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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on animals related to livestock production, especially ACE2 in pigs, and the biological function is still unclear

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Obtaining of ACE2 Target Fragment

[0031] According to the complete porcine ACE2 gene sequence (KX756982.1) published on NCBI, the sequence of the target gene was searched and primers were designed. Hind III and Xho I restriction sites were inserted at the 5' ends of the upstream and downstream primers, respectively, and a 6×His tag was added to the downstream primers to facilitate subsequent NI column purification of the protein. Primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd. Reverse transcribe RNA into cDNA, use cDNA as template for PCR amplification, reaction system: cDNA template 2uL, reaction conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 98°C for 10s, annealing at 57°C for 15s, extension at 72°C for 30s, 32 cycle; PCR amplification results such as figure 1 , the results showed that a single band appeared (see lane 2), and the band size was between 2000-3000bp, which was consistent with the expected (2418bp) siz...

Embodiment 2

[0032] Example 2 Construction and Identification of Eukaryotic Expression Plasmid pcDNA3.1(+)-ACE2

[0033]The ACE2 PCR purified product was ligated with the pcDNA3.1(+) plasmid and recovered. The ACE2 gene fragment was connected to the 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 place on a PCR machine at 22°C for 14 hours. 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, Xho I double enzyme digestion and identification, which was sent to Shanghai Handsome Biotechnology Co., Ltd. for sequencing . Monoclonal colony PCR verification results are as follows figure 2 , there is a single band between 2000-3000bp (swimming lane 1-5), and the fragment size is consistent with the expec...

Embodiment 3

[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. in a 5% incubator.

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

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

[0048] 2) CHO cells were seeded in a 6-well plate at a density of 20% to 30%. After the cells adhered to the wall, the solution was discarded, washed with PBS, and 2 mL of DMEM / F12 medium was added to each well. G418 of different concentrations was added and mixed evenly.

[0049] 3) Change the medium every 2 days, culture for 10-14 days, observe the state of the cells every day, and take the lowest concentration as the benchmark for all the cells to die.

[0050] After the 6th day of G418 selection, the death of CHO cells was as follows Figure 4 . With the increase ...

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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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