Coding gene of drosophilid MYC nano antibody, preparation method and application thereof

A technology of nano-antibody and coding gene, applied in the field of genetic engineering, can solve the problems of high production cost, affecting the application of MYC antibody, poor stability, etc., and achieve the effects of small molecular weight, easy penetration into tissue cells, high specificity and affinity

Active Publication Date: 2021-07-02
GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional MYC antibody is obtained by immunizing animals, which often has problems of poor stability and high production cost, which affects the application of MYC antibody

Method used

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  • Coding gene of drosophilid MYC nano antibody, preparation method and application thereof
  • Coding gene of drosophilid MYC nano antibody, preparation method and application thereof
  • Coding gene of drosophilid MYC nano antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of alpaca nanobody library after Drosophila MYC immunization

[0034] (1) Drosophila embryo protein lysate was mixed with an equal volume of adjuvant (purchased from GERBU), and an alpaca was immunized once every two weeks for a total of 3 times.

[0035] (2) After the 3 times of immunization, 50 ml of peripheral blood of the alpaca was extracted, the blood lymphocytes were separated by density gradient centrifugation (dextran-diatrizoate), and the total RNA of the lymphocytes was extracted by the Trizol method.

[0036] (3) According to TAKARA's PrimerScript TM Instructions for the 1st Strand cDNA Synthesis Kit, which reverse-transcribes the extracted RNA into cDNA.

[0037] (4) Utilize nested PCR (the enzyme used in PCR is TAKARA company Max DNA Polymerase) to amplify the VHH region of alpaca heavy chain antibody. The first round reaction conditions are: 98°C, 3min; 98°C, 10s, 55°C, 10s, 72°C, 1min, 30 cycles; 72°C, 10min. The PCR product...

Embodiment 2

[0041] Example 2: Enrichment process of Drosophila MYC protein-specific nanobodies

[0042] (1) According to the method of prokaryotic purification, the MYC protein fused with the His tag was purified, and the result figure 1 shown.

[0043] (2) Take 1 μg of purified MYC protein and incubate with 30 μl of His-coupled magnetic beads at room temperature for 30 min to bind MYC protein to the magnetic beads, and then wash with PBST for 3 times to wash away unbound MYC protein.

[0044] (3) Add 500 μl phage library (containing 5×10 12 A phage displaying the immune alpaca nanobody), and incubated at room temperature for 2h. Wash 25 times with PBST to wash away unbound or weakly binding phages.

[0045] (4) 500 μl trypsin (0.25 mg / ml) was used to dissociate the phages specifically bound to MYC, and 10 μl protease inhibitor cocktail (purchased from Roche Company) was added to the dissociated phage solution for neutralization.

[0046] (5) Take 300 μl of the dissociated phage solutio...

Embodiment 3

[0048] Example 3: Enzyme-linked immunosorbent assay (ELISA) screening of MYC-specific nanobody positive monoclonal

[0049] (1) Randomly select 10 bacterial monoclonal clones from the overnight cultured Nanobody bacterial library obtained after the third round of screening above, inoculate them into LB medium respectively, and cultivate them until the logarithmic phase of bacterial growth, adding a final concentration of 0.2mM IPTG was incubated overnight at 30°C to induce Nanobody expression.

[0050] (2) The bacteria were collected the next day, and CelLytic was used to TM B Cell Lysis Reagent (purchased from Sigma Company) was used to lyse the bacteria to obtain the nanobody crude extract. Take 100 μl antibody crude extract and add to the ELISA plate coated with MYC protein and blocked with 3% BSA, and incubate at room temperature for 1 h.

[0051] (3) Wash 3 times with PBST, 2 min each time, to wash away unbound protein.

[0052] (4) Anti-pIII antibody (1:1000, purchase...

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Abstract

The invention discloses a coding gene of a drosophilid MYC nano antibody, a preparation method and application thereof. According to the invention, a drosophilid MYC nano antibody library is constructed, a drosophilid MYC nano antibody is screened from the antibody library by utilizing a phage display technology, the nucleotide sequence of the screened coding gene of the drosophilid MYC nano antibody is shown as SEQ ID NO: 1, the corresponding amino acid sequence is shown as SEQ ID NO: 2, and the MYC nano-antibody gene is cloned to a modified expression vector and introduced into a TOP1 strain to obtain an expression vector and a strain of the MYC nano-antibody; and the expression vector of the drosophilid MYC nano-antibody can conveniently detect the nano-antibody, and a strain containing the expression vector of the drosophilid MYC nano-antibody is easy to culture and rapid in proliferation, can be amplified infinitely and stably, can infinitely provide the nano-antibody with stable functions, can produce the MYC nano-antibody on a large scale, and greatly reduces the production cost.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene encoding a fruit fly MYC nanobody, a preparation method and an application thereof. Background technique [0002] As a regulatory gene of nucleoproteins, MYC not only participates in important functional activities of cells, such as cell proliferation, differentiation and apoptosis, but also involves the formation and evolution of human tumors. A large number of studies have shown that the abnormality of MYC can be detected in human tumors. It can be seen that MYC has universal significance in tumor and other cell activities, and has great demand in subsequent basic research and clinical research. Currently, the most widely used antibody is the traditional antibody, which is a tetramer composed of two heavy chains and two light chains. Traditional antibodies have the advantages of high affinity and specificity, but the disadvantages of large molecular weight ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C07K16/18C07K16/00
CPCC07K16/18C07K16/005C07K2317/569
Inventor 方智新张智武文娇
Owner GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
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