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Primer pair, method for preparing cell cycle factor y and application thereof

A cell cycle and factor technology, applied in the biological field, can solve problems such as clinical application value to be studied, and achieve the effect of low cost, high sensitivity and convenient operation

Active Publication Date: 2019-01-25
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the clinical application value of the detection of cyclin factor Y antibody remains to be studied

Method used

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  • Primer pair, method for preparing cell cycle factor y and application thereof
  • Primer pair, method for preparing cell cycle factor y and application thereof
  • Primer pair, method for preparing cell cycle factor y and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The gene encoding CCNY was amplified by PCR method, as follows:

[0063] (1) Acquisition of sample DNA

[0064] Sample DNA was collected using A549 lung adenocarcinoma cells as raw materials. The specific method is as follows:

[0065] A. Collect A549 lung adenocarcinoma cells 1*10 6 indivual.

[0066] B. RNA samples of A549 lung adenocarcinoma cells were extracted using TRIzol RNA extraction reagent.

[0067] C. Using a reverse transcription kit III Cells Direct YM cDNA Synthesis Kit (Invitrogen TM ) to reverse transcribe the RNA sample to obtain the cDNA template of A549 lung adenocarcinoma cells.

[0068] (2) PCR reaction

[0069] Upstream primer: 5'-TAACCCATGGGGAACACTACCTCGTGC-3' (SEQ ID NO: 1)

[0070] Downstream primer: 5'-AATACTCGAGCTAAG AGATG ATGGC-3' (SEQ ID NO: 2)

[0071] PCR reaction system:

[0072] cDNA template 2μl;

[0073] 2U / μl pfuDNA polymerase 1μl;

[0074] 10mM dNTP 1μl;

[0075] 1 μl each of 10mM upstream and downstream primers;

[00...

Embodiment 2

[0096] Utilize embodiment 1 to obtain the coded gene of CCNY after amplifying, the operating steps of preparing His-CCNY recombinant protein are as follows:

[0097] (1) The amplified CCNY coding gene obtained in Example 1 was double-digested with NcoI / XhoI enzyme digestion system and then ligated into plasmid pET30a(+).

[0098] (2) Transform the recombinant plasmid into Escherichia coli E.coli BL21(DE3), and culture it with shaking. When the OD600 of the E. coli culture medium is 0.6, add IPTG to a final concentration of 0.4mM / L, induce expression for 3 hours, and collect the cells , sonicate, and collect the supernatant.

[0099] (3) Purify the collected supernatant according to the operating instructions of the HISTrap purification kit of GE Company. And chromatography column 50mM / L imidazole wash solution 20ml, sample loading speed is 0.5ml / min, affinity chromatography column 200mM / L imidazole eluent 10ml, sample loading speed is 0.5ml / min. According to OD 280 For the ...

Embodiment 3

[0104] Using the CCNY protein-coated microtiter plate obtained in Example 2, the steps are as follows

[0105] (1) Take the His-CCNY recombinant protein obtained in Example 2 and dilute it to 1 μg / ml with coating buffer to obtain the coated

[0106] solution, stored at 4°C, for later use.

[0107] (2) Add the coating solution to a 96-well flat-bottomed polystyrene reagent plate, add 100 μl to each well, and leave overnight at 4°C.

[0108] (3) Wash the plate three times, pat dry the liquid in the plate, add 200 μl of 5% fetal bovine serum blocking solution to each well, and block overnight at 4°C.

[0109] (4) Wash the plate three times, and pat dry the liquid in the plate to obtain the microplate plate coated with CCNY protein, and store it at 4°C for later use.

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Abstract

The invention discloses a primer pair, a method for preparing a cell cycle factor Y and a reagent kit and method for detecting a cell cycle factor Y antibody. The primer pair has a nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2. The primer pair is used for amplifying the coding gene of the cell cycle factor Y, then the cell cycle factor Y is synthesized, and the cell cycle factor Y is used for coating a reagent plate to obtain a solid phase carrier so as to prepare the reagent kit for detecting the cell cycle factor Y antibody in serum or detect the cell cycle factor Y antibody in serum. The reagent kit and the detecting method are high in sensitivity, good in accuracy, low in cost and easy to operate and can be applied to detecting the cell cycle factor Y antibody in serum or plasma.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, to the technical field of antibody detection, more specifically, to a primer pair, a method for preparing cell cycle factor Y, a kit for detecting cell cycle factor Y antibody, and a method for detecting cell cycle factor Y Y antibody method. Background technique [0002] CCNY is also called CyclinY, cyc-Y, cell cycle factor Y, cyclin box protein 1 (cyclin box protein 1), cyclin fold protein 1 (cyclin fold protein 1) or cyclin box carrying protein 1 (cyclin-boxcarrying protein 1) , is a newly discovered cell cycle protein in recent years. Its coding gene ccny is located on chromosome 10, with a total length of 3968bp, 10 exons and 9 introns. CCNY protein contains 341 amino acids with a molecular weight of about 39KD. Studies have shown that the change of CCNY gene level affects the process of cell cycle, and further affects the proliferation, migration and invasion ability of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/70G01N33/531G01N33/68
CPCC07K14/4738C12N15/11C12N15/70C12N2800/101G01N33/531G01N33/6872G01N2333/4739
Inventor 岳文涛马丽滕宇张丽娜顾勐王玥
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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