Orally-taken recombined fusion protein TAT-MAP30, preparation method and applications

A fusion protein and protein technology, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, peptide/protein components, etc., can solve the difficult and impossible problems of controlling and treating animal viral diseases, and achieve low cost, The effect of large amount of expression and simple operation

Active Publication Date: 2014-05-14
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0023] In summary, there have been many reports on the use of Momordica charantin to resist medical viruses, but there has been no report on the use of TAT to carry MAP30 transmembrane into the body to resist viruses. If MAP30 cannot enter the an...

Method used

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  • Orally-taken recombined fusion protein TAT-MAP30, preparation method and applications
  • Orally-taken recombined fusion protein TAT-MAP30, preparation method and applications
  • Orally-taken recombined fusion protein TAT-MAP30, preparation method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Preparation of engineering bacteria Escherichia coli BL21 (DE3) pET-28a-TAT-MAP30

[0086] 1. Obtaining of MAP30 gene and TAT sequence

[0087] The sequence of the artificially synthesized MAP30 gene is shown in SEQ ID NO.3.

[0088] TAT sequence according to Dietz GP et al. Application of a blood-brain-barrier-penetrating form of GDNF in a mouse model for Parkinson's disease. Brain Res .2006Apr12;1082(1):61-6. According to literature reports, a biological company was entrusted to synthesize the full sequence of the TAT gene and save it on the T vector. The TAT sequence is shown in SEQ ID NO.4.

[0089] 2. Acquisition of TAT-MAP30 fusion gene

[0090] Using fusion PCR technology, a TAT sequence was added to the 3' end of the MAP30 gene sequence to realize the fusion of two gene sequences. In order to add TAT at the MAP305' end by overlapping PCR technology, the primers are now designed as follows:

[0091]

[0092]

[0093] Explanation: The bolded part is ...

Embodiment 2

[0145] Obtaining of Recombinant Fusion Protein TAT--MAP30

[0146] 1. Identification of expression of recombinant fusion protein TAT--MAP30

[0147] The engineered strain Escherichia coli BL21 (DE3) pET-28a-TAT-MAP30 was induced by IPTG, and identified by SDS-PAGE gel and Western-blotting, which proved that the recombinant fusion protein TAT-MAP30 was effectively expressed. The specific method is as follows:

[0148] ①Inoculate 50uL of Escherichia coli BL21(DE3)pET-28a-TAT-MAP30 glycerol bacterium in 20mL LB liquid medium and culture overnight.

[0149] ②According to the proportion of 4%, inoculate 800uL overnight culture into 20ml rich medium (2×YT), and culture at 37℃200rpm until OD600 is about 0.6.

[0150] ③ Place the flask in ice bath for 10-20 minutes.

[0151] ④Take 1ML of the culture as a negative control, add 10ul of IPTG (the concentration of the mother solution is 1mol / ml) to the remaining culture medium, and incubate at 20°C and 200rpm for 12-18 hours.

[0152]...

Embodiment 3

[0182] ELISA detection of TAT-MAP30 protein's intestinal penetration function

[0183] 1. Enzyme-linked immunosorbent assay (ELISA) related solution

[0184] Chromogenic solution:

[0185] Weigh 0.47g citric acid, 0.92g Na 2 HPO 4 -12H 2 O, dilute to 100ml, which is prepared as substrate buffer. Just before use, add 40mg o-phenylenediamine, 0.1ml H 2 o 2 , mix well.

[0186] Stop solution: 2mol / L H 2 SO 4 .

[0187] PBS buffer:

[0188] Weigh 8.5g NaCl, 2.85g Na 2 HPO 4 -12H 2 O, 0.2g KCl, 0.27g KH 2 PO 4 , add deionized water to make the volume to 1000ml.

[0189] PBST buffer:

[0190] Add 0.5-2ml Tween-20 to 1000ml PBS buffer.

[0191] 5%BSA:

[0192] Weigh 0.5g BSA and dissolve in 10ml PBST buffer.

[0193] 2. Experimental materials

[0194] The Crayfish (Cambarus clarkii) used in the experiment was purchased from Wuhan Aquatic Products Market, with a weight of about 15-20 g. After the purchase, it was raised in our laboratory at a temperature of about ...

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Abstract

The invention discloses orally-taken recombined fusion protein TAT-MAP30, a preparation method and applications. The genetic engineering technology is used for combining cell-penetrating peptide TAT and elaterin MP30, Escherichia coli is converted, and a genetically engineered bacterium capable of producing the TAT-MAP30 fusion protein is obtained, CCTCC NO: M2013546. The obtained TAT-MAP30 fusion protein has the ability of quickly penetrating through a midgut cytomembrane, can lower protein loss caused by the organism biological barrier, the physical barrier and the chemical barrier, and can be used as oral drugs for prevention and treatment of bacterium resistance and virus resistance of vertebrate and invertebrate, and the fusion protein is high in expression index and easy to purify and has important application prospects and practical significance.

Description

technical field [0001] The present invention relates to the technical field of genetic bioengineering, in particular to an oral recombinant fusion protein TAT-MAP30, a method for preparing the oral recombinant fusion protein TAT-MAP30, and a method capable of expressing the oral recombinant fusion protein TAT-MAP30 Genetic engineering strains, and the application of an oral recombinant fusion protein TAT-MAP30 in antibacterial and antiviral applications. Background technique [0002] MAP30 protein [0003] MAP30 protein (mordtca anti-HIV protein of 30) consists of 286 amino acids. Its genome consists of 858 bases, which contains an independent ORF reading frame without introns. The first 23 amino acids are the leader peptide, and no leader peptide sequence was detected in the mature and functional MAP30 protein. [0004] At present, the specific mechanism by which MAP30 protein can inactivate the ribosomes of bacteria, viruses, and tumor cells has not been clearly elucida...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N1/21C12P21/02A61K38/16A61K47/48A61P31/04A61P31/20C12R1/19
Inventor 孟小林徐进平孟明翔王健潘娟
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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