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Preparation method and application of grass carp TNF-[alpha] recombinant protein

A recombinant protein and grass carp technology, which is applied in the field of preparation of grass carp TNF-α recombinant protein, can solve the economic loss of grass carp aquaculture and other problems, achieve a strict and reasonable preparation technology route, promote gene and protein expression, and enhance the immune function of grass carp Effect

Active Publication Date: 2021-05-28
FRESHWATER FISHERIES RES INST OF SHANDONG PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors cause the grass carp farming industry to suffer huge economic losses every year

Method used

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  • Preparation method and application of grass carp TNF-[alpha] recombinant protein
  • Preparation method and application of grass carp TNF-[alpha] recombinant protein
  • Preparation method and application of grass carp TNF-[alpha] recombinant protein

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preparation example Construction

[0039] In a specific embodiment of the present invention, a preparation method of grass carp TNF-α recombinant protein is provided, comprising the steps of: adopting a method based on PAS (PCR-based Accurate Synthesis), designing a full-length splicing primer, and at both ends of the primer The protective base synthesis gene TNF-α (Genbank: JQ040498.1) was designed for each, and connected into the BamHI(GGATCC)-HindIII(AAGCTT) site of the vector pCZN1, and into the vector pET-32a(+) respectively. between the seamless cloning site and the XhoI (CTCGAG) site, and between the NdeI (CATATG)-HindIII (AAGCTT) site of the vector pMAL-c5x; the obtained recombinant plasmids pCZN1-TNF-α, pET-32a ( +)-TNF-α, pMAL-c5x-TNF-α were transferred into Arctic-Express. Using IPTG to induce the expression of grass carp pIgR, after optimizing the expression conditions, analyze the target protein expressed by pCZN1-TNF-α, pET-32a(+)-TNF-α in the form of inclusion bodies, and the target protein expre...

Embodiment 1

[0052] Example 1: The optimized preparation method of TNF-α recombinant protein.

[0053] 1. Design of primers for grass carp TNF-α expression

[0054] According to the cDNA sequence of the open reading frame of grass carp TNF-α (Genbank: JQ040498.1) and the sequence of the multiple cloning site of the expression vector, NdeI(CATATG)-HindIII(AAGCTT) of pMAL-c5x and the non-cloning of pET-32a(+) were added. Slit clone XhoI (CTCGAG), BamHI (GGATCC)-HindIII (AAGCTT) of pCZN1, and designed specific primers.

[0055] TNF-α-F1: CATATG AACAAGAGCCAGAGTAATCAGGAAAGC (SEQ ID NO. 1)

[0056] TNF-α-R1: AAGCTT TTAGTGATGATGATGATGATGCAGGGCGA (SEQ ID NO. 2)

[0057] TNF-α-F2: CACCAT CATCATCATCATAACAAGAGCCAGAGTAATC (SEQ ID NO. 3)

[0058] TNF-α-R2: CTCGAG TTACAGGGCGAACACGCCGAAGAAGGTT (SEQ ID NO. 4)

[0059] TNF-α-F3: GGATCC AACAAGAGCCAGAGTAATCAGGAAAGCGCCAC (SEQ ID NO. 5)

[0060] TNF-α-R3: AAGCTT TTAATGATGATGATGATGATGATGCAGGGCGAACACG (SEQ ID NO. 6)

[0061] 2. Amplification, p...

Embodiment 2

[0110] Example 2: qPCR detection of changes in the expression level of pIgR gene after TNFα recombinant protein of the present invention stimulates grass carp L8824 cells.

[0111] 1. Recovery of L8824 cells

[0112] (1) Turn on the ultraviolet light to sterilize the cell culture room and ultra-clean workbench, and adjust the constant temperature water bath to 37°C.

[0113] (2) Take out the frozen L8824 cells from the -80°C refrigerator, place them in a water bath immediately, and shake the cryopreservation tubes to accelerate their thawing.

[0114] (3) Transfer the cell suspension in the cryopreservation tube to a new centrifuge tube on a clean bench, and centrifuge for 5 minutes.

[0115] (4) Discard the supernatant, add fresh medium, and repeatedly pipette with a micropipette to disperse the cells evenly.

[0116] (5) Transfer the cell suspension into a sterile cell culture bottle, add an appropriate amount of fresh medium, and culture in a precision constant temperatur...

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PUM

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Abstract

The invention provides a preparation method and application of grass carp TNF-[alpha] recombinant protein, and belongs to the technical field of fish molecular immunology. The target protein TNF-[alpha] is obtained by constructing recombinant plasmids and transferring the recombinant plasmids into an expression strain, using IPTG to induce expression of grass carp TNF-[alpha], optimizing expression conditions and purifying, and further verifying that the prepared TNF-[alpha] recombinant protein has the effect of promoting gene and protein expression of pIgR, so that the immune function of grass carp is enhanced, and the method has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of fish molecular immunology, and in particular relates to a preparation method and application of grass carp TNF-α recombinant protein. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Tumor necrosis factor α (TNF-α) is secreted by activated monocytes, macrophages and T cells, and is named for its ability to inhibit and kill tumor cells in vivo. In addition to causing hemorrhagic necrosis of tumor cells, TNF-α can also participate in the regulation of the body's inflammatory response and immune response, and has important functions such as promoting cell growth, differentiation, and apoptosis. So far, TN...

Claims

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Application Information

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IPC IPC(8): C07K14/525C07K14/46C12N15/28C12N15/70A61K38/19A61P37/04
CPCC07K14/525C07K14/461C12N15/70A61P37/04A61K38/00Y02A40/81
Inventor 许国晶张金路马汝芳朱永安巩俊霞杜兴华王志忠李壮张明磊
Owner FRESHWATER FISHERIES RES INST OF SHANDONG PROVINCE
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