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Method for detecting fowl PRL based on JAK-STAT 5 signal transduction pathway

A JAK-STAT5, signal transduction technology, applied in the field of bioengineering, can solve problems such as high detection limit, data distortion, and environmental hazards, and achieve the effects of good stability and repeatability, low cost of use, and high sensitivity

Inactive Publication Date: 2016-07-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological assay can detect the content of PRL with biological activity, but the detection limit of biological assay is high, the repeatability is poor, and it takes a long time; The method uses radioactive markers, enzymes, and luminescent agents as indicators. At present, most poultry-related literatures use the radioimmunoassay method to detect PRL, but its shortcomings are very obvious. There is a downward trend; the sensitivity of chemiluminescence method is higher than that of radioimmunoassay, but only a few hospitals and scientific research institutes use it when measuring human PRL. The results of the determination of samples are low, resulting in data distortion

Method used

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  • Method for detecting fowl PRL based on JAK-STAT 5 signal transduction pathway
  • Method for detecting fowl PRL based on JAK-STAT 5 signal transduction pathway
  • Method for detecting fowl PRL based on JAK-STAT 5 signal transduction pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Obtaining of Goose PRL R CDS Region Sequence

[0043] First take the goose pituitary tissue, after grinding with liquid nitrogen, use the TRIZol method to extract total RNA, and reverse transcribe it into cDNA; according to the goose PRLR sequence, the upstream primer PRLRf is designed by conventional methods as shown in SEQ ID No.1, and the downstream primer PRLRr sequence is shown in SEQ ID No. 2 shown;

[0044] Next, the CDS region of goose PRLR was amplified, and HindⅢ and XhoI restriction sites were introduced at the 5′ ends of the upstream and downstream primers. Using RT-PCR, the full-length 2496bp sequence of the CDS region of goose PRLR gene was obtained and carried out in genebank. Alignment (GeneID: DQ209271.2).

[0045] Finally, the obtained goose PRL receptor CDS region sequence was digested with HindⅢ and XhoI endonucleases, and (operated according to the instructions of the endonucleases) was ligated into the eukaryotic expression sequence that...

Embodiment 2

[0046] Example 2 Obtaining the STAT5 binding region sequence

[0047] According to the STAT5 binding sequence disclosed in the existing literature, it was synthesized by artificial synthesis, repeated 5 times, and two restriction sites KpnI and XhoI were introduced to obtain the sequence of the STAT5 binding region as shown in SEQIDNo.3.

[0048] The synthesized STAT5 binding region sequence was digested with KpnI and XhoI endonucleases, and then ligated into the eukaryotic expression vector pGL3-Enchancer (the reporter gene contained in the vector is fluorescent Sulfase gene), the sequence structure is constructed as figure 2 The indicated pGL3-STAT5 response vector.

Embodiment 3

[0049] The cell transfection of embodiment 3 transgenic vector and the stimulation of standard product PRL

[0050] The specific operation steps are as follows:

[0051](1) Human embryonic kidney 293T cells were added to modified DMEM medium and cultured at 37°C, 5% CO 2 Under the conditions, passaging once every 2-3 days, after culturing for 2 days, inoculate 293T cells in a 96-well plate, and when the cells grow to a confluence rate of 60%-70%, the pCMV6-PRLR constructed in Examples 1 and 2 The expression vector and pGL3-STAT5 response vector were transfected into cells with liposome LipofectamineLTX (the specific operation steps were carried out according to the instructions of the LipofectamineLTX kit), as the experimental group; the negative control group was transfected with eukaryotic expression vectors pCMV6-PRLR and pGL3-Enhancer ; With pRL-TK as the internal reference plasmid, 3 replicate wells were set for each group.

[0052] (2) Change the medium (improved DMEM ...

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Abstract

The invention provides a method for detecting fowl PRL based on a JAK-STAT 5 signal transduction pathway.The method comprises the specific steps that A, a CDS region sequence of a fowl PRL acceptor is extracted and inserted into an eukaryotic expression vector to form a signal reception vector; B, a signal response sequence STAT5 is inserted into the eukaryotic expression vector containing a reporter gene to form a signal response vector; C, human embryo kidney 293T cells are added into an improved DMEM culture medium for culture, transfection of the signal expression vector and the signal response vector is performed, and PRLs of different final concentrations are added in sequence; D, the activity of the reporter gene is detected, and a curve of the reporter gene activity along with PRL changes is obtained; E, the steps A-C are repeatedly executed, the activity of the reporter gene in a sample is detected, and the PRL concentration in the sample is obtained through calculation according to the curve of the reporter gene activity along with PRL changes.The method is high in operability, low in using cost, high in sensitivity, good in stability and repeatability and capable of meeting the requirement for scientific research and fowl breeding detection.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for detecting poultry PRL based on the JAK-STAT5 signal transduction pathway. Background technique [0002] Prolactin (PRL), also known as prolactin and luteinizing hormone, is named for its ability to promote pigeon crop epithelial hyperplasia and produce crop milk. In mammals, prolactin mainly stimulates and maintains the function of the corpus luteum, stimulates and guides the mucous membrane to secrete mucus, stimulates mammary gland development and lactation, etc., and has the functions of inhibiting the secretion of pituitary gonadotropin, promoting and maintaining nesting behavior and feeling The role of photoperiod in regulating poultry seasonality, regulating follicle development, and suppressing reproductive activity to bring poultry into a seasonally non-reproductive state. Studies have found that high concentrations of PRL promote and maintain nesting, inhibit ...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/65C12Q1/68
CPCC12N15/79C12N15/65C12Q1/6897
Inventor 施振旦李辉
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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