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Higher eukaryote non-intron nested gene NJx-1 and expression protein and application thereof

A technology of njx-1, 1.njx-1, applied in the field of eukaryotic non-intron nesting gene NJx-1 and its expressed protein, can solve the problem of only surgery, no early diagnosis method, narrow cranial cavity and unable to adapt to the brain problems with normal development

Pending Publication Date: 2021-12-10
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to premature closure of cranial sutures, the narrowing of the cranial cavity cannot accommodate the normal development of the brain
Craniostenosis is currently diagnosed, there is no suitable medicine, only surgery; there is no early diagnosis method

Method used

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  • Higher eukaryote non-intron nested gene NJx-1 and expression protein and application thereof
  • Higher eukaryote non-intron nested gene NJx-1 and expression protein and application thereof
  • Higher eukaryote non-intron nested gene NJx-1 and expression protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Discovery of NJx-1

[0024] The applicant finds compound QOA-8a (structural formula is figure 1 The structure marked in black in QOA-8aProbe) has a unique dual effect of inhibiting bone resorption and promoting bone formation, so Biotin was used to prepare the QOA-8a biotin probe (QOA-8a Probe). The synthesized probe is similar to QOA-8a at a concentration of 1 μM, and has a unique dual function of inhibiting bone resorption and promoting bone formation. Using this probe, the possible target of QOA-8a in osteoblasts was "fished". ".

[0025] Specific steps: collect the calvarial osteoblasts (2×107) of suckling mice by centrifugation, resuspend them with 1ml of pre-cooled PBS and transfer them to Eppendorf tubes; collect the cells by centrifugation at 6000rpm×0.5min at 4°C; discard the supernatant, and scatter the cells For precipitation, add 2ml of pre-cooled cell lysate (containing 1mM PMSF, added before use) and pipette repeatedly; let stand on ice for 30min; cen...

Embodiment 2

[0038] 1. Preparation of NJx-1 gene mutant mice

[0039] The NJx-1 gene knock-in strategy was used for the preparation of NJx-1 gene mutant mice. A synonymous codon replacement strategy was used to create NJx-1 knock-in mice (hereinafter referred to as NJx-1-ki mice). The 157Ser & 158Ser codons of Klf9 are edited by the CRISPR / Cas9 system after the fourth ATG (30Met) of the NJx-1 gene-TCC>TCA-The amino acid sequence of the K1f9 gene is still serine, and the antisense NJx-1 gene becomes two consecutive stop codon, which terminates the translation of NJx-1 gene (see the gene sequence mutation point Figure 4 representation of small and medium boxes).

[0040] The results of sequencing identification showed that the mutation of the NJx-1 gene was realized (due to two consecutive stop codons, 30 amino acids at the N-terminal of NJx-1 were retained, and 120 amino acids were removed); similarly, for genes similar to NJx-1 Sequence sequencing showed no mutations and no off-target ...

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Abstract

The present application discloses a higher eukaryote non-intron nested gene NJx-1 and expression protein and application thereof. A murine cDNA sequence of the non-intron nested gene NJx-1 is as shown by SEQ ID NO.3, and a human-derived cDNA sequence of the non-intron nested gene NJx-1 is as shown by SEQ ID NO.4, and the gene is a rare non-intron nested gene; a sequence of a coding murine protein of the gene is as shown by SEQ ID NO.1, and a sequence of a coding human-derived protein of the gene is as shown by SEQ ID NO.2. The NJx-1 gene disclosed by the present application plays a very important role in development of bones, and has a relatively good application prospect and application value in research and development of medicines for treating bone diseases such as craniosis, osteoporosis and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a non-intron nesting gene NJx-1 of higher eukaryotic organisms, its expressed protein and its application. Background technique [0002] Overlapping gene, also known as nested gene, means that two or more genes share a DNA sequence, or a DNA sequence becomes a component of two or more genes. Nested genes include two categories: intron-nested genes and non-intron-nested genes. Intron-nested genes, located within introns of external genes, are common in higher eukaryotes and most are predicted to encode proteins. Non-intron nested genes are extremely rare in eukaryotic genomes, and there is no report on the identification and biological functions of non-intron nested genes and their encoded products in higher eukaryotes (especially mammals) . [0003] Craniostenosis, also known as craniosynostosis early fusion or cranial stenosis deformity. Manifestations incl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00
CPCA61K48/005A61K38/1709C07K14/47A61P19/10A61P19/08
Inventor 吴静李建新
Owner NANJING UNIV
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