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Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and cloning method and application thereof in recombination

A technology of B lymphocytes and stimulators, applied in the field of biogenetic engineering

Inactive Publication Date: 2011-07-06
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The zebrafish B lymphocyte stimulating factor (zBAFF) cDNA has not been cloned yet, and the research on the zebrafish BAFF gene is still completely blank at home and abroad

Method used

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  • Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and cloning method and application thereof in recombination
  • Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and cloning method and application thereof in recombination
  • Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and cloning method and application thereof in recombination

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Zebrafish were purchased from the Model Animal Genetics Center of Nanjing University

[0041] (1) Primer design: Analyze the conserved region of rainbow trout, Atlantic salmon, and spotted green puffer BAFF cDNA by Clustal w software, and design degenerate primers zBAFF1: 5'-TTGTNCCNTGGCTTCTNAGCTTTAA-3'; zBAFF2: 5'-GCACCAAANAANGTNNCNTCTCC-3'.

[0042] (2) Extraction of total RNA: RNA extraction reagent TRIzol (Invitrogen Company) was used to extract total RNA from zebrafish spleen cells according to its operation manual, and its quality and purity were identified by formaldehyde-denatured agarose gel electrophoresis, and determined by ultraviolet spectrophotometer its concentration.

[0043] (3) RT-PCR: PCR amplification was performed using the above zBAFF1 and zBAFF2 as primers to obtain a 807bp fragment P1.

[0044] ① reverse transcription

[0045] In the DEPC-treated 1.5ml Eppendorf tube, sequentially add 3 μl of total RNA extract, Oligo d(T) 18 1μl, 10mmol / L dNT...

Embodiment 2

[0051] Construction of zebrafish BAFF recombinant expression vector and its induced expression in Escherichia coli

[0052] Primers Al, A2 and A3, A4 were designed according to the known zsBAFF sequence and EGFP sequence as templates. Wherein the 5' end of Al has Bam H Ⅰ Restriction site, the 5' end of A4 has Hind Ⅲ restriction site, while A2 and A3 contain coding between EGFP and zsBAFF (Gly 4 Ser) 2 The base sequence of the linker. The EGFP-zsBAFF gene fragment was amplified by over-lap PCR. After two rounds of PCR, the PCR product was recovered by tapping rubber and the pET-28a vector was used separately Bam H I and Hind Ⅲ digestion, T4 DNA Ligase ligation, ligation product conversion E. coli In DH5a, a recombinant strain containing pET28a-EGFP-zsBAFF was obtained. Select the positive recombinant strain, expand the culture to extract the pET28a-EGFP-zsBAFF recombinant plasmid, transform it into Escherichia coli BL21(DE3), and select the positive strain to indu...

Embodiment 3

[0066] receptor binding properties

[0067] BAFF can bind to its three receptors, which are TACI, BCMA and BAFF-R. TACI

[0068] Expressed on the surface of B cells and activated T cells, while BCMA and BAFF-R are mostly or absolutely expressed on the surface of B cells. Here we use confocal laser microscopy to detect the binding properties of EGFP-zsBAFF. Through laser confocal microscopy, we can see that EGFP-zsBAFF can bind to the surface of lymphocytes in mice, while the control EGFP has no binding properties ( Figure 7 ).

[0069] EGFP-zsBAFF promotes the survival of mouse lymphocytes

[0070] The pro-survival effect of EGFP-zsBAFF on mouse lymphocytes was detected by MTT cytotoxicity assay in vitro, such as Image 6 As shown, PBS is the blank control, 12 μg / ml EGFP-zsBAFF, 12 μg / ml EGFP+anti-IgM, 5 μg / ml EGFP and anti-IgM cultured cells are the negative control, 6 μg / ml zsBAFF+ anti-IgM and 2 μg / ml hsBAFF +anti-IgM is a positive control. The results showed that t...

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Abstract

The invention discloses a Danio rerio B-lymphocyte activating factor (zBAFF) cDNA (complementary deoxyribonucleic acid), and a cloning method and application thereof in recombination, and relates to the field of biological gene engineering. The cloning method of the zBAFF gene comprises the following steps: extracting total RNA (ribose nucleic acid) of the spleen of Danio rerio by a TRIzol method, and carrying out reverse transcription; and analyzing BAFF cDNAs of rainbow trout, Atlantic salmon and Tetraodon nigroviridis, and conversed regions of BAFF cDNAs of rainbow trout, Atlantic salmon and Ictalurus punctatus by Clustalw software, designing degenerate primers, and carrying out PCR (polymerase chain reaction) amplification to obtain the 807bp full-length cDNA. The Danio rerio B-lymphocyte activating factor and proliferation inducing ligand cDNA can be used for producing recombinant Danio rerio BAFF by the existing gene engineering method.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to cDNA of B lymphocyte stimulating factor and its cloning and recombination application. Background technique [0002] BAFF has strong B cell chemotaxis, and acts as a co-stimulator of B cell proliferation and differentiation in vitro, and can induce activated B cells to secrete a large amount of IgG, IgA, IgM, etc. For dormant B cells, although BAFF cannot be independently activated to enter the cell cycle, it can be activated by up-regulating the anti-apoptotic proteins Bcl-2 and Bcl-X on the cell surface. L The expression of can prolong the lifespan of dormant B cells. In vivo, it can promote the development of T1-B cells to T2-B cells and mature B cells in the spleen. The normal expression of BAFF is a necessary condition for GC formation, antibody affinity maturation, memory B cell formation and normal B cell development BAFF - / - B cells in the extracapsular and transi...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10A61K48/00A61P37/04C07K14/52
Inventor 张双全闵翠梁臻宁
Owner NANJING NORMAL UNIVERSITY
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