bath at 42 ℃ for 50 min; heating at 70 deg.C for 15min to terminate the reaction; the tube was inserted into ice, RNase H1. mu.l was added, incubated at 37 ℃ for 20 min, and residual RNA was degraded. Storing at-20 deg.C for use.
The primer for sequencing the light chain variable region gene of the casein monoclonal antibody of the cattle and the sheep comprises the following components:
VLF：5’-GACATTCAGCTGACCCAGTC-3’
VLR：5’-GTTAGATCTCCAGCTTGGTCC-3’
the light chain variable region gene sequence of the bovine and sheep casein monoclonal antibody 5B10-1G3 is shown as SEQ ID NO: 3, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 1 is shown.
The nucleotide sequence of SEQ ID NO: 3 has the following sequence structure:
gacattgagctcccccagtctccagcaatcttgtctgcctctctaggggaacgggtcaccatgacccgctctcccggcacacgtttacgtgccagttacttgcactggtaccagcagaagccaggatcctcccccaaactctggattcattgcccaaccgacatgcctactcgactcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgcgacaaggattatggtaccgcatccgcgttcggagcgggactaagctcgagatcaaacaat。
the nucleotide sequence of SEQ ID NO: 1 has the following sequence structure:
DIELPQSPAILSASLGERVTMTRSPGTRLRASYLHWYQQKPGSSPKLWIHCPTDMPTRLPARFSGSGSGTSYSLTISSMEAEDAATYYCDKDYGTASAFGAGLSSRSNN。
the sequencing primer for the heavy chain variable region gene of the casein monoclonal antibody of the cattle and the sheep comprises the following components:
VLF：5’-AGGTGAGCTGCAGCAGTCTGG-3’
VLR：5’-GTGCCGTGGTCCCTTGGCCCCAG -3’
the heavy chain variable region gene sequence of the bovine and sheep casein monoclonal antibody 5B10-1G3 is shown as SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, respectively.
The nucleotide sequence of SEQ ID NO: 4 has the following sequence structure:
caggtccaactgcaggagtcaggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcaccagtcataatgtccggaactggatccggcagtttccaggaaacaaactggagtggatgggccacgtaggctactgtcgtggccctacctacagcctatctctcaaaagtcgaatctctatcactcgagacacatcccagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagatctactatgattaccacaagaagggtcggctactggggccaagggaccacggtcaccgtctcctca。
the nucleotide sequence of SEQ ID NO: 2 has the following sequence structure:
QVQLQESGPGLVKPSQSLSLTCTVTGYSITSHNVRNWIRQFPGNKLEWMGHVGYCRGPTYSLSLKSRISITRDTSQNQFFLQLNSVTTEDTATYYCARSTMITTRRVGYWGQGTTVTVSS。
EXAMPLE 3 in vitro expression of bovine and ovine Casein monoclonal antibodies
The invention also provides a method for preparing the bovine and sheep casein monoclonal antibody by in vitro expression of genes of the variable regions of the heavy chain and the light chain of the obtained monoclonal antibody.
Constructing a eukaryotic expression vector of NabaiFC-Entry1, respectively introducing two enzyme cutting sites of SaLI/HindIII into two ends of a gene design primer of a heavy chain variable region of a bovine and sheep milk casein monoclonal antibody 5B10-1G3, and cloning to MCS2 to form a NabaiFC-Entry1-VH recombinant plasmid; introducing two ends of a design primer of a light chain variable region gene of a bovine and sheep milk casein monoclonal antibody 5B10-1G3 respectively into two restriction enzyme sites of SmaLI/HindIII, and cloning to MCS1-MCS2 to form a NabaiFC-Entry1-VL recombinant plasmid; and co-transfecting CHO cells with the two constructed NabaiFC-Entry1-VH and NabaiFC-Entry1-VL recombinant plasmids, and performing induction expression to obtain the bovine and sheep casein monoclonal antibodies. FIG. 1 shows a plasmid map of a NabaiFC-Entry1 eukaryotic expression vector: wherein Kozak is enhancer, MusFC is Fc fragment of mouse IgG; PolyA is an mRNA 3' end regulating sequence, AmpR is an ampicillin resistance gene, and CMV is a promoter; FIG. 2 is a diagram of a NabaiFC-Entry1-VH recombinant plasmid; FIG. 3 shows a diagram of the NabaiFC-Entry1-VH recombinant plasmid.
The specific operation of transfecting cells and inducing expression is as follows: adding OPTI-MEM 600 muL + 20 mug plasmid into a tube 1, uniformly mixing, adding OPTI-MEM 600 muL + 60 muL PEI (polyethyleneimine MW:40000) into a tube 2, uniformly mixing, adding the mixed solution in the tube 2 into the tube 1, uniformly mixing, standing for 20 minutes (uniformly mixing before adding into a flat dish)) The mixture was uniformly added to a plate having a diameter of 150 mm. The plate was shaken back and forth and left and right to distribute the added plasmids evenly in the plate, and then put in 5% CO at 37 deg.C2Culturing in an incubator, changing a culture medium containing a transfection reagent after 6 hours, culturing for 24 hours, and collecting supernatant or cells, wherein the PEI concentration is 2 mug/mug L, the OPTI-MEM is GBICO 31985-.
FIG. 4 is the identification chart of the prepared bovine and ovine casein monoclonal antibodies.
Example 4 preparation of a kit for detecting adulteration of cow's or sheep's milk in camel's milk
The kit for detecting the adulteration of the milk or the goat milk in the camel milk comprises: a test strip and a microporous strip;
the test strip comprises a back plate 1 and a sample pad 2, a nitrocellulose membrane 3 and a water absorption pad 4 which are sequentially arranged on the back plate 1; the nitrocellulose membrane 3 is provided with a detection line 5 and a quality control line 6, the detection line 5 is coated with a mixture of milk casein and goat milk casein, the quality control line 6 is coated with goat anti-mouse IgG, the detection line 5 is arranged at one side close to the sample pad 2, and the quality control line 6 is arranged at one side close to the water absorption pad 4;
the microporous battens are filled with the bovine and sheep milk casein monoclonal antibodies marked by the colloidal gold, and the bovine and sheep milk casein monoclonal antibodies are the bovine and sheep milk casein monoclonal antibodies.
The preparation method of the kit for detecting the adulteration of the milk or the goat milk in the camel milk comprises the following steps:
(1) taking cow and sheep milk casein antigen prepared in equal amount, diluting with PBS (0.01 mol/L) with pH7.2 to 2.4mg/mL for marking membrane on T line; taking goat anti-mouse IgG, diluting to 0.5mg/mL by 0.01mol/L PBS (phosphate buffer solution) with pH7.2, and using the diluted goat anti-mouse IgG for C line membrane scribing; the prepared colloidal gold labeled bovine and sheep milk casein monoclonal antibody 5B10-1G3 is diluted with 0.01mol/L PBS (phosphate buffer solution) with pH7.2, 5% BSA (bovine serum albumin) and 3% Tween-20 to 60 muL/well and freeze-dried for later use.
(2) On a nitrocellulose membrane with the size of 25mm multiplied by 300mm, a detection line is formed by transversely and linearly spraying casein antigen of cow and sheep by a film spraying machine at the concentration of 1.0 mu L/cm. Goat anti-mouse IgG is sprayed in a transverse linear way at an interval of 6mm and at a rate of 1.0 mu L/cm to form a quality control line.
(3) A sample pad made of clean non-woven fabric, an NC film coated with a detection line and a quality control line and absorbent paper are assembled on a PVC back plate according to the structure diagram of a test strip assembly, and the test strip assembly is cut into strips with the width of 4.05mm by a slitter to obtain the test strip finished product shown in figure 5.
Example 5 detection of adulteration of cow's or sheep's milk in camel milk
Weighing 1g (accurate to 0.01 g) camel milk powder, pouring into a centrifuge tube, adding 8 mL of purified water, and uniformly mixing to obtain a sample to be detected for later use. The sample to be detected must be uniform liquid, and no caking, fermentation and deterioration precipitate can exist, otherwise the detection result can be influenced. And (3) taking the micropore reagent filled with the colloidal gold labeled bovine and goat milk casein monoclonal antibodies and the test paper strips, and marking. And sucking 200 mu L of sample solution to be detected into the micropores by using a micropipettor, slowly sucking and fully mixing the sample solution with the reagent in the micropores uniformly, incubating for 5 minutes, and judging the result after inserting the test strip for 10 minutes, wherein the result does not exceed 20 minutes.
Determination of results
Negative (-): the C line and the T line are both developed, and the T line is far stronger in color development than the C line, which indicates that the sample does not contain cow milk or goat milk or is far below the detection limit.
Positive (+) position: c line color development; the color development of the T line is the same as that of the C line, the color development of the T line is weaker than that of the C line or the T line does not develop, and both indicate that the concentration of the cow milk and the goat milk in the sample is equal to or higher than the detection limit.
And (4) invalidation: the absence of a C-line indicates an incorrect procedure or the test strip has deteriorated and failed.
As shown in figure 6, the test paper can simultaneously detect the adulteration of the goat milk and the milk in the camel milk, and the product sensitivity can reach 0.2 percent by adopting the test paper to respectively test the test results of samples of the pure camel milk, the camel milk containing 0.2 percent of the goat milk, the camel milk powder containing 0.2 percent of the milk powder and the camel milk powder containing 0.2 percent of the goat milk powder.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
<110> Beijing gold Intelligent technology Limited
<120> bovine and goat milk casein monoclonal antibody, detection kit and application
<130>2020
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>109
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Asp Ile Glu Leu Pro Gln Ser Pro Ala Ile Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Met Thr Arg Ser Pro Gly Thr Arg Leu Arg Ala Ser
20 25 30
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp
35 40 45
Ile His Cys Pro Thr Asp Met Pro Thr Arg Leu Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Asp Lys Asp Tyr Gly Thr Ala
85 90 95
Ser Ala Phe Gly Ala Gly Leu Ser Ser Arg Ser Asn Asn
100 105
<210>2
<211>120
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser His
20 25 30
Asn Val Arg Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly His Val Gly Tyr Cys Arg Gly Pro Thr Tyr Ser Leu Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Gln Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Met Ile Thr Thr Arg Arg Val Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210>3
<211>327
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gacattgagc tcccccagtc tccagcaatc ttgtctgcct ctctagggga acgggtcacc 60
atgacccgct ctcccggcac acgtttacgt gccagttact tgcactggta ccagcagaag 120
ccaggatcct cccccaaact ctggattcat tgcccaaccg acatgcctac tcgactccca 180
gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240
gctgaagatg ctgccactta ttactgcgac aaggattatg gtaccgcatc cgcgttcgga 300
gcgggactaa gctcgagatc aaacaat 327
<210>4
<211>360
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
caggtccaac tgcaggagtc aggacctggc ctggtgaaac cttctcagtc tctgtccctc 60
acctgcactg tcactggcta ctcaatcacc agtcataatg tccggaactg gatccggcag 120
tttccaggaa acaaactgga gtggatgggc cacgtaggct actgtcgtgg ccctacctac 180
agcctatctc tcaaaagtcg aatctctatc actcgagaca catcccagaa ccagttcttc 240
ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aagatctact 300
atgattacca caagaagggt cggctactgg ggccaaggga ccacggtcac cgtctcctca 360
Claims (9)
1. The bovine and goat milk casein monoclonal antibody is characterized in that: the variable region of the light chain of the bovine and sheep casein monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region has an amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
2. A nucleotide sequence characterized in that: a heavy chain variable region and a light chain variable region encoding the bovine or ovine casein monoclonal antibody of claim 1.
3. The nucleotide sequence of claim 2, characterized in that: the nucleotide sequence of the variable region of the light chain of the bovine and sheep casein monoclonal antibody is shown as SEQ ID NO: 3, and the nucleotide sequence of the heavy chain variable region of the bovine and sheep casein monoclonal antibody is shown as SEQ ID NO: 4, respectively.
4. A carrier, characterized by: the vector comprising the nucleotide sequence of claim 2 or 3.
5. A host cell, characterized in that: the host cell comprising the nucleotide sequence of claim 2 or 3.
6. A milk casein detect reagent box of cow, sheep which characterized in that: comprising the bovine and ovine casein monoclonal antibody of claim 1.
7. A kit for detecting milk or goat milk adulteration in camel milk is characterized by comprising: a test strip and a microporous strip;
the test strip comprises a back plate and a sample pad, a nitrocellulose membrane and a water absorption pad which are sequentially arranged on the back plate; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a mixture of cow milk casein and goat milk casein, the quality control line is coated with goat anti-mouse IgG, the detection line is arranged near one side of the sample pad, and the quality control line is arranged near one side of the water absorption pad;
the microporous battens are filled with bovine and sheep milk casein monoclonal antibodies marked by colloidal gold, and the bovine and sheep milk casein monoclonal antibodies are the bovine and sheep milk casein monoclonal antibodies as claimed in claim 1.
8. The kit for detecting the adulteration of the milk or the goat milk in the camel milk according to claim 7, wherein: the mass ratio of the cow milk casein to the goat milk casein in the mixture of the cow milk casein and the goat milk casein is 1: 1.
9. The use of the bovine or ovine casein monoclonal antibody of claim 1 in the detection of adulteration of cow's milk or goat's milk in camel's milk.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010840294.4A CN111704667B (en) | 2020-08-20 | 2020-08-20 | Bovine and goat milk casein monoclonal antibody, detection kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010840294.4A CN111704667B (en) | 2020-08-20 | 2020-08-20 | Bovine and goat milk casein monoclonal antibody, detection kit and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111704667A true CN111704667A (en) | 2020-09-25 |
| CN111704667B CN111704667B (en) | 2020-12-01 |
Family
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| CN202010840294.4A Active CN111704667B (en) | 2020-08-20 | 2020-08-20 | Bovine and goat milk casein monoclonal antibody, detection kit and application |
Country Status (1)
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| CN (1) | CN111704667B (en) |
Cited By (4)
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| CN113311156A (en) * | 2021-04-09 | 2021-08-27 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of bovine milk protein and goat milk protein duplex detection card |
| CN114716531A (en) * | 2022-05-17 | 2022-07-08 | 中国农业科学院农业质量标准与检测技术研究所 | Casein polypeptide, polypeptide antigen, antibody, test strip and application thereof |
| CN114891101A (en) * | 2021-12-02 | 2022-08-12 | 吉林省农业科学院 | Bovine myostatin protein monoclonal antibody and application thereof |
| CN116333151A (en) * | 2022-08-12 | 2023-06-27 | 广西壮族自治区水牛研究所 | Monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113311156A (en) * | 2021-04-09 | 2021-08-27 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | Preparation method and application of bovine milk protein and goat milk protein duplex detection card |
| CN114891101A (en) * | 2021-12-02 | 2022-08-12 | 吉林省农业科学院 | Bovine myostatin protein monoclonal antibody and application thereof |
| CN114891101B (en) * | 2021-12-02 | 2023-06-16 | 吉林省农业科学院 | Bovine myostatin protein monoclonal antibody and application thereof |
| CN114716531A (en) * | 2022-05-17 | 2022-07-08 | 中国农业科学院农业质量标准与检测技术研究所 | Casein polypeptide, polypeptide antigen, antibody, test strip and application thereof |
| CN114716531B (en) * | 2022-05-17 | 2023-11-03 | 中国农业科学院农业质量标准与检测技术研究所 | Casein polypeptide, polypeptide antigen, antibody, test strip and application thereof |
| CN116333151A (en) * | 2022-08-12 | 2023-06-27 | 广西壮族自治区水牛研究所 | Monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and preparation method and application thereof |
| CN116333151B (en) * | 2022-08-12 | 2023-08-25 | 广西壮族自治区水牛研究所 | Monoclonal antibody for detecting adulterated common cow milk in buffalo milk, and preparation method and application thereof |
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Effective date of registration: 20211213 Address after: 100176 building 3, courtyard 11, Kechuang 14th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing Patentee after: BEIJING NABAI BIO-TECH CO.,LTD. Address before: 100000 room 730, unit 2, floor 7, building 18, No. 7, Tongji Middle Road, Beijing Economic and Technological Development Zone, Daxing District, Beijing Patentee before: Beijing jinzhizhun Technology Co.,Ltd. |
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