Cloning of goat ACE2 gene and preparation of mouse anti-goat polyclonal antibody

A technology of goats and genes, applied in genetic engineering, plant genetic improvement, anti-enzyme immunoglobulin, etc.

Pending Publication Date: 2021-10-29
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in livestock, ACE2 gene is rarely reported, ...

Method used

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  • Cloning of goat ACE2 gene and preparation of mouse anti-goat polyclonal antibody
  • Cloning of goat ACE2 gene and preparation of mouse anti-goat polyclonal antibody
  • Cloning of goat ACE2 gene and preparation of mouse anti-goat polyclonal antibody

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1 Amplification of ACE2 gene, and construction of connection vector

[0044] Primers were designed according to the bovine ACE2 gene sequence (NM_001024502) in GenBank, and the upstream and downstream primers were inserted into the BamHI and HindIII restriction sites respectively, and the primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd. Reverse transcribe RNA into cDNA, use cDNA as a template for PCR amplification, reaction system: cDNA template 2uL, reaction conditions: 95°C, 3min; 95°C, 10s; 59°C, 30s; 72°C, 1min30s; 35 cycles ; 72°C, 10 min. The results of PCR amplification were as figure 1 , a single band appeared (see lane 1), and the band size was about 3000bp, which was consistent with the expected (2954bp) size.

[0045] Purify and recover the PCR product using the DNA gel recovery kit, and refer to the instructions of the DNA gel recovery kit for specific steps. The recovered product was ligated with the pMD19T vector (16°C, over...

Embodiment 2

[0049] Expression experiment of embodiment 2 protein

[0050] The correct pMD19T-ACE2 recombinant plasmid and pET32a prokaryotic expression vector were identified by sequencing using HindIII / BamHI double enzyme digestion, and the results of pET28a plasmid double enzyme digestion are shown in the figure Figure 3A , The pET28a plasmid was verified by double digestion with BamHI and HindIII, and the digested product was identified by 1% agarose gel electrophoresis, and specific bands appeared at 5kb and 2kb, which proved that the digestion was successful. The result of double enzyme digestion of pET32a-ACE2 recombinant plasmid is as follows: Figure 4 , pET32a-ACE2 recombinant plasmid BamHI, HindIII double enzyme digestion showed that specific bands appeared at 6kb and 2kb, that is, the size of the vector pET32a and the inserted target gene ACE2 was consistent with the size of the vector and the target fragment, proving that the enzyme digestion was successful.

[0051] The rec...

Embodiment 3

[0054] Example 3 protein purification experiment

[0055] Prepare 10% separating gel and 5% stacking gel without inserting a comb. After the gel is solidified, add 500uL of inclusion body extract treated with loading buffer, and prepare the gel according to the conventional method, and add standard molecular weight protein as a reference. SDS-PAGE was carried out by conventional methods.

[0056]After electrophoresis, put the unloaded gel into a clean large plate, and perform 0.25mol / L KCl staining on a shaker for 10 minutes. After staining, cut off the silver-white stained target band with a scalpel, and wash 5 times with distilled water. Finally, transfer the gel into a clean pouch, add 500uLPBS to shake and mix, freeze and thaw three times at -20°C, 12000r / min, centrifuge for 3min, take the supernatant for SDS-PAGE electrophoresis, and check the purity by Western blot. Specific operation of Western blot identification of ACE2 recombinant protein: after SDS-PAGE electrophor...

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Abstract

The invention provides cloning of a goat ACE2 gene, expression and purification of a protein and preparation of a polyclonal antibody of the protein. An amino acid sequence and a gene sequence of the ACE2 protein are provided, an ACE2 prokaryotic expression vector is constructed, and the protein is purified. The purified protein can be used for preparing the polyclonal antibody with higher titer, and an antigen and the polyclonal antibody in the invention can lay a foundation for further development of a detection kit. Since the ACE2 is found, various functions of the ACE2 are studied, and the ACE2, as a powerful negative regulation molecule, balances various functions of ACE and shows a protective effect in all organs of a whole body through targeted degradation of AngII; secondly, as a necessary receptor of a SARS coronavirus, down-regulation of the ACE2 can induce serious lung failure; and both the ACE2 and homologue Collectin thereof are associated with an amino acid transporter and play an important role in amino acid absorption in kidneys and intestinal tracts respectively. Therefore, the invention has a great significance in both fundamental research and clinical application.

Description

technical field [0001] The invention belongs to the technical field of molecular cloning, and in particular relates to the cloning of a new goat gene ACE2 (Angiotensinconverting enzyme 2), the expression and purification of the protein and the preparation of polyclonal antibodies. Background technique [0002] Angiotensin-converting enzyme 2 (ACE2), a member of the ACE homologue discovered in 2000, is an important regulator of the renin-angiotensin system. Belonging to type I transmembrane glycoproteins, it is a zinc ion-containing monocarboxypeptide glycoproteinase. Widely present in the heart, liver, lung, kidney, testis and intestinal tract of animals. ACE2 degrades angiotensin II (AngII) into angiotensin (1-7) [Ang(1-7)], which has vasodilation and antiproliferative effects, and can also degrade angiotensin I (AngI) into Ang(1 -9). Ang(1-7) exerts anti-oxidation, anti-fibrosis, anti-inflammation and other effects through its receptor Mas (MasR), which has attracted ex...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12N15/66C07K16/40
CPCC12N9/485C12Y304/17023C12N15/66C07K16/40
Inventor 张源淑闫书平杨维维何晟谢娜娜陈希
Owner NANJING AGRICULTURAL UNIVERSITY
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