Construction method of recombinant plasmid of PD-L1 (programmed death-ligand 1) in chicken peripheral blood mononuclear lymphocytes, real-time gene abundance detection method and application of detection method

A technology of PD-L1 and lymphocytes, applied in the field of molecular pathology and immunology, to achieve the effect of enriching the mechanism of immunosuppression

Active Publication Date: 2014-05-14
XINXIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the establishment and application of the Real-Time PCR detection system for the ...

Method used

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  • Construction method of recombinant plasmid of PD-L1 (programmed death-ligand 1) in chicken peripheral blood mononuclear lymphocytes, real-time gene abundance detection method and application of detection method
  • Construction method of recombinant plasmid of PD-L1 (programmed death-ligand 1) in chicken peripheral blood mononuclear lymphocytes, real-time gene abundance detection method and application of detection method
  • Construction method of recombinant plasmid of PD-L1 (programmed death-ligand 1) in chicken peripheral blood mononuclear lymphocytes, real-time gene abundance detection method and application of detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of chicken PD-L1 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 target gene fragment of PD-L1.

[0049] The target gene fragment of PD-L1 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-L1 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

[0060] Example 2: Establishment of chicken PD-L1 real-time fluorescent quantitative PCR system

[0061] (1) Preparation of plasmid DNA template

[0062] The target gene fragment of PD-L1 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 PD-L1 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 104.6 μg / mL using a micronucleic acid protein spectrophotometer, and the initial standard plasmid solution was diluted 1:10 times.

[0063] (2) Calculation ...

Embodiment 3

[0077] Example 3: Sensitivity, specificity and repeatability analysis of chicken PD-L1 real-time fluorescent quantitative PCR system

[0078] The chicken PD-L1 recombinant plasmid constructed in Example 1 was used as the positive recombinant standard plasmid; the chicken PD-L1 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.

[0079] with 10-fold serial dilutions of 10 1 ~10 9 Copies / μL chicken PD-L1 positive recombinant standard plasmid for sensitivity test, the results showed that the detection limit of PD-L1 was 10 copies / μL.

[0080] Real-time-PCR melting curve for chicken PD-L1 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 fragment of...

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Abstract

The invention belongs to the technical field of molecular pathology and immunology and in particular relates to a construction method of recombinant plasmid of PD-L1 (programmed death-ligand 1) in chicken peripheral blood mononuclear lymphocytes, a real-time gene abundance detection method and application of the detection method. The construction and detection methods comprise the following steps: acquiring total RNA (ribonucleic acid) of chicken lymphocytes and carrying out reverse transcription on the total RNA to obtain cDNA (complementary deoxyribonucleic acid); carrying out common PCR (polymerase chain reaction) amplification on a PD-L1 target gene segment, detecting the target gene segment through agarose gel electrophoresis and recovering and purifying the target gene segment; connecting the PD-L1 target gene segment with a pMD18-T carrier, transforming the product into a competent cell DH5alpha and extracting the recombinant plasmid; carrying out sequencing analysis after cloning and screening, selecting positive plasmid with the same sequence as the target gene segment as standard plasmid and drawing a standard curve according to the copy concentration; and detecting the gene abundance of the PD-L1 according to fluorescence signal change and the standard curve. The real-time PD-L1 gene abundance detection method has the advantages of high detection flux, high sensitivity, strong specificity, simplicity and 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 specifically relates to the construction of a chicken peripheral blood mononuclear lymphocyte PD-L1 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 王选年冯春花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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