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Construction and real-time detection of gene abundance of chicken peripheral blood mononuclear lymphocytes pd-l2 recombinant plasmid and its application

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

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 chicken peripheral blood mononuclear cells has not been reported yet.

Method used

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

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] 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

[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 pMD18-T vector (TaKaRa, Dalian), and then transformed into a competent cell DH5α, and the recombinant plasmid was extracted; after the clone was screened, it was sequenced and analyzed. The results showed that it was 100% homologous to the sequence of the PD-L2 gene in GenBank, indicating that the obtained sequence 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 verified b...

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 ABI7500 produced by LifeTechnologies (USA). Fluorescent quantitative PCR instrument.

[0081] 10 with a 10-fold serial dilution 1 ~10 9 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 10copies / 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, and a specific fragm...

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Abstract

The invention belongs to the technical field of molecular pathology and immunology, and 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. By collecting the total RNA of chick 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; the PD-L2 target gene fragment was combined with The pMD 18-T vector 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 is measured according to the change of the fluorescence signal and the standard curve; the real-time detection method of the PD-L2 gene abundance of the present invention has the advantages of high detection throughput, high sensitivity, strong specificity, simple operation, low cost and quantitative Accurate and other advantages.

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] Chicken infectious bursal disease (infectious bursal disease, IBD) is caused by infectious bursal disease virus (infectious bursal disease virus, IBDV), an acute, highly contagious infectious disease of chicks. 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 mediat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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