Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Feline b-lymphocyte stimulating factor cdna and its encoded protein, cloning method and application

A technology of B lymphocytes and stimulators, applied in the field of biogenetic engineering, can solve the problems of inability to activate B cells, immune deficiency, etc., and achieve the effect of obviously promoting survival/value-added effects

Active Publication Date: 2021-11-12
NANJING NORMAL UNIVERSITY
View PDF4 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Feline b-lymphocyte stimulating factor cdna and its encoded protein, cloning method and application
  • Feline b-lymphocyte stimulating factor cdna and its encoded protein, cloning method and application
  • Feline b-lymphocyte stimulating factor cdna and its encoded protein, cloning method and application

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 degenerate primers cBAFF1: 5'-GTCNCCTGTNCAGNGTGGNCNTCCTGC-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℃, ice ...

Embodiment 2

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

[0066] Using the method for extracting RNA in Example 1, extracted respectively cat The total RNA of the heart, liver, spleen, lung, and intestine was reverse-transcribed into cDNA by reverse transcriptase, and GAPDH was used as an internal reference, and the real time-PCR method was used for cat The expression level of BAFF gene in various tissues was studied.

[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 reaction system is: Mix 1...

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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a cat B lymphocyte stimulating factor cDNA and its encoded protein, a cloning method and an application. The base sequence of the feline B lymphocyte stimulating factor cDNA is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is SEQ ID NO.2. The cDNA of the feline B lymphocyte stimulating factor cBAFF cloned from cats for the first time in the present invention, cBAFF is expressed in various detection tissues by fluorescence quantitative PCR analysis, the expression level is the highest in the spleen, and the lowest expression level is in the small intestine; it shows that BAFF is expressed in Plays an important role in the cat's immune system. The recombinant fusion protein csBAFF in the extracellular region of the gene performs the active function of cBAFF, and the protein has obvious pro-survival / proliferation effects on mouse lymphocytes. The cat B lymphocyte stimulating factor cDNA of the present invention can be used in the production of recombinant cat B lymphocytes. Cytostimulators as Immunopotentiators in Felines.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com