Fusion protein of PA4-Fc and coding gene and application thereof

A protein and protein technology, applied in the field of PA4-Fc fusion protein and its coding gene and application, can solve the problems of unknown natural function

Inactive Publication Date: 2013-06-26
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its natural function is unknown

Method used

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  • Fusion protein of PA4-Fc and coding gene and application thereof
  • Fusion protein of PA4-Fc and coding gene and application thereof
  • Fusion protein of PA4-Fc and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the acquisition of fusion protein PA4-Fc and its coding gene and expression vector containing the gene

[0053] 1. Acquisition of PA4-Fc gene

[0054] Referring to the anthrax toxin protective antigen PA protein sequence published on the NCBI website (NCBI referencesequence: YP_016495.2), the gene encoding the fourth domain of PA (596-735aa) was obtained by overlapping PCR. In order to facilitate the secretion and expression of PA in mammalian cells, The codon was optimized, the signal peptide sequence was the signal peptide of prourokinase, and the overlapping gene fragments were shown in Table 1. After the overlapping PCR test was completed, the DNA molecule shown in sequence 1 (can be artificially synthesized) was obtained, which was named PA4-Fc fragment. Among them, the 1-60bp nucleotide sequence from the 5' end in sequence 1 is the signal peptide; the 61-486bp nucleotide sequence from the 5' end in sequence 1 is the PA4 gene; The nucleotide sequenc...

Embodiment 2

[0066] Embodiment 2, the application of fusion protein PA4-Fc

[0067] 1. Expression, purification and identification of fusion protein PA4-Fc protein

[0068] 1. Expression, purification and identification of fusion protein PA4-Fc protein in CHO-K1 cells

[0069] 1) Transfection of recombinant plasmids into CHO-K1 cells and screening of positive clones

[0070] Lipofectamine produced by Invitrogen was used for transfection TM 2000 Cationic Liposome Transfection Kit. According to the instructions of the kit, CHO-K1 cells transfected with the recombinant vector pcDNA3.1-PA4-Fc constructed in Example 1 (Cell Bank of Typical Culture Collection Committee, Chinese Academy of Sciences, catalog number: GNHa7) were cultured in 5% CO 2 , 37 ℃ thermostat. Twelve hours after transfection, culture was replaced with D-MEM / F-12 medium (Gibco, product number 12500-096) containing 10% peptide bovine serum (Hangzhou Sijiqing Company, catalog number: 4033). After 48 hours, use D-MEM / F-12 m...

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Abstract

The invention discloses a fusion protein of PA4-Fc and a coding gene and application thereof. The protein provided by the invention is any one of 1) a protein formed by amino acid residue sequences of a sequence 2 in a sequence table and 2) a protein which is derived from 1), is obtained by subjecting the amino acid residue sequences of the sequence 2 in the sequence table to substitution and / or deletion and / or addition of one or more amino acid residues and has the same functions. Experiments prove that a DNA sequence expressing the fusion protein PA4-Fc is constructed by optimizing codons and is imported into cells via carriers to express and purify the fusion protein PA4-Fc; the fusion protein can suppress growth of tumor cells and has an antitumor effect; besides, as the fusion protein contains a human IgG1Fc fragment, the half-life period of the fusion protein in bodies can be lengthened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of PA4-Fc, its encoding gene and its application. Background technique [0002] Anthrax is an infectious disease caused by Bacillus anthracis. Bacillus anthracis is a Gram-positive bacillus, and its carriers are herbivores such as horses and deer. The incidents of human anthrax infection mainly occurred in herdsmen who often come into contact with animals and workers engaged in leather work. There are three ways to cause anthrax infection: one is through skin infection, mainly caused by the entry of anthrax spores from damaged skin. Anthrax bacillus often gathers in damaged skin, and this type of infection can often recover without causing serious consequences. The second is gastrointestinal infection, which is often caused by eating raw meat contaminated with spores. This type of infection is often fatal, and it is also the main mode of infection in sporadic cases...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12P21/02A61K39/07A61K47/48A61K48/00A61P31/04A61K38/16A61P35/00
Inventor 胡显文郗永义段海锋高丽华邵勇陈惠鹏胥照平张连成
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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