Nano-antibody aiming at H3K64Ac/H3K64 fragments and application thereof

A technology of H3K64 and nanobody, applied in the field of nanobody, can solve the problems of difficulty in obtaining, weak stability and high preparation cost, and achieve the effect of reducing research cost

Active 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

[0004] In order to overcome the technical defects of conventional antibodies against H3K64Ac / H3K64 in the prior art, such as large molecular weight, weak stability, difficulty in obtaining, and high production cost, the purpose of the present invention is to provide nanobodies against H3K64Ac / H3K64 fragments and their amino acid sequences , at the same time provide the DNA coding sequence of the Nanobody against the H3K64Ac / H3K64 fragment, construct the eukaryotic plasmid system of the Nanobody, and use it to replace the traditional antibody tools to study the biological function of this site

Method used

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  • Nano-antibody aiming at H3K64Ac/H3K64 fragments and application thereof
  • Nano-antibody aiming at H3K64Ac/H3K64 fragments and application thereof
  • Nano-antibody aiming at H3K64Ac/H3K64 fragments and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of a Nanobody library against H3K64Ac fragments

[0060] (1) Mix 200 μg of H3K64Ac fragment antigen with Freund’s adjuvant in equal volumes, and immunize a Xinjiang Bactrian camel once a week for a total of 6 consecutive immunizations. During the immunization process, B cells are stimulated to express specific nanobodies, among which The titer was determined after 6 times of immunization, and the result was 10 4 ( figure 1); (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 to construct the H3K64Ac fragment nanobody phage display library and measure the storage capacity, the size of the storage capacity is about 1.2×10 8 At the same time, th...

Embodiment 2

[0061] Example 2: Nanobody screening process against H3K64Ac fragments

[0062] (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) 500ng of H3K64Ac fragments were coupled to the 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, and blocked at room temperature for 2 hours; (4) After 2 hours, Add 100 μl of amplified phage (2×10 11 tfu immunized camel nanobody phage display gene library), and reacted at room temperature for 1 h; (5) washed off the bound phage with PBS+0.05% Tween-20; Under the dissociation of sex-binding phages, they infect Escherichia coli TG1 cells in the logarithmic growth phase, culture at 37°C for 1 hour, produce and collect phages for the next ...

Embodiment 3

[0063] Embodiment 3: use the enzyme-linked immunosorbent method (ELISA) of phage to screen specificity positive clone

[0064] (1) Select 300 single colonies from the cell culture plates after the above three rounds of screening and inoculate them in 96 deep-well plates containing 100 μg / 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 the unbound antibody with PBST, add 100μl mouse anti-HA tag antibody (Mouse anti-HA tag antibody) diluted 1:2000, and let it stand at room temperature for 1h; (4) Wash off the unbound antibody with PBST, add 100μl The goat anti-mouse alkaline phosphatase conjugate antibody (Anti-mouse alkaline phosphatase conjugate) diluted at 1:2000 was pla...

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Abstract

The invention relates to the field of biotechnology and biomedicine, and relates to a nano-antibody aiming at H3K64Ac / H3K64 fragments and application thereof. Specifically, a VHH chain of the nano-antibody of the anti-histone 3 64 lysine acetylation (H3K64Ac) fragment has an amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34 or SEQ ID NO:35 or SEQ ID NO:36. A VHH chain of the nano-antibody of the anti-histone 3 64 lysine non-modified (H3K64) fragment has an amino acid sequence shown in SEQ ID NO:37 or SEQ ID NO:58. The nano-antibody is efficiently expressed through microorganisms. The molecular weight of the nano-antibody does not reach 1 / 10 that of a conventional antibody, and therefore research on the nucleosome core region H3K64 acetylation site biological functions can be achieved.

Description

technical field [0001] The invention relates to the field of biotechnology or biomedicine, and relates to nanobodies directed at the acetylated and unmodified (H3K64Ac / H3K64) fragments of the 64th lysine of histone 3 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 hea...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/44C12N15/13C12N15/63C12N5/10G01N33/68
Inventor 谢维于笑笑苏志鹏
Owner REGENECORE BIOTECH CO LTD
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