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Preparation and application of African clawed frog proliferation induction ligand TNFSF13 gene and protein

A technology of Xenopus laevis and ligands, applied in DNA preparation, application, genetic engineering and other directions, can solve the problem of not being cloned

Inactive Publication Date: 2013-04-03
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] According to the search results of the three major international nucleic acid sequence databases, Genebank, EMBL, and DDBJ, the cDNAs of APRIL from humans, mice, pigs, dogs, cattle, sheep, salmon, and zebrafish have been cloned so far, and have been in The sequence number has been applied for in the GenBank database, but the cDNA of the African clawed frog APRIL (XlAPRIL), which is a representative of amphibians among the model organisms, has not been cloned yet, and the research on the African clawed frog APRIL gene is still completely blank at home and abroad

Method used

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  • Preparation and application of African clawed frog proliferation induction ligand TNFSF13 gene and protein
  • Preparation and application of African clawed frog proliferation induction ligand TNFSF13 gene and protein
  • Preparation and application of African clawed frog proliferation induction ligand TNFSF13 gene and protein

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Experimental program
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Effect test

Embodiment 1

[0042] African clawed frogs were purchased from the Model Animal Genetics Center of Nanjing University

[0043] (1) Extraction of total RNA: The total RNA of Xenopus frog spleen tissue was extracted using the animal tissue RNA extraction kit (TIANGEN) according to the operation manual, and its quality and purity were identified by formaldehyde-denatured agarose gel electrophoresis, and its quality and purity were determined by ultraviolet spectrophotometer. concentration. SMARTTM RACE Kit (TaKaRa) was reverse transcribed into the first strand of cDNA, and the templates of 5'RACE and 3'RACE were done separately. The reaction process of 5'RACE reverse transcription: 1.75μl RNA, 1.0μl 5'-CDS Primer A, 1.0μl sterile water, mix and centrifuge, incubate at 72°C for 3min, incubate at 42°C for 2min, after cooling, spin at 14000g for 10 seconds, then Add 1.0μl SMARTer Ⅱ Aoligo, 2.0μl 5×First-Strand Buffer, 1.0μl DTT (20mM), 1.0μl dNTP Mix (10mM), 0.25μl RNase Inhibitor (40U / μl), 1.0μl...

Embodiment 2

[0053] Construction of recombinant expression vector of Xenopus APRIL and its induced expression in Escherichia coli, purification and identification;

[0054] Primers Xs-F and Xs-R were designed according to the known sequence of APRIL to amplify the soluble segment of the gene. Among them, the 5' end of Xs-F has a StuI restriction site, and the 5' end of Xs-R has an XhoI restriction site. The first-strand cDNA of Xenopus frog is used as a template, and Xs-F and Xs-R are used as primers (Xs -F5'-TGACCTCACAGGAGCCAGGA-3'(SEQ ID NO.18), Xs-R5'-CCGCTCGAG TTAAAGCTTGACAAGACCAAGGA-3'(SEQ ID NO.19)), PCR amplification of the functional region of the gene (10×dreamTaq Buffer 5μl, dNTP ( 2.5mM) 8μl, Xs-F (10μmol / L) 2μl, Xs-R (10μmol / L) 2μl, H2O30.5μl, cDNA 2μl, dream Taq0.5μl) The reaction conditions are as follows: 94°C 5min, (94°C30s, 60 °C30s, 72°C1min), 30 cycles, and finally extended at 72°C for 7min. After that, the PCR product was recovered by tapping the gel. The recovered pro...

Embodiment 3

[0059] The effect of SUMO-XsAPRIL on promoting survival / proliferation of mouse B lymphocytes was detected in vitro;

[0060] Mouse B lymphocytes were isolated by magnetic beads (Miltenyi Biotech). Use RPMI1640 (10%FCS, 100U / ml Penicillin / streptomycin) medium to adjust the cell concentration to 2×10 6cells / ml, add 100 μl cell suspension to each well of a 96-well plate, place at 37°C, 5% CO 2 Adapt to the cell culture incubator for 3-4 hours. The recombinant protein SUMO-XsAPRIL was added in groups sequentially, and the final concentration of the recombinant protein was 0.5, 1, 2, 4, 6, 8, 10, 12 μg / ml in sequence, with PBS, SUMO, anti-IgM, mouse APRIL protein (msAPRIL) as comparison. Placed in an incubator for 48 hours. After 48 hours, 10 μl of WST-8 was added to each well of the 96-well plate and incubated for 1 hour. Measure the absorbance at 450 nm with a microplate reader. The results showed that SUMO-XsAPRIL could significantly promote the survival / proliferation of B...

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Abstract

The invention relates to preparation and application of an African clawed frog proliferation induction ligand TNFSF13 gene and protein. The African clawed frog proliferation induction ligand cDNA (complementary deoxyribonucleic acid) has the sequence disclosed as SEQ ID NO.1. The cloning method comprises the following steps: extracting spleen total RNA (ribonucleic acid) and carrying out reverse transcription; and designing primers to proliferate the intermediate segment of the gene, respectively designing primers according to the intermediate segment to proliferate 3' and 5' terminated sequences, sequencing properly, and carrying out sequence splicing, thereby obtaining the full-length cDNA sequence. Extracellular soluble segment is connected into the SUMO pronucleus plasmid and transferred into Escherichia coli Rosseta (DE3) to be expressed, and purified through an Ni+-NTA column to obtain the recombinant protein His6-XsAPRIL. The cDNA of the African clawed frog B lymphopoiesis induction ligand TNFSF13(APRIL) can be used for producing the recombinant protein by the existing gene engineering method, thereby being used for further research.

Description

technical field [0001] The invention relates to the field of biogenetic engineering, in particular to the B lymphocyte proliferation-inducing ligand cDNA and its cloning and expression technology. Background technique [0002] In view of the fact that members of the tumor necrosis factor superfamily (TNFSF) have the function of promoting cell growth, differentiation or death, and play important biological roles in the formation of lymphoid organs, the activation of immune cells and the maintenance of body homeostasis, so in recent years , more and more members of the TNFSF family were found. Proliferation-inducing ligand (APRIL) is a new member of the tumor necrosis factor superfamily, namely TNFSF13, also known as CD256, TALL-2, TRDL-1, which has a high homology with BlyS or TALL-1, 1998 First discovered and successfully cloned by Hahne et al. APRIL is expressed on the surface of dendritic cells, macrophages, and T and B lymphocytes. In addition, it is also expressed at h...

Claims

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Application Information

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IPC IPC(8): C12N15/28C12N15/10C12N15/70C12N15/66C12P21/02
Inventor 张双全刘霞
Owner NANJING NORMAL UNIVERSITY
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