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Construction method of porcine C1QTNF3 gene overexpression vector plasmid and detection of expression level of porcine C1QTNF3 gene

A technology of TY-C1QTNF3-F and TY-C1QTNF3-R is applied in the field of construction of porcine C1QTNF3 gene overexpression vector plasmids to achieve the effects of improving efficiency and accuracy, strong practicability, and improving specificity and accuracy

Inactive Publication Date: 2019-09-20
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current research on the C1QTNF3 gene is largely concentrated on humans and mice, and there is no research report on the pig C1QTNF3 gene

Method used

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  • Construction method of porcine C1QTNF3 gene overexpression vector plasmid and detection of expression level of porcine C1QTNF3 gene
  • Construction method of porcine C1QTNF3 gene overexpression vector plasmid and detection of expression level of porcine C1QTNF3 gene
  • Construction method of porcine C1QTNF3 gene overexpression vector plasmid and detection of expression level of porcine C1QTNF3 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 is used for screening and obtaining the primers constructed by pig C1QTNF3 gene overexpression vector plasmid

[0056] According to the porcine C1QTNF3 gene mRNA sequence published by NCBI (GenBank Accession No. NM_001145386.1), homologous recombination primers capable of amplifying the full length of the C1QTNF3 gene were designed.

[0057] After a large number of experimental studies, the present invention has found that it is not only necessary to meet the general primer design principles or use the primers designed by the primer design software to ensure that the amplified fragment can pass through the overexpression plasmid vector while ensuring the amplification effect of the porcine C1QTNF3 gene. Homologous recombination efficient ligation. On the basis of satisfying general design principles, the present invention aims at the affinity and mismatch rate between primers and target sequences, the secondary structure between primers and the secondary struc...

Embodiment 2

[0062] Example 2 Construction of pig C1QTNF3 gene overexpression vector plasmid

[0063] This embodiment provides a method for constructing a pig C1QTNF3 gene overexpression vector plasmid, which specifically includes the following steps:

[0064] 1. Collection of tissue samples

[0065] Three 1-month-old Mashen pigs came from a pig breeding farm in Datong City, Shanxi Province. Tissue samples were collected after slaughter, put into liquid nitrogen immediately, and then stored in a -80°C refrigerator for later use.

[0066] 2. Tissue RNA extraction and cDNA synthesis

[0067] The total RNA of each tissue was extracted with TaKaRa RNAiso Plus kit, and the extraction method was referred to the kit instructions. The integrity of total RNA was determined by agarose gel electrophoresis; the concentration of total RNA was determined by NanoDrop1000 micro-ultraviolet spectrophotometer.

[0068] The RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit with g...

Embodiment 3

[0084] Example 3 Screening and acquisition of primers for detection of pig C1QTNF3 gene expression level

[0085] According to the porcine C1QTNF3 gene mRNA sequence published by NCBI (GenBank Accession No. NM_001145386.1), primers that can be used to detect the expression level of porcine C1QTNF3 gene were designed.

[0086] After a large number of experimental studies, the present invention finds that the high-efficiency and specific detection of porcine C1QTNF3 gene expression level can not be realized only by meeting the general primer design principles or by using primer design software to design primers. On the basis of satisfying general design principles, the present invention aims at the affinity and mismatch rate between primers and target sequences, the secondary structure between primers and the secondary structure between primers and target sequences, and the GC content and Tm of primers. Value, length, and length of the amplified fragment have been manually optim...

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Abstract

The invention belongs to the technical field of molecular biology, in particular to a construction method of a porcine C1QTNF3 gene overexpression vector plasmid and detection of an expression level of the porcine C1QTNF3 gene. The invention provides a primer for constructing the porcine C1QTNF3 gene overexpression vector plasmid with a sequence shown in SEQ ID NO: 1-2, and a construction method of the porcine C1QTNF3 gene overexpression vector plasmid. The invention further provides a primer for detecting the expression level of the porcine C1QTNF3 gene with a sequence shown in SEQ ID NO: 3-4, and a method for detecting the expression level of the porcine C1QTNF3 gene. The invention provides an effective method and basis for functional research and an application of the porcine C1QTNF3 gene.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a pig C1QTNF3 gene overexpression vector plasmid and a method for detecting its expression level. Background technique [0002] C1QTNF3 (CORS26), also known as CTRP3 or Cartducin, is an adipokine discovered in 2003. The physiological functions of C1QTNF3 include: regulating chondrocyte proliferation and bone and joint diseases; regulating endocrine, glucose and lipid metabolism, mitochondrial biosynthesis, immunity, inflammatory response, apoptosis, angiogenesis, vascular calcification, and ventricular remodeling, etc. Physiological and pathological process; regulate the secretion of testosterone and adipokines in the body. C1QTNF3 belongs to the secreted protein family and can be divided into 4 structural domains: N-terminal signal peptide, variable domain, collagen repeat sequence and C-terminal gC1q globular domain. The N-terminus of human...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85C12N15/11C12Q1/6888C12Q1/686
CPCC07K14/47C12Q1/6888C12Q1/686C12Q2600/158C12Q2563/107C12Q2545/114
Inventor 张雪莲蔡春波曹果清秦本源吴怡琦李文霞王鹤洁郭玉龙刘亚丹高鹏飞郭晓红李步高
Owner SHANXI AGRI UNIV
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