Natural nanometer antibody library for Bactrian camel phage display as well as construction method and usage thereof

A phage display and nanobody technology, applied in the field of biomedicine or biopharmaceuticals, to achieve the effects of good quality, long time to solve, and rich diversity

Inactive Publication Date: 2015-03-11
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Method used

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  • Natural nanometer antibody library for Bactrian camel phage display as well as construction method and usage thereof
  • Natural nanometer antibody library for Bactrian camel phage display as well as construction method and usage thereof
  • Natural nanometer antibody library for Bactrian camel phage display as well as construction method and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Construction of a natural Bactrian camel-derived phage display nanobody library:

[0024] (1) Take 100 mL of blood samples from 20 Bactrian camels that have not been immunized with any antigen, and at the same time, kill another Bactrian camel that has not been immunized with any antigen, and remove its spleen. Lymphocytes were isolated from blood samples while the spleen was ground. Total mRNA was then extracted from lymphocytes and ground spleen. (2) Synthesize cDNA and use nested PCR to amplify VHH, the result is as follows figure 2 show. (3) 96 μg of pMECS phage display vector (supplied by Biovector) and 32 μg of VHH were digested with restriction endonucleases Pst I and Not I, and the two fragments were ligated. (5) The ligation product was electrotransformed into competent cell TG1 (Beijing Shenzhou Hongye Technology Co., Ltd.), and a natural Bactrian camel-derived phage display nanobody library was constructed and the storage capacity was determine...

Embodiment 2

[0046] Example 2: Screening process for Nanobodies against procalcitonin PCT or neutrophil gelatinase-associated lipocalin NGAL:

[0047] (1) Add 200 μL of transformed TG1 cells into 100 mL of 2×TY medium, and culture at 37° C. for 3 h. (2) Add 40 μL VCSM13 helper phage and let stand at room temperature for 30 minutes. (3) Centrifuge for 10 minutes, resuspend the cells precipitated by centrifugation into 250mL 2×TY medium, and culture overnight at 37°C; (4) Centrifuge the culture solution at 8000rpm for 30 minutes, take the supernatant, and use PEG / NaCl precipitation for amplification The precipitated phages were dissolved in PBS solution. (5) Dissolve in 100mM pH 8.2NaHCO 3 10 μg PCT and 10 μg NGAL were coupled to NUNC microtiter plates, placed overnight at 4°C, and a negative control was set up at the same time. (26) On the next day, 100 μL of 0.1% casein was added to each well, and blocked at room temperature for 2 hours. (7) After 2 hours, add 100 μL of collected phage...

Embodiment 3

[0048] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity single positive clone:

[0049] (1) From the cell culture dish containing phage after the above four rounds of screening, pick 96 single colonies and inoculate them in TB medium containing 100ug / mL ampicillin (1L TB medium contains 2.3g potassium dihydrogen phosphate, 12.52 g dipotassium hydrogen phosphate, 12 g peptone, 24 g yeast extract, 4 mL glycerol), grow to the logarithmic phase, add IPTG with a final concentration of 1 mM, and culture overnight at 28°C. (2) Obtain the crude antibody by infiltration method, transfer the antibody to the ELISA plate coated with the corresponding antigen, and place it at room temperature for 1 hour. (3) Unbound antibodies were washed away with PBST, and a primary 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) Unbou...

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Abstract

The invention discloses a natural nanometer antibody library for Bactrian camel phage display. The natural nanometer antibody library is prepared from the following steps: sampling Bactrian camel blood and spleen which are not immunized with any antigen, extracting total mRNA, reversely transcribing into cDNA, and amplifying VHH by nested PCR (Polymerase Chain Reaction); carrying out enzyme digestion on a pMECS phage display vector and the VHH by restrictive incision enzymes Pst I and Not I and connecting two fragments; electrically transforming a connected product into a competent cell TG1. The invention further discloses a construction method and application thereof in the sieving of PCT and NAGL nanometer antibodies. The constructed natural phage display library can obtain the nanometer antibodies with specificity and a detection function through sieving, and can solve the problem that as a camel cannot be immunized due to antigen factors, the corresponding phage display library cannot be constructed, and accordingly, the nanometer antibodies cannot be obtained. Meanwhile, the library can also solve the problem that the immunization of the camel spends long time.

Description

technical field [0001] The invention relates to the technical field of biomedicine or biopharmaceuticals, in particular to a natural Bactrian camel-derived phage display nanobody library, construction method and application. Background technique [0002] Screening Nanobodies from immune libraries is the most widely used method to obtain Nanobodies. Nanobody libraries generated from immunized camels maintained full diversity. Although high-affinity nanobodies can be screened in a short time, in some cases, it is impossible to achieve camel immunization, such as the immunogen is highly toxic, pathogenic or infectious, and the protein misfolds to form inclusion bodies and cannot be soluble Antigen, antigen self-immunity, and when the antigen is a small molecule compound that does not have immunity, etc. [0003] Procalcitonin (PCT) is a protein with an in vivo reference value of <0.5ug / L. Its plasma levels are elevated in severe bacterial, fungal, and parasitic infections...

Claims

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Application Information

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IPC IPC(8): C40B40/10C40B50/06C12N15/13C12N15/70C07K16/26C07K16/18G01N33/68G01N33/74
Inventor 万亚坤严俊荣朱敏
Owner SOUTHEAST UNIV
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