Anti-CTLA-4 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof

A CTLA-4, nanobody technology, applied in the field of immunology, can solve the problem of no nanobody report

Pending Publication Date: 2022-05-31
NINGXIA MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] No nanobodies targeting CTLA-4 have been reported so far

Method used

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  • Anti-CTLA-4 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof
  • Anti-CTLA-4 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof
  • Anti-CTLA-4 nano antibody, coding gene, recombinant nano antibody, recombinant vector, recombinant bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] For the construction of natural camel-derived nanobody gene library:

[0068] To extract total RNA from camel spleen tissue, the specific steps are as follows:

[0069] ① Take out the camel spleen tissue from -80°C, add liquid nitrogen to grind the sample, add 1ml Trizol per 100mg of ground tissue, and homogenize it with a glass homogenizer until no tissue sample particles are visible to the naked eye. Then transferred to a 1.5ml centrifuge tube and placed at room temperature for 15min.

[0070] ②According to the ratio of adding 0.2ml of chloroform per 1ml of Trizol, shake vigorously for 15s, and let stand for 3min at room temperature.

[0071] ③ Centrifuge the sample at 12,000 rpm and 4°C for 15 min, and transfer the upper layer to a new Ep tube.

[0072] ④Add an equal volume of isopropanol and mix on ice for 20min.

[0073] ⑤ Centrifuge the solution at 12000rpm and 4°C for 10min, and discard the supernatant.

[0074] ⑥ Add 1ml of DEPC water to prepare 75% ethanol ...

Embodiment 2

[0121] Expression and purification of nanobodies in host bacteria Escherichia coli:

[0122] (1) The nanobody sequence obtained by sequencing analysis was connected to the human IgGFc segment, subcloned into the pCZN1 plasmid vector, and transformed into Escherichia coli Arctic Express. In a test tube of 3 mL of LB medium, shake at 37°C at 220 rpm overnight; (2) inoculate 30 mL of 50 μg / mL Amp in 30 mL of LB medium at 1:100 the next day, shake at 37° C. at 220 rpm until the OD600 of the cells is 0.6 to 0.8, IPTG was added to a final concentration of 0.5 mM, shaken at 20°C and 220 rpm overnight to induce fusion protein expression; (3) the cells were collected and sonicated to obtain a bold liquid inclusion body protein, which was then subjected to Ni column affinity purification to obtain fusion protein.

[0123] Figure 4 It is the western blot of the purified recombinant nanobody: lane 1 is the protein molecular standard, lane 2 is the purified anti-CTLA-4 nanobody (the prim...

Embodiment 3

[0125] Specificity Verification of Recombinant Nanobodies:

[0126] The CTLA-4 antigen and activated human T cells were used for western blotting (the primary antibody was purified recombinant nanobody, and the secondary antibody was anti-human IgG Fc / HRP). Figure 5 Middle lane 1 is the whole protein of activated human T cells, lane 2 is the whole protein of 293T cells without CTLA-4 expression, and lane 3 is the CTLA-4 antigen. This shows that the anti-CTLA-4 nanobody E5 provided by the present invention can specifically recognize CTLA-4 antigen.

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Abstract

The invention provides an anti-CTLA-4 nano antibody, a coding gene, a recombinant nano antibody, a recombinant vector, recombinant bacteria and application thereof, and belongs to the technical field of immunology. The anti-CTLA-4 nano antibody E5 provided by the invention is designed aiming at a heavy chain of CTLA-4, and the anti-CTLA-4 nano antibody E5 comprises a framework region FR and an antigenic determinant complementary CDR. The nano antibody E5 provided by the invention can specifically recognize a CTLA-4 antigen. According to the present invention, the coding gene for coding the anti-CTLA-4 nanometer antibody E5 and the human IgG Fc segment gene are subjected to fusion expression to obtain the recombinant nanometer antibody, and the recombinant nanometer antibody can specifically recognize the CTLA-4 antigen, and can be used for tumor molecular diagnosis and / or antitumor drug preparation.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to an anti-CTLA-4 nanobody, an encoding gene, a recombinant nanobody, a recombinant vector, a recombinant bacteria and applications thereof. Background technique [0002] Cytotoxic T lymphocyte associated protein-4 (CTLA-4) is another immunosuppressive receptor expressed on T cells. After binding with B7 molecule, it induces T cell anergy and participates in immunity. Negative regulation of responses. Blocking the CTLA-4 molecule can improve the anti-tumor response of T cells and kill tumor cells. [0003] Nanobody was first reported by Belgian scientists in Nature in 1993. It is a natural light chain-deficient antibody (VHH) that exists in the peripheral blood of alpaca. Found in fish, it is the smallest unit known to bind the target antigen. The molecular weight of VHH is only 15KD, so it is also called nanobody (Nanobody, Nb). Nanobodies have unique advantages,...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/70C12N1/21G01N33/574G01N33/68C12R1/19
CPCC07K16/2818C12N15/70G01N33/574G01N33/68C07K2317/569C07K2317/565Y02A50/30
Inventor 张爱君张艳婷徐广贤蒋丹王丽燕李艳宁
Owner NINGXIA MEDICAL UNIV
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