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(cBAFF)cDNA as well as encoded protein, cloning method and applications thereof

A technology of B lymphocytes and stimulators, applied in the field of biogenetic engineering, can solve problems such as immune deficiency and inability to activate B cells

Active Publication Date: 2019-02-15
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient levels of BAFF will fail to activate B cells to produce sufficient immunoglobulins and lead to immunodeficiency

Method used

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  • (cBAFF)cDNA as well as encoded protein, cloning method and applications thereof
  • (cBAFF)cDNA as well as encoded protein, cloning method and applications thereof
  • (cBAFF)cDNA as well as encoded protein, cloning method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 8-month-old Chinese domestic cat; purchased from Nanjing Red Sun Farm

[0050] (1) Primer design: The conserved regions of human, mouse and bovine BAFF cDNA were analyzed by Clustal w software, and the degenerate primers cBAFF1: 5'-GTCNCCTGTNCANGTGGNCNTCCTGC-3'; cBAFF2: 5'-GGANCAANTTCNCCAGNCTCANTTCGT-3' were designed.

[0051] (2) extracting total RNA: use RNA extraction reagent TRIzol (Invitrogen Company) to extract the total RNA of cat spleen cells according to its operating manual, and identify its quality and purity by formaldehyde-denatured agarose gel electrophoresis, and measure its quality and purity by ultraviolet spectrophotometer concentration.

[0052] (3) RT-PCR: PCR amplification was performed using the above-mentioned cBAFF1 and cBAFF2 as primers to obtain a 530bp fragment P1.

[0053] ① reverse transcription

[0054] Add 3 μl of total RNA extract to DEPC-treated 1.5ml Eppendorf tubes, Oligod(T) 18 1μl, 10mmol / L dNTP 2μl, DEPC water 5μl placed; 65℃, i...

Embodiment 2

[0065] Analysis of the expression level of feline BAFF gene (feline B lymphocyte stimulating factor cDNA sequence) in various tissues:

[0066] Adopt the method for extracting RNA among the embodiment 1, extract the total RNA of cat heart (heart), liver (liver), spleen (spleen), lung (lung), intestine (intestine) respectively, and use reverse transcriptase to reverse transcribe into cDNA, using GAPDH as an internal reference, using real time-PCR method to study the expression level of cat BAFF gene in various tissues.

[0067] The primers for the amplification of the target gene are

[0068] Q1 (SEQ ID NO.11): 5'-CGGGCAGGTTTTATACACGG-3';

[0069] Q2 (SEQ ID NO.12): 5'-GATGCCAGCGGAATAACAGG-3';

[0070] The primers for the amplification of GAPDH are

[0071] Q5 (SEQ ID NO.13): 5'-CATTGCCC TCAACGACCACTTTGTC-3';

[0072] Q6 (SEQ ID NO.14): 5'-CTCCTTGGAGGCCATGTGGACCATG-3';

[0073] With DEPC water as the blank control, three replicate holes were made for each sample. The reac...

Embodiment 3

[0087] The feline B lymphocyte stimulating factor cDNA obtained in Example 1 was used to produce recombinant cat BAFF as a cat immune enhancer through existing genetic engineering methods.

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Abstract

The invention discloses a (cBAFF)cDNA as well as an encoded protein, a cloning method and applications thereof. The base sequence of the (cBAFF)cDNA is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the (cBAFF)cDNA is shown as SEQ ID NO.2. The invention clones the cDNA of the cBAFF from a cat for the first time, and fluorescence quantitative polymerase chain reaction (PCR) analysis shows that cBAFF is expressed in all detected tissues, among which the expression in spleens is the highest and the expression in small intestines is the lowest, thereby indicating thatBAFF plays an important role in the immune system of the cat. Protein csBAFF recombined and fused at the extracellular domain of a gene exerts the active function of the cBAFF and has obvious effectsin promoting mouse lymphocyte with survival rate and cell proliferation. The (cBAFF)cDNA provided by the invention can be applied to the production of recombinant (cBAFF)cDNA which serves as an immunopotentiator for the cat.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to a cat B lymphocyte stimulating factor cDNA, its encoded protein, its cloning method and its application. Background technique [0002] B-lymphocyte-stimulating factor of the TNF family (BAFF, also known as BLyS, TALL-1, THANK, zTNF4 or TNFSF13b) is a transmembrane protein consisting of 285 amino acids that is capable of functioning in a membrane-bound and extracellular soluble form It plays a major role in B cell survival, proliferation and differentiation. BAFF is a type II transmembrane protein that forms biologically active trimers. BAFF interacts with three cell surface receptors of the TNF receptor superfamily (TNFRSF): BAFF receptor (BAFF-R, also known as BR3, CD268 and TNFRSF17), B cell maturation antigen (BCMA and TNFRSF13C), transmembrane Activators and calcium modulators and cyclophilin ligands (TACI, CD267 and TNFRSF13B), which are all predominantly ...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12C12N15/10C12N15/70C07K19/00A61K38/19A61P37/04
CPCA61K38/19A61P37/04C07K14/70575C07K2319/00
Inventor 张嘉鑫武小龙张双全
Owner NANJING NORMAL UNIVERSITY
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