Nano antibody for HDAC6-cat 1 region and application thereof

A nanobody and domain technology, applied in the field of biotechnology or biomedicine, can solve the problem of lack of selective inhibitors

Inactive Publication Date: 2019-05-10
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of lack of selective inhibitors (antibody drugs) for HDAC6 in the prior art, the present invention prepares and screens out specific nanobodies that can recognize HDAC6. Based on the advantages of nanobodies over ordinary antibodies, it is HDAC6 in Provide effective theoretical support in the fields of drug development and disease treatment

Method used

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  • Nano antibody for HDAC6-cat 1 region and application thereof
  • Nano antibody for HDAC6-cat 1 region and application thereof
  • Nano antibody for HDAC6-cat 1 region and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Example 1: Construction of a Nanobody library for the HDAC6-cat1 region:

[0074] (1) 200 μg HDAC6-cat1 antigen ( figure 1 ) mixed with Freund's adjuvant in equal volume, immunized a Xinjiang Bactrian camel, once a week, a total of 6 consecutive immunizations, during the immunization process stimulated B cells to express specific nanobodies, and the antibody titer ended at the end of 6 immunizations After the determination, the result is 10 4 ( figure 2 ); (2) After 6 times of immunization, extract 100ml of camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Use restriction enzymes PstI and NotI Cut 20 μg pMECS phage display vector and 10 μg VHH and connect the two fragments; (5) Transform the ligated product into electroporation competent cell TG1, construct HDAC6-cat1 region nanobody phage display library and measure the storage capacity, the size of the storage capacity is about 1.2 ×10 8 At the same t...

Embodiment 2

[0075] Example 2: Nanobody screening process for the HDAC6-cat1 region:

[0076] (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) 200 μg of HDAC6-cat1 recombinant protein dissolved in 100 mM pH 8.4 NaHCO3 was coupled to a microtiter plate, placed overnight at 4 ° C, and a negative control was set up at the same time; (3) 100 μl of 3% BSA was added the next day, Block at room temperature for 2 h; (4) After 2 h, add 100 μl of amplified phage (2×10 11 tfu immunized camel nanobody phage display gene library), and acted at room temperature for 1h; (5) washed off the bound phage with PBS+0.05% Tween-20; (6) combined with HDAC6-cat1 with trypsin at a final concentration of 25mg / ml Under the dissociation of specific binding phage, infect E. coli TG1 cells in...

Embodiment 3

[0077] Embodiment 3: Screen specific positive clone ( Figure 4 ):

[0078] (1) Select 175 single colonies from the cell culture plates after the above three rounds of screening and inoculate them in 96 deep-well plates containing 100ug / mL ampicillin in TB medium, and set up a blank control, and culture at 37°C until logarithmic After the period, add IPTG with a final concentration of 1mM and incubate overnight at 28°C; (2) Obtain the crude antibody by osmotic bursting method, transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 hour; (3) Wash off unbound antibody with PBST, add 100ul of Mouse anti-HA tag antibody (mouse anti-HA antibody) diluted 1:2000, and let stand at room temperature for 1h; (4) wash off unbound antibody with PBST, add 100ul Anti-mouse alkaline phosphatase conjugate (goat anti-mouse alkaline phosphatase-labeled antibody) diluted at 1:2000, placed at room temperature for 1 hour; (5) Wash unbound antibody with PBST,...

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Abstract

The invention discloses a nano antibody for an HDAC6-cat 1 region and application thereof, particularly relates to a nano antibody for a histone deacetylase 6 catalase 1 region (HDAC6-cat1)and application thereof, discloses an amino acid sequence of a framework region FR and a complementarity determining region CDR of a VHH chain of the nano antibody, and also discloses a gene sequence encoding the heavy-chain nano antibody and a host cell expressing the nano antibody. By means of the gene sequence and the host cell of the single-region heavy-chain nano antibody, the nano antibody can be efficiently expressed in escherichia coli, can be recognized and specifically identified with a target antigen after being expressed and purified, and can be used for analyzing the epitope of the target antigen. Meanwhile, a pcDNA5 / FRT recombinant plasmid is constructed, so that the influence of the nano antibody on the biological function of histone deacetylase 6 (HDAC6) is explored.

Description

technical field [0001] The invention relates to the field of biotechnology or biomedicine, and relates to a nanobody targeting the HDAC6-cat1 region and applications thereof. Background technique [0002] Antibodies (Abs), also known as immunoglobulins (Igs), are large Y-shaped proteins produced primarily by plasma cells used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. Antibodies are secreted by B cells of the adaptive immune system, mostly by differentiated B cells called plasma cells, glycoproteins belonging to the immunoglobulin superfamily that make up most of the gamma globulin portion of blood proteins. At present, the most commonly used antibody is IgG antibody, which is also the antibody with the highest content in the human body, about 9.5-12.5g / L. Conventional antibodies are usually composed of two large heavy chains and two small light chains, and the heavy chains and light chains are combined by disulfide bonds to form a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C40B50/06C12N15/70C12N1/21A61K39/395C12R1/19
Inventor 谢维于笑笑苏志鹏
Owner REGENECORE BIOTECH CO LTD
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