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Construction of pd-l2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application

A technology of PD-L2 and peripheral blood list, applied in the field of molecular pathology and immunology, to achieve the effect of enriching the mechanism

Active Publication Date: 2016-01-13
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the establishment and application of the Real-TimePCR detection system for the abundance of PD-L2 gene in porcine peripheral blood mononuclear cells has not been reported yet.

Method used

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  • Construction of pd-l2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application
  • Construction of pd-l2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application
  • Construction of pd-l2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, real-time detection method of gene abundance and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of porcine PD-L2 real-time fluorescence quantitative PCR positive standard recombinant plasmid

[0043] The peripheral blood of 45-day-old piglets was collected, and the lymphocytes in the peripheral blood were separated according to the instructions of the lymphocyte separation medium, and the total RNA was extracted according to the instructions of the Invitrogen TRIzol kit and related literature. The extracted total RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit, and stored at -20°C for use as a template for amplifying the PD-L2 target gene fragment.

[0044] Amplify the PD-L2 target gene fragment by common PCR method, detect the target gene fragment by agarose gel electrophoresis, recover and purify; then connect the purified PD-L2 target gene fragment with the pMD18-T vector, and transform it into a competent state Extract the recombinant plasmid from DH5α cells, perform sequencing analy...

Embodiment 2

[0055] Example 2: Establishment of porcine PD-L2 real-time fluorescent quantitative PCR system

[0056] (1) Preparation of plasmid DNA template

[0057] The target gene fragment of PD-L2 was amplified by ordinary PCR amplification method, and the target gene fragment was detected by 2% agarose gel electrophoresis, recovered and purified, and then transformed into competent cells DH5α after ligation with pMD18-T vector (TaKaRa, Dalian) In , the recombinant plasmid was extracted; after clone screening, sequencing analysis was performed, and the sequencing results showed that it was 100% homologous to the sequence of the porcine PD-L2 gene in GenBank, indicating that the obtained sequence was correct, and the positive plasmid could be used for the production of plasmid standards. The standard plasmid DNA concentration was determined to be 84.7 μg / mL using a micronucleic acid protein spectrophotometer, and the initial standard plasmid solution was diluted 1:10 times.

[0058] (...

Embodiment 3

[0072] Example 3 : Sensitivity, specificity and repeatability analysis of porcine PD-L2 real-time fluorescent quantitative PCR system

[0073] The porcine PD-L2 recombinant plasmid constructed in Example 1 was used as the positive recombinant standard plasmid; the porcine PD-L2 real-time fluorescent quantitative PCR detection system established in Example 2 was used; the real-time fluorescent quantitative PCR instrument was produced by LifeTechnologies (USA) ABI7500 fluorescent quantitative PCR instrument was used.

[0074] 10 with a 10-fold serial dilution 1 ~10 9 The copies / μL porcine PD-L2 positive recombinant standard plasmid was used for the sensitivity test, and the results showed that the detection limit of PD-L2 was 10 copies / μL.

[0075] Real-time-PCR melting curve for porcine PD-L2 molecule (see attached Figure 4 ) and product agarose gel electrophoresis for specificity analysis, the results showed that a single narrow peak appeared in the melting curve, and a ...

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Abstract

The invention belongs to the technical field of molecular pathology and immunology, and relates to the construction of a porcine peripheral blood mononuclear lymphocyte PD-L2 recombinant plasmid, a real-time detection method for gene abundance and an application thereof. By collecting the total RNA of porcine peripheral blood mononuclear lymphocytes, the total RNA was reverse-transcribed into cDNA; the PD-L2 target gene fragment was amplified by ordinary PCR, detected by agarose gel electrophoresis, and recovered and purified; PD-L2 Target gene fragment and pMD? 18-T carrier was connected, transformed into competent cells DH5α, and the recombinant plasmid was extracted; after cloning and screening, sequencing analysis was performed, and the positive plasmid with the same sequence as the target gene fragment was selected as the standard plasmid, and a standard curve was drawn according to the copy concentration; The gene abundance of PD-L2 was measured by the change of fluorescence signal and the standard curve. The real-time detection method of pig PD-L2 gene abundance of the present invention has the advantages of high detection throughput, high sensitivity, strong specificity, simple operation, low cost, accurate quantification, and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular pathology and immunology, and specifically relates to the construction of a PD-L2 recombinant plasmid in porcine peripheral blood mononuclear lymphocytes, a real-time detection method for gene abundance and an application thereof. Background technique [0002] Classical swine fever (CSF) is an acute, febrile, highly contagious infectious disease caused by classical swine fever virus (CSFV). Studies have shown that CSFV mainly infects the monocyte-macrophage system, causing severe lymphopenia, platelet aggregation and coagulation dysfunction, and atrophy of central immune organs such as thymus and bone marrow. CSFV can also cause immune function damage by reducing the body's antiviral response, inhibiting the apoptosis of infected cells, and promoting the apoptosis of uninfected cells. [0003] Programmed death ligand (PD-L2) and its programmed death receptor-1 (PD-L2) are members of the CD28 / B7...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/66
CPCC12Q1/6851C12Q2561/113C12Q2563/107C12Q2545/101
Inventor 王选年朱艳平岳锋张艳芳孙国鹏李鹏张万方杨媛王爱国李博文王军
Owner XINXIANG UNIV
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