Takifugu obscures B lymphocyte stimulating factor cDNA and cloning method and recombination application thereof

A technology of B lymphocytes and puffer puffer, applied in the field of genomics, bioinformatics, protein and cell, gene manipulation

Inactive Publication Date: 2011-07-13
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fugu obscurus B lymphocyte stimulating factor (fTALL-1) cDNA has not been cloned, and the research on TALL-1 gene of puffer obscura is still completely blank at home and abroad

Method used

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  • Takifugu obscures B lymphocyte stimulating factor cDNA and cloning method and recombination application thereof
  • Takifugu obscures B lymphocyte stimulating factor cDNA and cloning method and recombination application thereof
  • Takifugu obscures B lymphocyte stimulating factor cDNA and cloning method and recombination application thereof

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Experimental program
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Embodiment 1

[0030] Fugu obscurus ( Takifugu obscures ),Captivity.

[0031](1) Primer design: The TALL-1 sequence of the green puffer was used as the seed sequence to find the full-length cDNA sequence of B lymphocyte stimulating factor in the genome database of puffer puffer. Sequence alignment of the green puffer and redfin puffer carefully selected two highly conserved regions to design the sense oligonucleotide primer fTALL-11 (5'-ATGGGACCAGTGAGGGTAGGTTT-3' for homologous clones, as shown in SEQ ID NO .3) and antisense oligonucleotide primer fTALL-12 (5'- TTAACCCAGTTTGATAGCACCCA-3' as shown in SEQ ID NO.4).

[0032] (2) Extraction of total RNA: Use the RNA extraction kit (Tiangen Company) to extract about 0.5 g of total RNA from the spleen cells of Fugu obscurus according to its operation manual, and identify its quality and Purity was determined by UV spectrophotometer.

[0033] (3) Using the RT-PCR method, using the above-mentioned fTALL-11 and fTALL-12 as specific primers, ampli...

Embodiment 2

[0036] Analysis of the expression level of fTALL-1 gene in various tissues:

[0037] Using the method for extracting RNA in Example 1, the total RNA of the liver (liver), kidney (kidney), spleen (spleen), heart (heat), gill (gill), and intestine (intestine) of puffer puffer obscura was extracted respectively , using quantitative RT-PCR method to study the expression level of fTALL-1 gene in various tissues, the results are as follows: Figure 4 As shown, the fTALL-1 gene is mainly expressed in the immune organs of puffer pufferfish, including spleen and kidney. In the experiment, Fugu obscura GAPDH was used as an internal reference, and the primers used were RT-G1 (5’- CACCTCCAAGAAGGTGGAAA-3’, SEQ ID NO.5) and RT-G2 (5’-

[0038] CTCTCGTGGAAAACGGTGAT-3', SEQ ID NO. 6). The RT-PCR system is as in Example 1.

[0039] Construction of soluble fTALL-1 recombinant vector and its induced expression in Escherichia coli:

[0040] Using the fTALL-1 full-length cDNA obtained in Examp...

Embodiment 3

[0048] The cDNA of the puffer puffer B lymphocyte-stimulating factor obtained in Example 1 was used to produce recombinant fTALL-1 as an immune enhancer for puffer puffers through existing genetic engineering methods.

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Abstract

The invention relates to a takifugu obscures B lymphocyte stimulating factor (fuguTALL-1) cDNA and a cloning method and recombination application thereof, and belongs to the field of biological gene engineering. The gene of the takifugu obscures B lymphocyte stimulating factor has a sequence shown as SEQ ID No.1. The cloning method for the stimulating factor comprises the following steps of: designing primers according to the conserved sequence of the fuguTALL-1; extracting total RNA from the spleen of takifugu obscures; and amplifying a full-length cDNA sequence by a two-step reverse transcription-polymerase chain reaction (RT-PCR) method, cloning the full-length cDNA sequence into a pMD19-T vector, and picking positive clones to perform sequencing. The recombinant fuguTALL-1 serving as a fugu immunity enhancer can be produced from the cDNA of the takifugu obscures B lymphocyte stimulating factor by the conventional gene engineering method.

Description

technical field [0001] The invention relates to the fields of genomics, bioinformatics, gene manipulation, protein and cells, in particular to cDNA of puffer pufferfish obscura B lymphocyte stimulating factor and its cloning, expression and identification technology. Background technique [0002] Human B lymphocyte-stimulating factor (hTALL-1) is a member of the tumor necrosis factor family that was newly discovered in 1999 and is closely related to human immune regulation. It is mainly expressed in peripheral blood mononuclear cells, spleen, lymph nodes and bone marrow. hTALL-1 has strong B cell chemotaxis, in vitro it acts as a co-stimulatory factor for B cell proliferation and differentiation, and can induce activated B cells to secrete a large amount of IgG, IgA, IgM, etc. For dormant B cells, hTALL-1 Although it cannot be independently activated to enter the cell cycle cycle, it can be activated by up-regulating the anti-apoptotic proteins Bcl-2 and Bcl-X on the cell su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/09C07K14/52A61K38/19A61P37/04
Inventor 张双全艾洪新
Owner NANJING NORMAL UNIVERSITY
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