Making method and use of non-human mammal B lymphocyte defect animal model

A non-human mammal, B lymphocyte technology, applied in the biological field, can solve the problems of unknown physiological functions of G protein-coupled receptors, no animal model G protein-coupled receptors physiological functions, etc.

Active Publication Date: 2013-11-13
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, the physiological function of G protein-coupled receptors is still unknown, and...

Method used

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  • Making method and use of non-human mammal B lymphocyte defect animal model
  • Making method and use of non-human mammal B lymphocyte defect animal model
  • Making method and use of non-human mammal B lymphocyte defect animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Comparison of amino acid sequence homology between human GPR97 protein and mouse GPR97 protein

[0046] The amino acid sequence of human GPR97 protein is shown in SEQ ID NO.1.

[0047] The amino acid sequence of the mouse GPR97 protein is shown in SEQ ID NO.2.

[0048] The above-mentioned amino acid sequences of mouse GPR97 protein and human GPR97 protein were compared with NCBI's blastp software (http: / / www.ncbi.nlm.nih.gov / BLAST / ), and the calculation results showed that the mouse GPR97 protein and The amino acid sequence homology of human GPR97 protein is as high as 68%. The specific comparison data are as follows:

[0049] HGPR97 SGQEKPTEGPRNTC--LGSNNMYDIFNLNDKALCFTKCRQSGSDSCNVENLQRYWLNYEA 76

[0050] + E+ TE PRN C L + YD F+LND A CFTKC QS C+V NLQRYWLNYE+

[0051] MGPR97 TSDEETTEEPRNVCRRLQEGHEYDTFDLNDTAQCFTKCGQSEHSPCDVGNLQRYWLNYES 75

[0052] HGPR97 HLMKEGLTQKVNTPFLKALVQNLSTNTAEDFYFSLEPSQVPRQVMKDEDKPPDRVRLPKS 136

[0053] +L++ + + V+ PF+...

Embodiment 2

[0076] Example 2, Tissue expression profile of Gpr97 gene at RNA level

[0077] The relative expression level of Gpr97 in mouse tissues was detected by cDNA samples from 12 mouse tissues, including brain, thymus, heart, lung, liver, spleen, kidney, stomach, testis, epididymis, uterus, and bone marrow. RNA was extracted with Trizol reagent (Invitrogen) and operated according to the instructions. 2 μg of total RNA was reverse transcribed into cDNA using MMTV reverse transcriptase (Promega, Madison, WI). cDNA is amplified with specific primer pairs. The primers for β-actin and Gpr97 in reverse transcription PCR are: β-actin (forward: 5'-GCCTTCCTTCTTGGGTATG-3'SEQ ID NO.:3 and reverse: 5'-ACGCAGCTCAGTAACAGTCC-3'SEQ ID NO.: 4), Gpr97 (forward: 5'-CACCTTCGACTTGAATGACACTGCTC-3' SEQ ID NO.: 5 and reverse: 5'-TGCTGATGTTCTGGATCAATGCCTT-3' SEQ ID NO.: 6). Amplified products were separated by agarose gel electrophoresis and observed by EB staining. Double-stranded DNA-specific fluoresc...

Embodiment 3

[0078] Example 3, Preparation and identification of Gpr97 gene inactivated mice

[0079] In order to make the Gpr97 gene inactivation of mice, the inventor has designed such as figure 2 The targeting strategy shown, figure 2 The design of the targeting vector is shown in . The mouse Gpr97 gene has a total of 12 exons. Using bioinformatics, the inventors deleted exons 1 (E1) and 2 (E2) of the Gpr97 gene, totaling 3368 bp, and replaced them with the neo gene. Exon 1 was deleted because it contained the start codon (ATG). The lengths of its 5' homology arm and 3' homology arm are 6116bp and 3485bp, respectively.

[0080]In a specific embodiment of the present invention, the ET cloning method is used to construct the Gpr97 gene targeting vector. The specific process is as follows: two pairs of primers A1, A2 and B1, B2 are used to amplify the A and B homology arms respectively from the bacterial artificial chromosome (BAC) containing the Gpr97 genome sequence. The primer se...

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Abstract

The invention provides a non-human mammal B lymphocyte defect animal model, a making method of the animal model and a use of the animal model. The Gpr97 gene of the animal model is inactive, so the animal model can be used for researching the biological functions of the Gpr97 gene, and screening or identifying substances capable of increasing the percentage contents of B lymphocytes in the immune system. The invention also provides a use of the G protein coupled receptor 97 protein or a stimulant thereof in the preparation of medicines for increasing the percentage contents of B lymphocytes in the immune system.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a preparation method and application of a non-human mammal B lymphocyte deficient animal model. Background technique [0002] G protein-coupled receptor (GPCR) is the largest family of membrane receptor proteins in the human body. It is a class of transmembrane protein receptors with seven transmembrane helices. It is widely involved in the physiological and For sensory functions, there are more than 800 GPCRs in the human genome, equivalent to 2-3% of human genes. The structural characteristics of GPCR and the important role in signal transduction determine that it can be used as a good drug target. About 50% of the target proteins in drug development are G protein-coupled receptors, and about 25% of the drug targets of more than 100 best-selling drugs are members of this protein family (Klabunde T, Hessler G.Drug design strategies for ta...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/09A01K67/027A61K49/00A61K38/17A61K45/00A61P37/04
Inventor 王铸钢王津津张良梁费俭匡颖
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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