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A kind of genetically engineered bacteria expressing membrane-penetrating peptide fusion protein and its application

A technology of genetically engineered bacteria and fusion proteins, applied to genetically engineered bacteria expressing penetrating peptide fusion proteins and application fields, can solve the problems of asynchrony of virus speed, increase of virus titer, decrease of virus activity, etc. The effect of simple production and operation process and low cost

Inactive Publication Date: 2018-05-18
武汉市畜牧兽医科学研究所
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  • Summary
  • Abstract
  • Description
  • 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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  • A kind of genetically engineered bacteria expressing membrane-penetrating peptide fusion protein and its application
  • A kind of genetically engineered bacteria expressing membrane-penetrating peptide fusion protein and its application
  • A kind of genetically engineered bacteria expressing membrane-penetrating peptide fusion protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0033] According to the sequence of CD46 (Gene ID: 396922) published in GeneBank and the sequence of PEP-1, the PEP-1-CD46 gene was artificially synthesized, and EcoR I and BamHI enzyme cleavage sites were added at both ends. The sequence comprises 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 cleavage reaction conditions were 37°C, and the reaction time was 2 to 3 hours. The digested products were subjected to agarose gel electrophoresis, and the open-circular pET-32a and PEP-1-CD46 cDNA digested fragments were recovered with a gel recovery kit.

[0040] The PEP-1-CD46 cDNA digested 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, and the ligation reaction was carried out at 22°C for 2 hours, and stored at 4°C for later use.

[0043] The calcium chloride method prepares Escherichia coli competent cells, and its steps are:

[0044] 1) Pick a sing...

Embodiment 3

[0064] Example 3: Preparation of Escherichia coli genetically engineered bacteria

[0065] The plasmid pET-32a-PEP-1-CD46 (1ul) was transformed into Escherichia coli BL21 (DE 3 ) competent cells. The positive transformants were identified and screened by PCR, and the obtained positive clone was the recombinant genetic engineering strain Escherichia coli BL21 (DE 3 ) pET-32a-PEP-1-CD46. The bacteria has been deposited on December 5, 2014 and sent to the China Center for Type Culture Collection for preservation. The deposit number is CCTCC NO: M2014631. 32a – CSFR.

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PUM

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Abstract

The present invention provides a genetically engineered bacterium expressing a membrane-penetrating peptide fusion protein and its application. The present invention provides a genetically engineered bacterium, E. coli BL21 / pET‑32a-CSFR, CCTCC NO: M2014631. The strain can express protein transduction domain polypeptide (PEP‑1) and classical swine fever virus receptor (CD46) genetically engineered fusion protein PEP‑1‑CD46. Its advantages are large expression, soluble protein, low cost and good safety. The expressed PEP‑1‑CD46 protein can be used as a mediating protein for CSFV to cross the cell membrane, improve the virus titer of CSFV in cell culture, and solve the problem that the current CSFV cell vaccine has a low virus content and cannot meet the vaccine standard Shortcomings.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a genetically engineered bacterium expressing a penetrating peptide fusion protein and its application. technical background [0002] Swine fever (classical swine fever, CSF) is an acute, febrile and lethal infectious disease of pigs caused by classical swine fever virus (CSFV). The disease is highly contagious and once widespread internationally, with a high morbidity rate and a mortality rate of over 90%, which is extremely harmful and caused huge economic losses to the pig industry at that time. Included in the list of important livestock and poultry infectious diseases. At present, my country is the country with the largest production and use of swine fever vaccines in the world. The total annual consumption is about 4 billion copies, and the total market price is about 2 billion yuan. [0003] Long-term vaccine immunization and the interference of other diseases, pigs ar...

Claims

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Application Information

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Patent Type & Authority Patents(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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