Expression vector pEGFP-N1-p53/MAR capable of improving transfection efficiency, construction method and transfection method

A technology of pegfp-n1-p53 and pegep-n1-p53, which is applied in the construction method and transfection field of the expression vector pEGFP-N1-p53/MAR, can solve the problem of slow progress in chicken transgenic research

Inactive Publication Date: 2016-04-27
HENAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • 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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  • Expression vector pEGFP-N1-p53/MAR capable of improving transfection efficiency, construction method and transfection method
  • Expression vector pEGFP-N1-p53/MAR capable of improving transfection efficiency, construction method and transfection method
  • Expression vector pEGFP-N1-p53/MAR capable of 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 Primera and Primerb 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 Primera: 5'-CCCAAGCTTATGGAGGAGCCGCAGTC-3' (SEQ ID NO.2)

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

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

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 the Escherichia coli E.coliJM110 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-coo...

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 (Primerc): 5'-TTTAGCGGCCGCCACTGTAGCCCTTA-3' (SEQ ID NO.4)

[0189] Downstream primer sequence (Primerd): 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 50mg of chicken liver tissue and put it into a homogenizer, add 100μL of BufferDigestion, grind it vigorously and quickly, transfer the ground tissue fluid into a 1.5ml centrifuge tube, and...

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Abstract

The invention discloses an expression vector pEGFP-N1-p53 / MAR capable of improving the transfection efficiency, a construction method and a transfection method. The expression vector pEGFP-N1-p53 / MAR is established through the manner that the human cancer suppressor gene p53 and the matrix attachment region MAR gene of chickens are inserted into an expression vector. According to the invention, the human cancer suppressor gene p53 and the regulatory sequence MAR of chickens are obtained from the peripheral blood of the early lung cancer patient and the liver tissue of the chickens respectively through RT-PCR and PCR methods, and the recombinant eukaryotic expression vector pEGFP-N1-p53 / MAR containing the EGFP gene is constructed through the means including enzyme digestion, connection, conversion and the like. Meanwhile, according to the invention, the chicken blastoderm is transfected with the expression vector pEGFP-N1-p53 / MAR, the result shows that the hatching rate of the obtained transgenic chicken achieves 56.4%. In the survival transgenic chickens, target gene p53 bands only exist in seven chickens shown by the testing, and the positive rate is 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 Applications(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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