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Construction of PD-L2 recombinant plasmid of chick peripheral blood mononuclear lymph cell, gene abundance real-time detecting method and application thereof

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

Active Publication Date: 2014-05-28
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-L2 gene in chicken peripheral blood mononuclear cells has not been reported yet.

Method used

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  • Construction of PD-L2 recombinant plasmid of chick peripheral blood mononuclear lymph cell, gene abundance real-time detecting method and application thereof
  • Construction of PD-L2 recombinant plasmid of chick peripheral blood mononuclear lymph cell, gene abundance real-time detecting method and application thereof
  • Construction of PD-L2 recombinant plasmid of chick peripheral blood mononuclear lymph cell, gene abundance real-time detecting method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 : Construction of chicken PD-L2 real-time fluorescent quantitative PCR positive standard recombinant plasmid

[0048] The peripheral blood of 4-week-old chicks 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.

[0049] The target gene fragment of PD-L2 is amplified by common PCR method, and the target gene fragment is detected by agarose gel electrophoresis, recovered and purified; then the purified fragment of the PD-L2 target gene is connected to the pMD 18-T vector, and transformed into a Extract the recombinant plasmid from DH5α cells in state, ...

Embodiment 2

[0061] Example 2: Establishment of real-time fluorescent quantitative PCR system for chicken PD-L2

[0062] (1) Preparation of plasmid DNA template

[0063] The PD-L2 target gene fragment recovered from the agarose gel in Example 1 was connected to the pMD 18-T vector (TaKaRa, Dalian), and then transformed into competent cells DH5α, and the recombinant plasmid was extracted; after clone screening, sequencing analysis was carried out. The sequencing results showed that it was 100% homologous to the sequence of the PD-L2 gene in GenBank, indicating that the sequence obtained was correct, and the positive plasmid could be used to make plasmid standards. The standard plasmid DNA concentration was determined to be 152.1 μg / mL using a micronucleic acid protein spectrophotometer, and the initial standard plasmid solution was diluted 1:10 times.

[0064] (2) Calculation of standard plasmid concentration and creation of PD-L2 standard curve

[0065] The plasmids of positive clones ...

Embodiment 3

[0079] Example 3: Sensitivity, specificity and repeatability analysis of chicken PD-L2 real-time fluorescent quantitative PCR system

[0080] The chicken PD-L2 recombinant plasmid constructed in Example 1 was used as the positive recombinant standard plasmid, the chicken PD-L2 real-time fluorescent quantitative PCR detection system established in Example 2 was used, and the real-time fluorescent quantitative PCR instrument was produced by Life Technologies (USA). ABI 7500 fluorescent quantitative PCR instrument was used.

[0081] with 10-fold serial dilutions of 10 1 ~10 9 The copies / μL chicken PD-L2 positive recombinant standard plasmid was used for sensitivity test, and the results showed that the detection limit of PD-L2 was 10 copies / μL.

[0082] Real-time-PCR melting curve for chicken 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, a...

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Abstract

The invention belongs to the technical field of molecular pathology and immunology, and relates to construction of a PD-L2 recombinant plasmid of a chick peripheral blood mononuclear lymph cell, a gene abundance real-time detecting method and an application thereof. The method comprises the following steps: collecting the total RNA of the chick lymph cell, and reversely transcribing the total RNA into a cDNA; augmenting a PD-L2 target gene segment by common PCR, detecting through agarose gel electrophoresis, recovering and purifying; connecting the PD-L2 target gene segment with a pMD18-T vector, converting into a competent cell DH5alpha, and extracting the recombinant plasmid; after clone selecting, carrying out sequence analysis, selecting a positive plasmid having the same sequence with the target gene segment as a standard plasmid, and drawing a standard curve according to replication concentration; measuring the gene abundance of the PD-L2 according to fluorescent signal variation and the standard curve. The PD-L2 gene abundance real-time detecting method has the advantages of high detection flux, high sensitivity, strong specificity, simplicity and convenience in 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 chicken peripheral blood mononuclear lymphocyte PD-L2 recombinant plasmid, a real-time detection method for gene abundance and an application thereof. Background technique [0002] Infectious bursal disease (IBD) is an acute, highly contagious disease of chickens caused by infectious bursal disease virus (IBDV). Studies have shown that IBDV infection can cause the body's immunosuppressive state and persistent infection. The target organ of virus infection is the bursa, and virus replication in B cells leads to damage and destruction of bursa lymphoid follicles and lysis of B lymphocytes. At the same time, virus replication in the mononuclear-macrophage system in the bursa of Fabricius leads to a large amount of secretion of inflammatory mediators, virus spread and aggravated damage, forming septic shock syndrome, leadin...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12Q1/68
Inventor 王选年孙国鹏王爱国张艳芳朱艳平李鹏岳锋张万方李博文杨媛阮涛王军
Owner XINXIANG UNIV
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