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Single domain antibody against bovine serum albumin bsa and its derivative protein

A bovine serum albumin and single-domain antibody technology, which is applied in the field of biotechnology or immunology, can solve the problems of limited single-batch preparation, inability to obtain antibodies for a long time, and difficult quality control, etc., to achieve easy implementation and high affinity , Good stability between batches

Active Publication Date: 2021-06-18
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the minimum limit for the detection of BSA content in biological products at home and abroad is 5 ng / mL, and the antibodies used in these detection reagents are all polyclonal antibodies (rabbit origin). Compared with mouse monoclonal antibodies, although rabbit polyclonal antibodies are prepared In addition to the advantages of short cycle time and slightly stronger affinity, its disadvantages are also very obvious: 1) The amount of preparation in a single batch is limited, and antibodies with good effects for a single batch cannot be obtained for a long time; 2) There is too much difference between batches; 3) Group The classification is relatively complicated, and the quality control is difficult to control

Method used

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  • Single domain antibody against bovine serum albumin bsa and its derivative protein
  • Single domain antibody against bovine serum albumin bsa and its derivative protein
  • Single domain antibody against bovine serum albumin bsa and its derivative protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Construction of a single domain antibody library against BSA protein:

[0073] (1) Mix 1 mg of BSA antigen with Freund's adjuvant in equal volumes, and immunize an Inner Mongolia Alxa Bactrian camel, once a week, for a total of 7 consecutive immunizations. During the immunization process, B cells are stimulated to express specific nanobodies; ( 2) After the immunization, extract 100ml of camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Digest 20 μg pMECS with restriction enzymes Pst I and Not I Phage display vector and 10 μg VHH and connect the two fragments; (5) transform the ligated product into electroporation competent cell TG1, construct the BSA protein phage display library and measure the storage capacity, the size of the library storage capacity is about 2×10 9 ; At the same time, the correct insertion rate of the target fragment in the built library was detected by colony PCR, figure 1 ...

Embodiment 2

[0074] Example 2: Screening against BSA protein single domain antibody:

[0075] (1) Take 200 μL of recombinant TG1 cells and culture them in 2×TY medium, add 40 μL of helper phage VCSM13 to infect TG1 cells, and culture overnight to amplify the phages, use PEG / NaCl to precipitate the phages the next day, and centrifuge to collect the amplified phages ; (2) NaHCO diluted in 100mM pH 8.3 3 500ug of neutravidin protein was coupled to the microtiter plate, placed overnight at 4°C, and a negative control well was set up at the same time; (3) the next day, add 100 μL of biotin-labeled BSA protein (BSA-Biotin) and incubate at room temperature After 2 hours, 100 μL of PBS was added to the negative control well; (4) 2 hours later, 100 μL of 3% skim milk was added, and blocked at room temperature for 2 hours; (5) After the blocking was completed, 100 μl of the amplified phage library (about 11 phage particles), at room temperature for 1 h; (6) after 1 hour of action, wash 5 times with...

Embodiment 3

[0076] Embodiment 3: use the enzyme-linked immunoassay (ELISA) of phage to screen specific positive clones:

[0077] (1) According to the above single domain antibody screening method, BSA protein was screened for 3 rounds. After the screening, the phage enrichment factor for BSA protein reached more than 10 (about 10000), and 400 single clones were selected from the positive clones obtained by screening. The colonies were respectively inoculated in 96 deep-well plates containing 100 μg / mL ampicillin in TB medium, and a blank control was set up. After culturing to logarithmic phase at 37°C, IPTG with a final concentration of 1mM was added and cultured overnight at 28°C; (2 )Use the osmotic bursting method to obtain the crude antibody; dilute the neutravidin protein to 100mM NaHCO with pH 8.3 3 Neutralize and coat 100μg of neutravidin protein in the microtiter plate overnight at 4°C, and add 100ug of BSA-Biotin protein to the microtiter plate the next day; (3) Transfer 100uL of...

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Abstract

The invention relates to the technical field of biotechnology or immunology, and relates to a single-domain antibody against bovine serum albumin (BSA) and its derivative protein. The amino acid sequence of the CDR1 of the single domain antibody is shown in SEQ ID NO:77-SEQ ID NO:95; the amino acid sequence of the CDR2 is shown in SEQ ID NO:96-SEQ ID NO:124; the The amino acid sequence of the CDR3 is shown in SEQ ID NO:125-SEQ ID NO:141 and SEQ ID NO:195-SEQ ID NO:206. The beneficial effects of the present invention are: the single-domain antibody, as a BSA detection antibody, has higher affinity than the traditional monoclonal antibody; compared with the polyclonal antibody in the existing detection kit, its composition is more single, and the stability between batches Better yet, quality control is easier to implement, resulting in greater confidence in the actual BSA content test results.

Description

technical field [0001] The invention relates to the technical field of biotechnology or immunology, and relates to a single-domain antibody against bovine serum albumin (BSA) and its derivative protein. Background technique [0002] Antibody (Ab) or Immunoglobulin (Ig) is a type of glycoprotein in blood and tissue fluid. It is produced by plasma cells that proliferate and differentiate after receiving antigen stimulation from B cells. It mainly exists in serum and other body fluids. It specifically binds to the corresponding antigen and is an important effector molecule that mediates humoral immunity. In addition to being an important effector molecule that mediates specific humoral immune responses, antibodies play an important role in the prevention and treatment of diseases, especially infectious diseases. Behring created serum therapy and thus won the Nobel Prize in Medicine and Physiology. Since then, antibodies artificially prepared by polyclonal, monoclonal and genet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21
CPCC07K16/18C07K2317/565C07K2317/569C07K2317/92C12N15/70
Inventor 苏志鹏赵泽英姚尧
Owner REGENECORE BIOTECH CO LTD
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