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Genetically engineered bacterium capable of expressing cell-penetrating peptide fusion protein and application

A technology for genetically engineering bacteria and proteins, applied in the field of genetic engineering, can solve the problems of asynchrony of virus speed, decrease of virus activity, increase of virus titer, etc., and achieve the effects of convenient large-scale production, low cost and simple operation process.

Inactive Publication Date: 2015-04-22
武汉市畜牧兽医科学研究所
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This has resulted in two results: 1. In the initial stage of culture, there are fewer cells involved in amplifying the virus, and it is difficult to increase the virus titer. 2. The speed of cell amplification of the virus is not synchronized. The viability of the virus released in the medium decreases, which prolongs the time for the culture medium to reach the standard virulence

Method used

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  • Genetically engineered bacterium capable of expressing cell-penetrating peptide fusion protein and application
  • Genetically engineered bacterium capable of expressing cell-penetrating peptide fusion protein and application
  • Genetically engineered bacterium capable of expressing cell-penetrating peptide fusion protein and application

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: Artificial synthesis of PEP-1-CD46 gene

[0033] According to the sequence of CD46 (Gene ID: 396922) and PEP-1 published in GeneBank, the PEP-1-CD46 gene was artificially synthesized, and EcoR I and BamHI restriction sites were added to its two ends, and the synthetic gene sequence Contains the coding region sequence shown in SEQ ID NO.1.

Embodiment 2

[0035] Construction of recombinant plasmid pET-32a-PEP-1-CD46

[0036] The vector pET-32a plasmid and PEP-1-CD46 cDNA were digested with restriction endonucleases EcoR I and BamHI. The enzyme digestion system is as follows:

[0037]

[0038]

[0039] The enzyme digestion reaction condition is 37° C., and the reaction time is 2 to 3 hours. The digested products were subjected to agarose gel electrophoresis, and the open-loop pET-32a and PEP-1-CD46 cDNA digested fragments were recovered with a gel recovery kit.

[0040] The PEP-1-CD46 cDNA fragment was ligated with the open-circle pET-32a to obtain the recombinant plasmid pET-32a-PEP-1-CD46. The connection system is as follows:

[0041]

[0042] The ligation reaction solution was mixed well, ligated at 22°C for 2 hours, and stored at 4°C for use.

[0043] Calcium chloride method prepares Escherichia coli competent cell, and its steps are:

[0044] 1) Pick a single colony of DH5α on a fresh solid LB plate, inoculate...

Embodiment 3

[0068] Embodiment 3: the preparation of escherichia coli genetic engineering bacterium

[0069] Plasmid pET-32a-PEP-1-CD46 (1ul) was transformed into Escherichia coli BL21 (DE 3 ) competent cells. Positive transformants were screened out through PCR identification, and the resulting positive clone was the recombinant genetic engineering strain Escherichia coli BL21 (DE 3 ) pET-32a-PEP-1-CD46. The bacterium was deposited on December 5, 2014 and sent to the China Center for Type Culture Collection for preservation, the preservation number is CCTCC NO: M2014631, address: Wuhan University, Wuhan, China, classification name: Escherichia coli BL21(DE3) / pET- 32a – CSFR.

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Abstract

The invention provides a genetically engineered bacterium capable of expressing cell-penetrating peptide fusion protein and application. The genetically engineered bacterium E.coli BL21 / pET-32a-CSFR with CCTCC NO: M2014631. The strain can be used for expressing protein transduction domain polypeptide (PEP-1) and hog cholera virus acceptor (CD46) genetically engineered fusion protein PEP-1-CD46. The genetically engineered bacterium has the advantages of large expression amount, soluble protein, low cost and high safety. The expressed PEP-1-CD46 protein can be used as mediated protein of hog cholera virus penetrating a cell membrane, so that the virus titer of hog cholera virus can be improved, and the defect that the virus content in a current hog cholera virus cell vaccine is low and cannot achieve the standard of vaccine preparation can be overcome.

Description

Technical field [0001] The present invention is a genetic engineering field. Specifically, a genetic engineering bacteria and applications expressing the messemid peptide fusion protein. technical background [0002] Classical Swine Fever (CSF) is a kind of acute, fever, and mortality infectious disease caused by the Classical Swine Fever Virus (CSFV).The disease is highly contagious and has been widely popular internationally. The incidence rate is high and the mortality rate is as high as 90 %, which is extremely harmful.Entering important livestock and poultry infectious diseases.At present, my country is the country with the most production and use of swine fever vaccines in the world. The total consumption of about 4 billion yuan each year is about 4 billion yuan, and the total market price is about 2 billion yuan. [0003] The long -term vaccine immunity and the interference of other epidemic diseases have gradually developed tolerance to swine fever vaccines. The response ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N1/21C12N7/00A61K39/12A61P31/14
Inventor 周莉谭本忠钱运国陈志华高其双刘武卢顺彭霞
Owner 武汉市畜牧兽医科学研究所
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