Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof

A nanobody and sequence technology, applied in the field of biomedicine or biopharmaceuticals, to achieve high-efficiency expression

Inactive Publication Date: 2015-03-25
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary antibody proteins are composed of two heavy chains and two light chains, while the new antibodies discovered from camel blood have only two heavy chains and no light chains. These HCAbs can tightly bind to targets such as antigens like normal antibodies, But unlike single-chain antibodies that stick to each other and aggregate into clumps

Method used

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  • Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof
  • Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof
  • Bactrian camel-derived ApoE nano antibody as well as coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of the Nanobody Library against ApoE:

[0019] (1) First mix 1 mg ApoE with Freund's adjuvant in equal volumes, and immunize a Xinjiang Bactrian camel once a week, a total of 7 times to stimulate B cells to express antigen-specific nanobodies; (2) 7 times of immunization After the end, extract 100mL camel peripheral blood lymphocytes and extract total RNA; (3) synthesize cDNA and amplify VHH by nested PCR; (4) digest 20ug pComb3 phage display vector with restriction enzymes Pst I and Not I (supplied by Biovector) and 39ug VHH and connect the two fragments; (5) transform the ligated product into electroporation competent cell TG1, construct the ApoE nanobody library and measure the storage capacity, the storage capacity is 4.9×10 9 CFU / mL; (6) 24 clones were randomly selected, and the insertion rate of the built library was detected by colony PCR. The insertion rate was 83.3%. figure 1 The colony PCR results are shown. The DNA bands in the gel we...

Embodiment 2

[0042] Embodiment 2: Nanobody screening process against ApoE:

[0043] (1) Dissolve in 100mM NaHCO 3 , 20ug ApoE in pH 9.5 was coupled to the NUNC microtiter plate, and placed overnight at 4°C; (2) the next day, 100uL 0.1% casein was added, and blocked at room temperature for 2h; (3) after 2h, 100uL phage (5× 10 11tfu immunized camel nanobody phage display gene library), and acted at room temperature for 1h; (4) washed 5 times with PBS+0.05% Tween-20 (PBST) to wash off unbound phage; (5) washed with 100mM triethanolamine TEA ( triethylamine) to dissociate the phage that specifically binds to ApoE, and infect Escherichia coli TG1 in logarithmic phase growth, culture at 37°C for 1 h, produce and purify the phage for the next round of screening, and repeat the same screening process for 3-4 Rounds, gradually enriched.

Embodiment 3

[0044] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity single positive clone:

[0045] (1) From the cell culture dish containing phage after the above 3-4 rounds of screening, select 96 single colonies and inoculate them in TB medium containing 100ug / mL ampicillin (1L TB medium contains 2.3g potassium dihydrogen phosphate , 12.52g dipotassium hydrogen phosphate, 12g peptone, 24g yeast extract, 4mL glycerol), after growing to the logarithmic phase, add IPTG with a final concentration of 1mM, and culture overnight at 28°C. (2) The crude antibody was obtained by infiltration method, and the antibody was transferred to an antigen-coated ELISA plate, and left at room temperature for 1 hour. (3) Unbound antibodies were washed away with PBST, and mouse anti-HA tag antibody (mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) was added, and left at room temperature for 1 hour. (4) Unbound antibodies w...

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Abstract

The invention discloses a nano antibody corresponding to apolipoprotein ApoE, and also discloses a gene sequence for coding the nano antibody and a host cell capable of expressing the nano antibody. By virtue of the nano antibody gene sequence and the host cell disclosed by the invention, the efficient expression of the nano antibody can be achieved in escherichia coli; and the nano antibody is applicable to the research and development of ApoE molecular detection reagents.

Description

technical field [0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a nanobody targeting ApoE molecules, its coding sequence and application. Background technique [0002] Apolipoprotein E (Apolipoprotein E, ApoE) is a basic protein rich in arginine. Human ApoE is composed of 299 amino acid residues, with a molecular weight of 34145D and contains 32 Arg and 12 Lys. Chylomicron, very low-density lipoprotein, high-density lipoprotein and other lipoprotein components can bind to low-density lipoprotein receptors and play a role in the metabolism of plasma cholesterol and triglycerides. The plasma Apo E concentration of normal people is 0.03-0.05g / L, and the concentration of Apo E is positively correlated with the plasma triglyceride content. The physiological functions of ApoE are as follows: ①It is the ligand of LDL receptor and the ligand of chylomicron remnant receptor in hepatic cells, which is closely related ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N1/21G01N33/92C12R1/19
Inventor 万亚坤严俊荣
Owner SOUTHEAST UNIV
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