Construction, identification and expression method of porcine ACE2 eukaryotic expression recombinant plasmid vector

A technology of recombinant plasmids and expression methods, applied in the biological field, can solve problems such as unclear biological functions, achieve the effects of stabilizing physiological metabolic capabilities, perfecting gene transfer and vector expression systems, and accurately modifying exogenous proteins

Pending Publication Date: 2021-01-26
NANJING AGRICULTURAL UNIVERSITY
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  • Claims
  • Application Information

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Problems solved by technology

However, there are few studies on animals related to livestock productio

Method used

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  • Construction, identification and expression method of porcine ACE2 eukaryotic expression recombinant plasmid vector
  • Construction, identification and expression method of porcine ACE2 eukaryotic expression recombinant plasmid vector
  • Construction, identification and expression method of porcine ACE2 eukaryotic expression recombinant plasmid vector

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Experimental program
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Example Embodiment

[0030]Example 1 Obtaining the ACE2 target fragment

[0031]According to the complete porcine ACE2 gene sequence published on NCBI (KX756982.1), search the sequence of the target gene and design primers. Insert Hind III and Xho I restriction sites at the 5'ends of the upstream and downstream primers, and add 6×His tags to the downstream primers to facilitate subsequent purification of the protein by NI column. The primers were synthesized by Nanjing Kinco Biotechnology Co., Ltd. Reverse transcription of RNA into cDNA, PCR amplification using cDNA as template, reaction system: cDNA template 2uL, reaction conditions: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10s, 57℃ annealing for 15s, 72℃ extension for 30s, 32 pieces Cycle; PCR amplification results such asfigure 1 , The result shows that a single band appears (see lane 2), and the band size is between 2000-3000bp, which is consistent with the expected (2418bp) size.

Example Embodiment

[0032]Example 2 Construction and identification of eukaryotic expression plasmid pcDNA3.1(+)-ACE2

[0033]The ACE2 PCR purified product and pcDNA3.1(+) plasmid were ligated and recovered. The ACE2 gene fragment was ligated to pcDNA3.1(+) eukaryotic expression vector by T4 DNA ligase. Ligation system (10uL): Including 1uL pcDNA3.1(+) vector, 7uL target sequence, 1uL T4 DNA ligase, 1uL 10×Buffer in a 200uL EP tube, mix well and ligate on a PCR machine at 22°C for 14h. The ligation product was transformed into DH5α competent cells, and the positive recombinant expression vector pcDNA3.1(+)-ACE2 was obtained by Hind III single enzyme digestion and Hind III and Xho I double enzyme digestion. The positive recombinant expression vector pcDNA3.1(+)-ACE2 was obtained and sent to Shanghai Handsome Biotechnology Co., Ltd. for sequencing . The monoclonal colony PCR verification results are as followsfigure 2 , There is a single band between 2000-3000bp (lanes 1-5), and the size of the fragment is ...

Example Embodiment

[0043]Example 3 Transfection and expression of eukaryotic expression plasmid pcDNA3.1(+)-ACE2

[0044]1. CHO cell culture

[0045]The medium used is DMEM / F12 medium, and the culture condition is 37°C, 5% incubator.

[0046]2. Determination of the minimum lethal concentration of G418

[0047]1) Set the lethal concentration of G418 to 0, 500ug / mL, 600ug / mL, 700ug / mL, 800ug / mL, 900ug / mL, 1000ug / mL.

[0048]2) Inoculate CHO cells in a 6-well plate at a density of 20%-30%. After the cells adhere to the wall, discard the solution and wash with PBS. Add 2mL DMEM / F12 medium to each well, add G418 of different concentrations, and mix well.

[0049]3) Change the medium once every 2 days, culture for 10-14 days, observe the cell status every day, and take the lowest concentration of cell death as the benchmark.

[0050]After G418 screening on the 6th day, the death of CHO cells is as follows:Figure 4 . With the increase of G418 treatment days, the degree of CHO cell death intensified. After G418 was stimulated to ...

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Abstract

The invention discloses a construction, identification and expression method of a porcine ACE2 eukaryotic expression recombinant plasmid vector. The method comprises the following steps: constructingan ACE2 eukaryotic expression vector, transfecting CHO cells, and screening stably-expressed pcDNA 3.1 (+)-ACE2 cell strains through G418 to obtain ACE2 protein with biological activity. The method has the advantages that (1) the reaction is quick, and only one hour is needed; (2) simplicity: the method is not influenced by fragment enzyme digestion sites, and enzyme digestion is not needed; (3) high efficiency: the positive rate reaches 95% or above; and (4) seamlessness: extra sequences are not introduced. ACE2 exerts various body protection functions such as vasodilation, inflammation inhibition and fibrosis resistance by targeted degradation of AngII. Besides, as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), the porcine ACE2 eukaryotic expression recombinant plasmid vector has extraordinary clinical significance. The invention lays a foundation for deeply researching the biological function and the molecular mechanism of the porcine ACE2 servingas a potential virus receptor in porcine coronavirus infection and developing the ACE2 serving as a coronavirus targeted therapy drug.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing and identifying a porcine ACE2 (Angiotensin converting enzyme2) eukaryotic expression recombinant plasmid vector and obtaining a cell line stably overexpressing the ACE2 gene. Background technique [0002] Angiotensin converting enzyme 2 (Angiotensin converting enzyme 2, ACE2) is a monocarboxypeptidase first reported in 2000. As a key regulatory protein of the renin-angiotensin system, it plays an important role in the maintenance of various tissues and organs throughout the body or locally. Functional homeostasis is of great significance. Studies in recent years have found that ACE2 is of great significance in inhibiting intestinal inflammation, assisting intestinal material transport, and maintaining intestinal homeostasis. ACE2 can promote the expression of substance transporters such as amino acids and glucose, and assist their absorption and transport in...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N9/48C12N15/57C12N15/66
CPCC12N15/85C12N15/65C12N15/66C12N9/485C12Y304/17023
Inventor 李志强张源淑王换换张崇昊曹西月谢娜娜付亚丽
Owner NANJING AGRICULTURAL UNIVERSITY
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