An expression vector pegfp‑n1‑p53/mar for improving transfection efficiency, construction method and transfection method

A technology of pegfp-n1-p53 and expression vector, applied in the field of bioengineering, can solve the problem of slow progress in chicken transgenic research

Inactive Publication Date: 2018-03-06
HENAN UNIV OF SCI & TECH
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  • Abstract
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  • Application Information

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Problems solved by technology

However, due to the particularity of poultry's physiological development, the progress of chicken transgenic research has been relatively slow.

Method used

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  • An expression vector pegfp‑n1‑p53/mar for improving transfection efficiency, construction method and transfection method
  • An expression vector pegfp‑n1‑p53/mar for improving transfection efficiency, construction method and transfection method
  • An expression vector pegfp‑n1‑p53/mar for improving transfection efficiency, construction method and transfection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Cloning of embodiment 1 human tumor suppressor gene p53

[0118] Experimental Materials

[0119] Human peripheral blood: The peripheral blood of patients with early-stage lung cancer was drawn in the First Affiliated Hospital of Henan University of Science and Technology, and ACD anticoagulant was added to the fresh peripheral blood and stored in liquid nitrogen at low temperature.

[0120] 1. Design and synthesis of primers for human tumor suppressor gene p53 gene cloning

[0121] The upstream and downstream primers Primer a and Primer b were designed with reference to the complete sequence of the human tumor suppressor gene p53 in GenBank (accession number: NW_926584.1), and the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0122] Upstream primer Primer a: 5'-CCCAAGCTTATGGAGGAGCCGCAGTC-3' (SEQ ID NO.2)

[0123] Downstream primer Primer b: 5'-CGCGGATCCCAGTCTGAATCAGGCCCT-3' (SEQ ID NO.3)

[0124] 2. Extraction of total RNA ...

Embodiment 2

[0131] Example 2 Construction of recombinant plasmid pMD18-T-p53

[0132] For a schematic diagram of the construction process of the recombinant plasmid pMD18-T-p53, see figure 2 .

[0133] 1. Preparation of Competent Cells

[0134] (1) Take out Escherichia coli E.coli JM110 strain from the refrigerator at -70°C, inoculate it in 5mL LB liquid medium, and cultivate it gently overnight at 37°C;

[0135] (2) Take 1 mL of the above-mentioned bacterial cell culture solution cultured overnight in a 1.5 mL centrifuge tube, centrifuge at 1500 r / min at 4°C for 5 min, and discard the supernatant (note that the supernatant should be discarded as much as possible);

[0136] (3) Repeat step (2) 3 to 4 times;

[0137] (4) Add 100 μL of Solution A pre-cooled in ice to each centrifuge tube, flick the centrifuge tube gently to suspend the precipitate, and violent shaking is prohibited;

[0138] (5) Centrifuge at 1500r / min at 4°C for 5min;

[0139](6) Add 100 μL of Solution B pre-cooled i...

Embodiment 3

[0185] Cloning of embodiment 3 chicken MAR gene

[0186] 1. Design and synthesis of primers for cloning of chicken MAR gene

[0187] Referring to the full sequence of the GenBank chicken MAR sequence (accession number: M58748.1GI:211883), the upstream and downstream primers to be amplified were designed and synthesized by Jiangsu Jinweizhi Engineering Technology Service Co., Ltd.

[0188] Upstream primer sequence (Primer c): 5'-TTTAGCGGCCGCCACTGTAGCCCTTA-3' (SEQ ID NO.4)

[0189] Downstream primer sequence (Primer d): 5'-CATCTTCTAGAGCTGGAAATGGCAAAC-3' (SEQ ID NO.5)

[0190] 2. Extraction of MAR gene in chicken liver tissue

[0191] The MAR sequence in chicken liver tissue was extracted using the Shanghai Animal Genome DNA Extraction Kit. The specific steps are as follows:

[0192] (1) Take 50 mg of chicken liver tissue and put it into a homogenizer, add 100 μL of Buffer Digestion, grind it vigorously and quickly, transfer the ground tissue fluid into a 1.5ml centrifuge tube...

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Abstract

The invention discloses an expression vector pEGFP-N1-p53 / MAR for improving transfection efficiency, a construction method and a transfection method. The expression vector pEGFP-N1-p53 / MAR attaches human tumor suppressor gene p53 and chicken nuclear matrix The region MAR gene was inserted into the expression vector to construct. The present invention obtains human tumor suppressor gene p53 and chicken regulatory sequence MAR from the peripheral blood of early lung cancer patients and chicken liver tissue by RT-PCR and PCR respectively, and constructs the EGFP gene containing Eukaryotic recombinant expression vector pEGFP‑N1‑p53 / MAR. At the same time, the present invention transfected the chicken germinal disc with the expression vector pEGFP-N1-p53 / MAR, and found that the hatching rate of the transgenic chicken of the present invention reached 56.4%. In 7 of the surviving transgenic chickens, the presence of the target gene p53 band was detected, and the positive detection rate was 18.9%.

Description

technical field [0001] The invention relates to an expression carrier pEGFP-N1-p53 / MAR for improving transfection efficiency, and also relates to a construction method and a transfection method of the expression carrier pEGFP-N1-p53 / MAR, belonging to the technical field of bioengineering. Background technique [0002] Cancer is recognized as one of the deadliest killers of human beings. According to the report of the International Cancer Research Center (IARC), the number of cancer cases is increasing at an average annual rate of 3% to 5%, and the number of cases and deaths has increased by 24.7% and 19.2% respectively compared with 10 years ago. It is estimated that by 2020 the world will There are 20 million new cases and 12 million deaths. However, the treatment of cancer is still a worldwide problem. Surgical treatment alone can easily cause the metastasis of lesions, while chemotherapy and radiotherapy bring great pain to patients and at the same time inhibit bone marr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66A01K67/027
Inventor 雷雪芹徐廷生史明艳宋祯李振红王攀林
Owner HENAN UNIV OF SCI & TECH
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