Bovine and goat cheese protein monoclonal antibody, detection kit and application

A monoclonal antibody, sheep cheese technology, applied in the field of genetic engineering, can solve the problems of complex operation, cumbersome operation, long detection time, etc., and achieve the effects of high detection sensitivity, simple detection operation, and simple operation.

Active Publication Date: 2020-09-25
北京纳百生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-immunological methods include gas chromatography, liquid chromatography, capillary electrophoresis and other techniques. These methods usually require professionals to operate, and the operation is complicated and the detection time is long
In the immunological method, there is bovine casein monoclonal antibody or goat casein monoclonal antibody to detec...

Method used

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  • Bovine and goat cheese protein monoclonal antibody, detection kit and application
  • Bovine and goat cheese protein monoclonal antibody, detection kit and application
  • Bovine and goat cheese protein monoclonal antibody, detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Preparation of bovine and sheep casein monoclonal antibodies

[0037]1. Preparation of Bovine and Sheep Casein Antigens

[0038] Low-temperature high-speed centrifugation (12000rpm, 15min, 4°C) to remove fat and impurity precipitates in the milk; the obtained supernatant was dialyzed overnight in 0.01M PBS (pH=7.2) buffer solution to obtain bovine casein. Goat cheese protein was extracted by the same method.

[0039] 2. Immunization of mice with bovine and sheep casein antigens

[0040] 5-6 week old Balb / c female mice were immunized with purified bovine casein as immunogen. For the first immunization, 100 μL of complete Freund's adjuvant was used to emulsify with 100 μL of phosphate buffered saline (0.01M PBS pH7.4) containing 60 μg of bovine and sheep casein, and the dosage was 10 μg per mouse, and then incomplete Freund's adjuvant was used for each booster immunization Emulsify with bovine casein phosphate buffer (0.01M PBS pH7.4), the dose is the same...

Embodiment 2

[0056] Example 2 The sequencing of bovine and sheep casein monoclonal antibodies

[0057] According to the determination of the titer of the purified monoclonal antibody, 5B10-1G3 was selected as the candidate monoclonal antibody, and the mRNA was extracted from the 5B10-1G3 hybridoma cells. The mRNA was extracted using the RNaeasy Mini KiT extraction kit from QIAGEN, and operated according to the kit instructions. The extracted nucleic acid was frozen and stored at -80°C for later use. Reverse transcription PCR amplification was carried out with SuperScriptTM Preamplification System for First Strand cDNA Synthesis kit from ThermoFisher Company.

[0058] The specific steps are as follows: In a 0.2 mL microcentrifuge tube, add 1 µg of total RNA, supplement with an appropriate amount of DEPC H 2 O to bring the total volume to 11 µL. Add 1 µL of 10 uM Oligo (dT) 12-18 to the tube, mix gently, and centrifuge; heat at 70°C for 10 min, and immediately insert the microcentrifuge tu...

Embodiment 3

[0075] Example 3 In vitro expression of bovine and sheep casein monoclonal antibodies

[0076] The present invention also provides a method for preparing the bovine and sheep casein monoclonal antibodies based on the in vitro expression of the genes of the heavy chain and light chain variable regions of the monoclonal antibodies obtained above.

[0077]The eukaryotic expression vector of NabaiFC-Entry1 was constructed, and the two ends of the heavy chain variable region gene design primers of bovine and sheep casein monoclonal antibody 5B10-1G3 were respectively introduced into two enzyme cutting sites of SaLI / HindIII, and cloned into MCS2 to form NabaiFC -Entry1-VH recombinant plasmid; the two ends of the light chain variable region gene design primers of bovine and sheep casein monoclonal antibody 5B10-1G3 were respectively introduced into SmaLI / HindIII two restriction sites, and cloned into MCS1-MCS2 to form NabaiFC- Entry1-VL recombinant plasmid; the two constructed NabaiF...

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PUM

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Abstract

The invention provides a bovine and goat cheese protein monoclonal antibody, a detection kit and an application. A light chain variable region of the bovine and goat cheese protein monoclonal antibodyhas an amino acid sequence as shown in SEQ ID NO:1, and a heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:2. The monoclonal antibody is a monoclonal antibody which is prepared and screened out by a hybridoma technology, can be combined with bovine cheese protein and goat cheese protein, is not combined with camel cheese protein, is combined with the bovine cheese protein and the goat cheese protein and has the highest affinity, and can be used for simultaneously detecting trace bovine milk and goat milk doped in camel milk. The detection operation is simple and convenient and the sensitivity is high.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bovine and sheep casein monoclonal antibody, a detection kit and its application. Background technique [0002] The nutritional value of camel milk is very high. Camel milk does not contain allergens and will not cause allergic reactions such as vomiting and diarrhea. 903 mg of calcium, which is very high in calcium. Camel milk also contains a large amount of unsaturated fatty acids, iron and vitamin B needed by the human body. Studies have shown that camel milk contains "insulin-like" small molecular substances, which have a certain therapeutic effect on type 2 diabetes. It is precisely because camel milk has these advantages that some unscrupulous businesses add milk or goat milk to camel milk, and even use cow milk or goat milk to pass off as camel milk. [0003] At present, there are mainly non-immunological methods and immunological methods for de...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13G01N33/68G01N33/577G01N33/558
CPCC07K16/18G01N33/558G01N33/577G01N33/68G01N2333/4731
Inventor 于在江李月冯小宇孙海霞陈曼利朱琳
Owner 北京纳百生物科技有限公司
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