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A kind of Songjiang perch creatine kinase tfm-ck gene, Songjiang perch tfm-ck recombinant protein and application thereof

A technology for recombining proteins and genes, which is applied in the field of genetic engineering and can solve problems such as functional studies that have not yet been carried out

Inactive Publication Date: 2019-08-02
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although different types of CK genes have been found in fish, their functional studies are basically in the undeveloped stage

Method used

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  • A kind of Songjiang perch creatine kinase tfm-ck gene, Songjiang perch tfm-ck recombinant protein and application thereof
  • A kind of Songjiang perch creatine kinase tfm-ck gene, Songjiang perch tfm-ck recombinant protein and application thereof
  • A kind of Songjiang perch creatine kinase tfm-ck gene, Songjiang perch tfm-ck recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining the full-length cDNA sequence of Songjiang perch creatine kinase TfM-CK gene:

[0027]The skin mucus of Songjiang perch stimulated by V. anguillarum (experimental group) and the skin mucus of Songjiang perch under normal conditions (control group) were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), by comparing the two groups The protein bands up-regulated in the experimental group compared with the control group were analyzed by mass spectrometry. The results showed that the M-type creatine kinase of Songjiang perch contained FEEILTR, QKRGTGGVDT, and ASVGGVFDIS peptides. According to the known protein sequence corresponding to the nuclear Nucleotide sequences and other species downloaded from NCBI website: spothead fish (Hexagrammos agrammus, AY583132.1), ten-line six-line fish (Hexagrammos decagrammus, AY583138.1), long-line six-line fish (Hexagrammos lagocephalus, AY583136.1) , Hexagrammos otakii, AY583133.1), Hexagrammosoctogrammus,...

Embodiment 2

[0030] Example 2: Real-time fluorescent quantitative PCR (qRT-PCR) detection of tissue distribution of TfM-CK gene and expression pattern changes after Vibrio anguillarum stimulation

[0031] The qRT-PCR primers CKRTF and CKRTR were designed according to the cloned TfM-CK gene cDNA sequence, and the qRT-PCR was carried out using the cDNA at each time point of the control group and the stimulation group as templates. Specific reaction system and steps refer to Premix Ex Taq TM manual.

[0032]

[0033] From the above experiments, it can be concluded that the TfM-CK gene of Songjiang perch was up-regulated in muscle, skin, spleen and head kidney after being stimulated by V. anguillarum, and the expression level in the muscle began to increase to that of the control group until 24h This may be due to the fact that after the body is invaded by external pathogens, it needs to adjust the immune function in the body to deal with bacterial infection. After the fish adapts to thi...

Embodiment 3

[0034] Example 3: Expression, purification and concentration determination of Songjiang perch creatine kinase TfM-CK recombinant protein

[0035] 1) Screening of expression strains

[0036] Extract the plasmid in the DH5α bacteria that successfully constructed the expression vector pet30a, and transfer it into the expression strain BL21(DE3) to screen positive clones. The prokaryotic expression strain of TfM-CK was screened out, and cultured with shaking at 37°C and 200rpm for 12-16h.

[0037] 2) Trial expression of recombinant protein

[0038] The TfM-CK prokaryotic expression strain that had been cultured for 12-16 hours was transferred to culture for about 3-4 hours according to the ratio of 1:100, added thiogalactopyranoside (IPTG) with a final concentration of 0.5mM for induction, and then carried out SDS-PAGE. Add 1 / 10 volume glycerol (80%) to the strains with better expression, and store them at -70°C.

[0039] 3) Large-scale expression and purification of recombinan...

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Abstract

The invention provides a Songjiang perch creatine kinase TfM-CK gene, Songjiang perch TfM-CK recombinant protein and application thereof, which belong to the technical field of genetic engineering, and not only obtain the full-length cDNA sequence of Songjiang perch creatine kinase TfM-CK gene , also obtained Songjiang perch TfM‑CK recombinant protein through prokaryotic expression. The invention provides a Songjiang perch creatine kinase TfM-CK gene, the full-length sequence of which is 1474bp, including a gene open reading frame of 1146bp. The invention can be used as an immune additive for fish feed to enhance fish disease resistance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a full-length cDNA sequence of Songjiang perch creatine kinase TfM-CK gene, a Songjiang perch TfM-CK recombinant protein expressed through a prokaryotic expression system and an application thereof. Background technique [0002] Songjiang perch (Trachidermus fasciatus), also known as four-gill perch, belongs to the order Scorpaeniforme, the family Cottidae, and the genus Trachidermus. The whole growth period faces two more complicated living environments of seawater and freshwater, and was once widely distributed in my country's coastal areas. In recent years, due to water pollution and the construction of river water conservancy and other reasons, the habitat, reproduction and migration routes of Songjiang perch have been destroyed, resulting in a sharp decline in the population of this species, and the distribution area has also shrunk sharply. Significa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12A23K50/80A23K20/147A23K20/153
CPCC12N9/1223C12Y207/03002
Inventor 刘莹莹祝茜陈学昭于珊珊宋翠蒋东方王琛琪
Owner SHANDONG UNIV
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