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

A lymphocyte, PD-1 technology, applied in the field of molecular pathology and immunology, to achieve high detection throughput, enrich the mechanism of immunosuppression, and quantitatively accurate results

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

AI Technical Summary

Problems solved by technology

[0004] In recent years, fluorescent quantitative PCR (Real-Time PCR) technology has provided a new method for gene abundance detection. Compared with traditional methods, this method has high sensitivity, strong specificity, easy operation, low cost and accurate quantification. It can achieve the purpose of real-time monitoring by detecting the fluorescence signal intensity of PCR amplification of the target sequence; however, the establishment and application of a Real-Time PCR detection system for the abundance of PD-1 gene in chicken peripheral blood mononuclear cells research has not yet been reported

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  • Construction method of recombinant plasmid of PD-1 (programmed death-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-1 (programmed death-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-1 (programmed death-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-1 real-time fluorescence quantitative PCR positive standard recombinant plasmid

[0048] Collect the peripheral blood of 4-week-old chicks, separate the lymphocytes in the peripheral blood according to the instructions of the lymphocyte separation medium, and extract the total RNA according to the instructions of the Invitrogen TRIzol kit and related literature, and refer to the instructions of the reverse transcription kit for the extracted total RNA. Reverse transcribed into cDNA, stored at -20°C, and used as a template for amplifying the PD-1 target gene fragment.

[0049] The target gene fragment of PD-1 is amplified by common PCR method, detected by agarose gel electrophoresis, recovered and purified, and then the purified fragment of the target gene of PD-1 is connected to the pMD 18-T vector and transformed into a Extract the recombinant plasmid from DH5α cells in state, and perform sequencing analysis after cloning and scr...

Embodiment 2

[0061] Example 2 Establishment of Real-time Fluorescent Quantitative PCR System for Chicken PD-1

[0062] (1) Preparation of plasmid DNA template

[0063] The PD-1 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 performed , the sequencing results showed that it was 100% homologous to the sequence of the PD-1 gene in GenBank, indicating that the obtained sequence was correct, and the positive plasmid could be used to make plasmid standards. The DNA concentration of the standard plasmid was measured by a micronucleic acid protein spectrophotometer to be 135.5 μg / mL, and the initial standard plasmid solution was diluted 1:10 times.

[0064] (2) Calculation of standard plasmid concentration and preparation of chicken PD-1 standard curve

[0065] The plasmids of positive...

Embodiment 3

[0079] Example 3 : Sensitivity, specificity and repeatability analysis of chicken PD-1 real-time fluorescent quantitative PCR system

[0080] The chicken PD-1 recombinant plasmid constructed in Example 1 was used as a positive recombinant standard plasmid, and the chicken 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 750 fluorescent quantitative PCR instrument. 10 with 1:10 serial dilution 1 ~10 9 Copies / μL chicken PD-1 positive recombinant standard plasmid for sensitivity test. The results showed that the detection limit of PD-1 was 10 copies / μL.

[0081] Real-time-PCR melting curve for chicken PD-1 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 the same size as the ex...

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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-1 (programmed death-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-1 target gene segment, detecting the target gene segment through agarose gel electrophoresis and recovering and purifying the target gene segment; connecting the PD-1 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 sequences 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-1 according to fluorescence signal change and the standard curve. The real-time PD-1 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 in particular relates to the construction of a chicken peripheral blood mononuclear lymphocyte PD-1 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): C12Q1/68C12N15/63C12N15/66
CPCC12Q1/6851C12N15/66C12N15/70C12Q2531/113C12Q2561/113C12Q2545/113
Inventor 王选年孙国鹏王爱国朱艳平张艳芳岳锋李鹏张万方李博文杨媛阮涛王军
Owner XINXIANG UNIV
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