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Insect cpcfc family epidermal protein, coding nucleotide sequence and application thereof

A nucleotide sequence and epidermal protein technology, applied in the field of bioengineering, can solve problems such as unreported research

Active Publication Date: 2022-04-15
AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the spatiotemporal expression pattern of CPCFC protein in Diptera and Lepidoptera has been studied, there is no report on the study of CPCFC protein at the protein level

Method used

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  • Insect cpcfc family epidermal protein, coding nucleotide sequence and application thereof
  • Insect cpcfc family epidermal protein, coding nucleotide sequence and application thereof
  • Insect cpcfc family epidermal protein, coding nucleotide sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Cloning and expression purification of Tdcpcfc1 gene

[0086] 1. Synthetic SEQ ID Nucleotide sequence shown in NO.5

[0087] (1) Extract total RNA from the horns of the pupal stage of the two-fork rhinoceros beetle, and use reverse transcriptase to synthesize the first strand of cDNA;

[0088] (2) using cDNA as a template, using upstream primer 1 and downstream primer 2 to perform PCR (polymerase chain reaction) amplification;

[0089] Upstream primer 1: 5'-ATGTTTGTAAAACTGGCAGTATTAGCTTG-3' (SEQ ID NO.7),

[0090] Downstream primer 2: 5'-TTAATGGCAGAATGGGTAATTTGGACA-3' (SEQ ID NO.8).

[0091] PCR reaction system: 1μL cDNA template, 25uL 2×exTaq Premix, 1.5uL upstream primer 1, 1.5uL downstream primer 2, supplemented with ddH 2 O (double distilled water) to total reaction system is 50uL;

[0092] The PCR reaction conditions are ①98°C, 10s; ②55°C, 30s; ③72°C, 1min; ④Repeat steps ①-③30 cycles;

[0093] (3) After the PCR product is purified and recovered,...

Embodiment 2

[0109] Example 2 Detection of TdCPCFC1 protein binding properties of metal ions

[0110] Binding of Cu element during the culture of recombinant Escherichia coli

[0111]The recombinant Escherichia coli obtained in Example 1 was harvested after being induced by LB medium. During the process of expressing protein after adding the inducer, Escherichia coli would automatically absorb the Cu element in the medium, and the bacteria appeared brick red. The isolated 1.9mL, 2.63mg / mL pure protein was subjected to ICP-AES (Inductively Coupled Plasma Emission Spectroscopy), and it was measured that TdCPCFC1 protein bound 0.023mg Cu element, and the molar ratio of protein to Cu was about 1:1.

Embodiment 3

[0112] Example 3 Detection of TdCPCFC1 Protein Reduction

[0113] 1. Quantity determination of TdCPCFC1 protein Cys

[0114] Theoretically, one TdCPCFC1 protein contains 4 Cys (excluding the signal peptide region). The standard curve was prepared with Cys standards, and the UV absorption at 412nm was detected by Ellman's Reagent reaction. It was measured that the completely reduced state was 4 Cys, but it was not treated. The protein contains 2 Cys. The results showed that in the purified protein TdCPCFC1, 2 of the 4 Cys formed disulfide bonds, and the other two were free. The protein was reductive.

[0115] 2. TdCPCFC1 protein reduces Cu(II)

[0116] Use Bcs to detect the production of Cu(I), Bcs can specifically bind Cu(I), and there is ultraviolet absorption at 483nm, respectively use purified protein and EDTA+TCEP (chelating agent+reducing agent treatment to make protein without Cu element, And after reducing agent treatment, reduce all Cys as a control) Treated pur...

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Abstract

The invention provides an insect CPCFC family epidermal protein, a coding nucleotide sequence and application thereof, and relates to the technical field of bioengineering. The present invention takes the coleopteran insect, the two-pronged rhinoceros beetle as the research object, from which two CPCFC family protein genes were discovered, named TdCPCFC1 and TdCPCFC2, and the recombinant proteins of the two genes were successfully obtained, which is helpful for understanding the properties of CPCFC-like proteins. As well as understanding the role of CPCFC family epidermal proteins in the assembly of insect epidermis, it provides a new gene resource for the study of CPCFC family epidermal proteins. The invention provides recombinantly expressed insect CPCFC family epidermal protein TdCPCFC1, which can bind metal ions, has reducing properties, can combine with chitin, and can self-assemble to form aggregates, gels, etc., and the formed aggregates and gels have adhesive properties. And other properties, can be used to develop the corresponding biological products.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to insect CPCFC family epidermal proteins, encoding nucleotide sequences and applications thereof. Background technique [0002] Arthropods are the largest and most abundant group in the animal kingdom, and insects are one of the most important components of arthropods. Insects have excellent adaptability in the survival, reproduction and development of aquatic and terrestrial environments. The structure of their cuticle plays an important role in their successful environmental adaptation. Insect epidermis is an extracellular matrix secreted and organized by epithelial cells, which can protect insects from water loss in the body; at the same time, it serves as a barrier for biological toxins, pathogens and predators; it can also serve as an insect exoskeleton to help insect movement. Insect skin is mainly composed of chitin and protein, which can provide many research dire...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/11C12N15/70C12N1/21C12Q1/6888C12R1/19
CPCC07K14/43563C12N15/70C12Q1/6888
Inventor 杨青包涵
Owner AGRI GENOMICS INST CHINESE ACADEMY OF AGRI SCI
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