阅读说明：本技术 一种牛、羊奶酪蛋白单克隆抗体、检测试剂盒及应用 (Bovine and goat milk casein monoclonal antibody, detection kit and application ) 是由 于在江 李月 冯小宇 孙海霞 陈曼利 朱琳 于 2020-08-20 设计创作，主要内容包括：本发明提供一种牛、羊奶酪蛋白单克隆抗体、检测试剂盒及应用,所述牛、羊奶酪蛋白单克隆抗体的轻链可变区具有如SEQ ID NO：1所示的氨基酸序列,重链可变区具有如SEQ ID NO：2所示的氨基酸序列。该单克隆抗体为经杂交瘤技术制备并筛选出的可与牛奶酪蛋白、羊奶酪蛋白结合而不与骆驼奶酪蛋白结合,且与牛奶酪蛋白、羊奶酪蛋白结合的亲和性最高的单克隆抗体,利用该单克隆抗体可同时检测驼奶中掺杂微量的牛奶和羊奶,检测操作简便,且灵敏度高。(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.)

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.

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.