Full-length cloning method for pig NLRC5 gene and fluorescent quantitative detection method for expression quantity of pig NLRC5 gene

A cloning method and expression technology, which is applied in the field of fluorescent quantitative detection of NLRC5 gene cloning and expression in porcine tissue, can solve the problems of increasing the difficulty of infectious disease treatment, pathogenic biological characteristics, antigenicity, drug resistance changes, etc. , to achieve the effect of simple operation, high detection sensitivity and good repeatability

Inactive Publication Date: 2014-05-28
SOUTHWEST UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, due to the extensive use of vaccines and the abuse of antibiotics, the biological characteristics, antigenicity, and drug resistance of pathogens have undergone great changes, which has increased the difficulty of treating infectious diseases.

Method used

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  • Full-length cloning method for pig NLRC5 gene and fluorescent quantitative detection method for expression quantity of pig NLRC5 gene
  • Full-length cloning method for pig NLRC5 gene and fluorescent quantitative detection method for expression quantity of pig NLRC5 gene
  • Full-length cloning method for pig NLRC5 gene and fluorescent quantitative detection method for expression quantity of pig NLRC5 gene

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Experimental program
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Effect test

Embodiment 1

[0085] The extraction of embodiment 1RNA:

[0086] Weigh 100 mg of small intestine tissue, extract RNA by grinding with liquid nitrogen, dissolve in DEPC water, and detect RNA integrity by 1% agarose gel electrophoresis.

Embodiment 2

[0087] Embodiment 2RT-PCR:

[0088] (1) Prepare a 0.2ml centrifuge tube and add the following ingredients:

[0089] Total RNA: 6ul

[0090] 50um oligo(dT) 20 1ul

[0091] 10mM dNTP mix 1ul

[0092] DEPC-treated H 2 O 2ul

[0093] (2) Incubate at 65°C for 5 minutes, and place on ice to cool.

[0094] (3) Add the following ingredients in order:

[0095] 10×RT buffer 2ul

[0096] 25mM Mgcl 2 4ul

[0097] 0.1mM DTT 2ul

[0098] RNase OUT TM (40U / ul) 1ul

[0099] 200U / ul III RT 1ul

[0100] Total volume: 20ul

[0101] (4) Briefly centrifuge and mix well.

[0102] (5) React at 50°C for 50 minutes.

[0103] (6) React at 85°C for 5 minutes, and cool on ice.

[0104] (7) Add 1ul RNase H, centrifuge and mix, and react at 37°C for 20min.

[0105] (8) The product is stored in a -20°C low-temperature refrigerator for later use.

Embodiment 3c

[0106] Embodiment 3cDNA clone:

[0107] Firstly, referring to NLRC5 genes of human, mouse and other species, primers for segmentally amplifying pig NLRC5 gene are designed. The polymerase used in PCR amplification is TaKaRa LA Add the following components to the 50uL reaction system: TaKaRa LA Taq0.5uL; 10×LA PCR Buffer II (Mg 2+ Free) 5uL; Mgcl 2 (25mM) 5uL; dNTP Mixture (2.5mM each) 8uL; template 1uL; upstream and downstream primers 1uL; sterilized distilled water 28.5uL. The PCR reaction conditions were: 94°C for 5min, 35 cycles (94°C for 30s, 58°C for 30s, 72°C for 90s), and 72°C for 10min. The PCR product was analyzed by 1% agarose gel electrophoresis, the recovered target fragment was ligated with PMD-19, transformed into DH5a, and the bacteria solution was identified by PCR and sent to Shanghai Invitrogen for sequencing, and the sequencing results were spliced.

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Abstract

The invention relates to a full-length cloning method for pig NLRC5 gene and a fluorescent quantitative detection method for the expression quantity of pig NLRC5 gene. An NLRC5 gene is obtained through treatment and preparation of materials, extraction of RNA (Ribonucleic Acid), synthesis of cDNA and an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and 3'RACE technology; the full length of the gene cDNA is 6638bp; the open reading frame of the gene is 5538bp; 1,846 amino acids are encoded. Bioinformatics analysis shows that the homologies of pig NLRC5 gene nucleotide sequence, with cattle, sheep, macaque and human beings are about 80%. Through optimization on parameters of a real-time fluorescent quantitative PCR reaction system, an SYBR Green I fluorescent quantitative detection method for detecting the expression quantity of NLRC5 gene is established, and based on the fluorescent quantitative PCR method, the NLRC5 gene expression in pig tissue is quantitatively analyzed for the first time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fluorescent quantitative detection method for NLRC5 gene cloning and expression in pig tissues. Background technique [0002] NLRC5 is one of the newly discovered NOD-like receptors (NOD-like receptors, NLRs) family members, and its role as a new natural immune regulatory molecule has received extensive attention in recent years. In various cell types of humans and mice, it has been confirmed that NLRC5 can significantly regulate the NF-κB pathway, type I IFN production pathway, MHC-I gene transcription, and formation of inflammasomes. Play an important role in innate immunity to pathogenic microorganisms. NLRC5 protein, as a new natural immune regulatory molecule, can provide an effective target for future regulation of immunity in related diseases such as microbial infection and immune inflammation. At present, the cDNA sequence cloning and identification methods of human, mouse ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 兰道亮陈亚冰李键熊显荣林宝山王树茂
Owner SOUTHWEST UNIVERSITY FOR NATIONALITIES
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