Multimeric protein having effect of brain targeting, and preparation method and usage thereof

A protein and brain-targeted technology, which is applied to peptide/protein components, medical preparations containing active ingredients, chemical instruments and methods, etc., can solve the problems of increased canceration, short time, unstable curative effect, etc., and achieve transformation efficiency High, easy-to-observe effect

Inactive Publication Date: 2015-03-04
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The short duration of gene expression, unstable curative effect, unclear impact on the microenvironment of the transplant

Method used

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  • Multimeric protein having effect of brain targeting, and preparation method and usage thereof
  • Multimeric protein having effect of brain targeting, and preparation method and usage thereof
  • Multimeric protein having effect of brain targeting, and preparation method and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Construction of Engineering Bacteria Encoding CTB Gene

[0060] The natural cholera toxin subunit B consists of 372 nucleotides encoding 124 amino acids. PCR technology is used to remove 63 nucleotides at the front end of subunit B of cholera toxin. These 21 amino acids are the signal peptide of CTB. The expression level of CTB does not affect its biological activity after removal, and its DNA and amino acid sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 2, respectively. The pET-28a-CTB vector construction process is as follows figure 1 shown. The specific steps are as follows:

[0061] 1. Use upstream primer: 5'-CATG CCATGG GAACACCTCAAAATATTACT-3' SEQ ID NO: 8,

[0062] Downstream primer: 5'-CCG CTCGAG ATTTGCCATACTAATTGC-3' SEQ ID NO: 9;

[0063] Carry out PCR amplification, PCR reaction system: 50ul system contains 10×PCR buffer 5ul (excluding Mg2+), dNTP 1ul, upstream and downstream primers 1ul, template DNA 1ul (about 0.7ug), pfu DNA polyme...

Embodiment 2

[0065] Example 2 Construction of Engineering Bacteria Encoding EGFP-CTA2-TAT Gene

[0066] 1. The pET-22b-EGFP-CTA2-TAT vector construction process is as follows Figure 4shown. The specific process is: using the natural amino acid sequences of CTA2, EGFP, and TAT as templates, the gene sequence obtained through codon optimization is shown in SEQ ID NO: 3, which is fully synthesized by the company. The above-mentioned gene sequence is carried out PCR amplification, amplification primer is as shown in SEQ ID NO:10 and SEQ ID NO:11:

[0067] Use upstream primer: 5'-GGGAATTC CATATG GTGAGCAAGG-3' SEQ ID NO: 10

[0068] Downstream primer: 5'-CCG CTCGAG CTGTGGTGGAC-3' SEQ ID NO: 11

[0069] 2. PCR reaction system: 5ul of 10×PCR buffer (without Mg2+) in the 50ul system, 1ul of dNTP, 1ul of upstream and downstream primers, 1ul of template DNA (about 0.5ug), 1ul of pfu DNA polymerase, Mg2+ added to 1.0 mmol / l, add ddH2O to 50ul. The reaction conditions are: pre-...

Embodiment 3

[0071] Example 3 Step-by-step transformation to obtain target protein expression engineering bacteria

[0072] Transform PET-28a-CTB into Escherichia coli BL21(DE3) competent, select stable single colony under 30mmol / lKan antibiotic pressure, induce overnight at 37℃, IPTG final solubility 0.75mmol / l, 200rpm , and select the engineering bacteria with stable expression. Make the above-mentioned engineering bacteria into a competent state, transform the recombinant plasmid PET-22b-EGFP-CTA2-TAT into the competent state, and screen the engineering bacteria that can be stably passed down under the double resistance selection pressure of 100mmol / l Amp and 30mmol / lKan . Plasmid and bacterial liquid PCR identification such as Figure 7 shown. Enzyme digestion identification such as Figure 8 shown.

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Abstract

The invention discloses a multimeric protein having the effect of brain targeting, and a preparation method and usage thereof. The method comprises the steps: firstly, expressing cholera toxin B subunit and a fusion protein EGFP-CTA2-TAT of three proteins: an enhanced green fluorescent protein, a cholera toxin A subunit and cell-penetrating peptide in escherichia coli by incompatible double plasmid systems to obtain CTB gene by PCR amplification; cloning the gene segment into a carrier pET-28a to obtain a recombinant plasmid pET-28a-CTB; using wild type CTA2, EGFP and TAT amino acid sequences as templates, inserting 3 enzyme cutting sites and linkers between EGFP and CTA2, and optimizing to obtain codons suitable for expression in escherichia coli; cloning the gene segment into a carrier PET-22b (+) to obtain recombinant plasmid PET-22b-EGFP-CTA2-TAT; using different resistances of PET-28a-CTB and PET-22b-EGFP-CTA2-TAT, and co-transforming the different resistances of the PET-28a-CTB and the PET-22b-EGFP-CTA2-TAT into escherichia coli BL21, to obtain engineering bacteria after screening under double resistance selection pressure of penbritin and kanamycin. CTB5/EGFP-CTA2-TAT chimeric proteins can be obtained after inducible expression of the engineering bacteria by IPTG. The invention further discloses the preparation method and usage of the protein.

Description

technical field [0001] The present invention relates to a multi-subunit protein and its preparation method and use, in particular to a multi-subunit protein with brain-targeting effect and its preparation method and use. A double-plasmid system is used to directly express a brain Target protein CTB 5 / EGFP-CTA2-TAT belongs to the related fields of genetic engineering technology, protein separation and purification technology and medical bioengineering technology; in the medical field, the present invention is a brain-targeted protein, which can be used for non-invasive drug delivery of brain-targeted protein . Background technique [0002] Protein drugs have high activity, low toxicity and strong specificity, and have become an important part of pharmaceutical products. In recent years, many protein drugs for the treatment of brain diseases have come out. The biggest obstacle to the application of protein drugs to brain diseases is the blood-brain barrier. Without the ab...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K38/16A61P25/00A61P25/28A61P25/16
CPCY02A50/30
Inventor 王华倩周小芬林卫萍张焜王真史杜志云郑希方岩雄赵肃清黄宝华
Owner GUANGDONG UNIV OF TECH
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