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Nanometer antibody, encoding sequence and application of H2A.Z variant

A nanobody, sequence technology, applied to the nanobody against H2A.Z variant, its coding sequence and application field, can solve the problem of nanobody lacking light chain and so on

Inactive Publication Date: 2014-01-08
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The constant region CH1 is the site anchored to the light chain, which exists in the genome of the Nanobody, but is cut out during mRNA formation, so the Nanobody lacks the light chain

Method used

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  • Nanometer antibody, encoding sequence and application of H2A.Z variant
  • Nanometer antibody, encoding sequence and application of H2A.Z variant
  • Nanometer antibody, encoding sequence and application of H2A.Z variant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of nanobody library against histone H2A.Z:

[0030](1) Mix 1 mg H2A.Z with Freund's adjuvant in equal volume, immunize a Xinjiang dromedary, once a week, immunize 7 times in total, and stimulate B cells to express antigen-specific nanobodies; (2) 7 After the first immunization, 100 mL camel peripheral blood lymphocytes were extracted and total RNA was extracted; (3) cDNA was synthesized and VHH was amplified by nested PCR; (4) 20 ug of pMECS vector and 10 ug VHH and ligated the two fragments; (5) Transformed the ligated product into electroporation-competent TG1 cells to construct the H2A.Z nanobody library and measure the library capacity, which was 1.15×10 8 .

Embodiment 2

[0032] H2A.Z nanobody screening process:

[0033] (1) Dissolve in 100 mM NaHCO 3 20 ug H2A.Z in (pH=8.2) was coupled to NUNC plate,

[0034] Place overnight at 4°C; (2) add 100 uL 0.1% casein the next day, block at room temperature for 2 h; (3) add 100 uL phage (5×10 11 tfu immunized camel nanobody phage display gene library) and reacted at room temperature for 1 h; (4) Washed 5 times with 0.05% PBS+Tween-20 to wash off unbound phages; (5) Washed with 100 mM TEA (triethylamine) Dissociate the phage that specifically binds to H2A.Z, infect Escherichia coli TG1 in the logarithmic growth phase, culture at 37 °C for 1 h, produce and purify the phage for the next round of screening, repeat the same screening process for 3-4 rounds, gradually enriched.

Embodiment 3

[0036] Use phage enzyme-linked immunosorbent assay (ELISA) to screen specific single positive clones:

[0037] (1) After 3-4 rounds of screening, select 96 single colonies from the cell culture plate containing phage and inoculate them in a 24-well cell culture plate. After growing to the logarithmic phase, add 1 mM IPTG and culture overnight at 28 °C ; (2) Obtain crude antibody by permeation method, transfer the antibody to an antigen-coated ELISA plate, and place it at room temperature for 1 h; (3) Wash unbound antibody with PBST, add mouse anti-HA tag antibody at room temperature for 1 h; (4) wash off unbound antibodies with PBST, add anti-mouse alkaline phosphatase conjugate, and place at room temperature for 1 h; (5) wash off unbound antibodies with PBST, add chromogenic solution, and use ELISA On the instrument, read the absorbance value at a wavelength of 405 nm; (6) When the OD value of the sample well is more than 2 times greater than the OD value of the control well,...

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Abstract

The invention discloses a nanometer antibody, an encoding sequence and an application of H2A.Z variant, and meanwhile, discloses a gene sequence encoding the nanometer antibody, a method for preparing the nanometer antibody and a host cell for expressing or capable of expressing the nanometer antibody. A nanometer antibody VHH chain of the histone H2A.Z variant comprises framework regions and a complementary determining region, wherein four framework regions are arranged and are respectively FR1-FR4; the complementary determining region is amino acid sequences of CDR1-CDR3, wherein CDR1 refers to amino acid sequences as shown in SEQNO.16-SEQNO.18; CDR2 refers to amino acid sequences as shown in SEQNO.19-SEQNO.21; CDR3 refers to amino acid sequences as shown in SEQNO.22-SEQNO.24. The nanometer antibody can be efficiently expressed in Escherichia coli and can be applied in research on high expression of cancer associated protein and related transcription regulation mechanism.

Description

[0001] technical field [0002] The present invention is a nanobody (or called single domain antibody fragment, single domain antibody fragment) directed at the epitope of a histone variant H2A.Z, and also announces the amino acid sequence encoding the nanobody, and prepares the nanobody Methods of antibodies, host cells expressing or capable of expressing the Nanobody. The nanobody can be highly expressed in Escherichia coli, and can be applied to the research of the mechanism of high expression of cancer-related proteins and the mechanism of related transcriptional regulation. Background technique [0003] Eukaryotic genomes are highly condensed in cells to form chromatin. The basic unit of chromatin structure is the nucleosome, which is composed of histone octamers formed by four core histones H2A, H2B, H3 and H4. Each histone is formed by two molecules, about About 147bp of DNA is wound on the histone octamer to form a nucleosome unit. Histone H1 binds between two adja...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/574C12N1/21
Inventor 万亚坤母亚雯孙燕燕
Owner SOUTHEAST UNIV
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