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Construction of pig peripheral bloodmononuclear lymphocytePD-1 (programmed death-1) recombinantplasmids, real-time gene abundance detection method and application of method

A peripheral blood list, PD-1 technology, applied in the field of molecular pathology and immunology, to achieve the effect of enriching the mechanism

Active Publication Date: 2014-08-06
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-Time PCR detection system for the abundance of PD-1 gene in porcine peripheral blood mononuclear cells has not been reported yet.

Method used

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  • Construction of pig peripheral bloodmononuclear lymphocytePD-1 (programmed death-1) recombinantplasmids, real-time gene abundance detection method and application of method
  • Construction of pig peripheral bloodmononuclear lymphocytePD-1 (programmed death-1) recombinantplasmids, real-time gene abundance detection method and application of method
  • Construction of pig peripheral bloodmononuclear lymphocytePD-1 (programmed death-1) recombinantplasmids, real-time gene abundance detection method and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of porcine PD-1 real-time fluorescence quantitative PCR positive standard recombinant plasmid

[0044] 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 target gene fragment of PD-1.

[0045] The target gene fragment of PD-1 is amplified by ordinary PCR method, and the target gene fragment is detected by agarose gel electrophoresis, recovered and purified; then the purified fragment of the PD-1 target gene is connected to the pMD 18-T vector, and transformed into a competent Extract the recombinant plasmid from DH5α cells...

Embodiment 2

[0056] Example 2: Establishment of porcine PD-1 real-time fluorescent quantitative PCR system

[0057] (1) Preparation of plasmid DNA template

[0058] The target gene fragment of PD-1 was amplified by ordinary PCR amplification method, and the target gene fragment was detected by 2% agarose gel electrophoresis, recovered and purified, and transformed into competent cells after ligation with pMD 18-T vector (TaKaRa, Dalian) In DH5α, the recombinant plasmid was extracted; after clone screening, sequencing analysis was performed, and the sequencing result showed that it was 100% homologous to the sequence of the porcine PD-1 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 95.4 μg / mL using a micronucleic acid protein spectrophotometer, and the initial standard plasmid solution was diluted 1:10 times.

[0059] (2) Calcul...

Embodiment 3

[0073] Example 3 : Sensitivity, specificity and repeatability analysis of porcine PD-1 real-time fluorescent quantitative PCR system

[0074] The porcine PD-1 recombinant plasmid constructed in Example 1 was used as the positive recombinant standard plasmid; the porcine PD-1 real-time fluorescent quantitative PCR detection system established in Example 2 was used; the real-time fluorescent quantitative PCR instrument was produced by Life Technologies (USA) ABI 7500 fluorescent quantitative PCR instrument was used.

[0075] with 10-fold serial dilutions of 10 1 ~10 9 The copies / μL porcine PD-1 positive recombinant standard plasmid was used for the sensitivity test, and the results showed that the detection limit of PD-1 was 10 copies / μL.

[0076] Real-time-PCR melting curve for porcine PD-1 molecule (see attached Figure 4 ) and product agarose gel electrophoresis (see attached Figure 5 ) for specificity analysis, the results showed that a single narrow peak appeared in t...

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Abstract

The invention belongs to the technical field ofmolecular pathology andimmunology, and relates to construction of pig peripheral bloodmononuclear lymphocytePD-1 (programmed death-1) recombinantplasmids, a real-time gene abundance detection method and an application of the method. TotalRNA of pig peripheral bloodmononuclear lymphocytes is collected and reverselytranscribed into cDNA (complementary DNA); PD-1 target gene fragmentsare amplified through a general PCR (polymerase chain reaction), detected throughagarose gel electrophoresis, recovered and purified; the PD-1 target gene fragments are connected with pMD18-T carriers and converted into competent cells DH5alpha, and the recombinantplasmids are extracted; sequencing analysis is performed after cloning and screening, positive plasmids with the same sequence as the targetgene fragments are selected to serve as standard plasmids, and a standard curve is drawn according to the copy concentration; thePD-1 gene abundance is measured according to fluorescence signal changes and the standard curve. The real-time pig PD-1 gene abundancedetection method has the advantages of high detection throughput, high susceptibility, high specificity, convenience in operation, low cost, accuracy in quantification and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular pathology and immunology, and in particular relates to the construction of a PD-1 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-1) and its programmed death receptor-1 (PD-1) are members of the CD28 / B7 s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/66
CPCC12Q1/6851C12Q2561/113C12Q2563/107C12Q2545/101
Inventor 王选年岳锋朱艳平冯春花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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