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M1 type MAP (macrophage activation peptide) and IL-2 (interleukin-2) fusion protein and fusion gene as well as expression vector and construction method of expression vector

A technology of fusion genes and expression vectors, applied in the field of genetic engineering, can solve problems such as inability to perform functions at the same time, poor treatment effects of diseases, etc., and achieve the effects of promoting acquired immune response, promoting intestinal health, and restoring immunosuppression

Active Publication Date: 2016-06-08
成都虎标行生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The fusion protein in the literature cannot function and play a role in the intestinal mucosa and mesenteric lymph nodes at the same time, and the therapeutic effect on the disease is not good

Method used

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  • M1 type MAP (macrophage activation peptide) and IL-2 (interleukin-2) fusion protein and fusion gene as well as expression vector and construction method of expression vector
  • M1 type MAP (macrophage activation peptide) and IL-2 (interleukin-2) fusion protein and fusion gene as well as expression vector and construction method of expression vector
  • M1 type MAP (macrophage activation peptide) and IL-2 (interleukin-2) fusion protein and fusion gene as well as expression vector and construction method of expression vector

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Embodiment 1

[0052] This embodiment is a fusion protein of M1 type macrophage activating peptide and IL-2, the amino acid sequence is as SEQ ID NO: 1, which is obtained by expressing the fusion gene of M1 type macrophage activating peptide and IL-2.

Embodiment 2

[0054] This embodiment is the fusion gene of M1 macrophage activating peptide and IL-2, the nucleotide sequence of which is as SEQ ID NO:2.

[0055] When eukaryotic gene clones are expressed in prokaryotes, it is necessary to change the codons preferred by eukaryotes to those preferred by prokaryotes (Bacillus subtilis, lactic acid bacteria) to achieve high-efficiency expression. The present invention optimizes the codons of the gene sequence according to the codon preference of the corresponding cells, designs and obtains the nucleotide sequence SEQ ID NO: 2, and then artificially synthesizes the gene according to the gene sequence.

[0056] The fusion gene of this example uses deoxyribonucleotides as substrates, and a nucleotide fragment of the target sequence is synthesized on a DNA synthesizer (Dr. Oligo-192 series).

Embodiment 3

[0058] The method for constructing the fusion gene expression vector of M1 macrophage activating peptide and IL-2 of the present invention, the specific steps are as follows:

[0059] 1) Amplify the fusion gene by PCR to obtain an amplified fragment of the fusion gene;

[0060] 2) Double digestion vector pHT43 and the amplified fragment, using T4 ligase (purchased from Fermentals) to ligate the double digested product to the pHT43 plasmid (purchased from MoBiTEC);

[0061] 3) Transform the ligation product into Escherichia coli to obtain positive clones, extract the plasmid, and obtain the expression vector of the fusion gene after sequencing verification.

[0062] Vector pHT43 was purchased from MoBiTEC agent in Sichuan, China (Chuanshi Kewei Biotechnology Co., Ltd.).

[0063] PCR amplification conditions:

[0064] 94℃1min

[0065] (94°C 10s, 52°C 10s, 68°C 10s) 5cycles

[0066] (94°C 10s, 68°C 15s) 30cycles

[0067] 68℃1min

[0068] Enzyme cutting system:

[0069] Sma...

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Abstract

The invention provides an M1 type MAP (macrophage activation peptide) and IL-2 (interleukin-2) fusion protein and fusion gene as well as an expression vector and a construction method of the expression vector. The amino acid sequence of the fusion protein is shown as SEQ ID NO:1. A multi-expression M1 type MAP and an IL-2 gene are adopted for splicing coexpression and constitute key factors for activating immune response. The fusion protein can perform a function and play a role simultaneously in intestinal mucosa and mesenteric lymph nodes and has remarkable treatment and regulation functions on PRRSV and PCV2, the quantity of M2 type macrophages is reduced by stimulating proliferation and activation of M1 type macrophages, and immunosuppression is recovered effectively.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion protein of M1 macrophage activating peptide and IL-2, a fusion gene, an expression vector and a construction method thereof. Background technique [0002] Diarrhea caused by drug-resistant bacteria in animals, Salmonella, Escherichia coli, streptococcus, PRRSV (PRRSV), circovirus (PCV2), swine fever (CSFV), poultry diarrhea, coccidiosis and other bacterial and viral diseases , and animal parasites, fungal infections, etc. have long been bottlenecks in animal husbandry and poultry and egg breeding, seriously hindering the development of animal husbandry. At present, in animal husbandry, antibiotics are mainly used to solve clinical problems. With the abuse of drugs, the micro-ecological balance of the animal digestive tract is seriously damaged. After long-term use, the residues and enrichment in animals will directly affect the quality and Indire...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/75C12N15/74C12N1/21C12N15/66C12R1/225C12R1/125
CPCC07K14/4723C07K14/55C07K2319/00C12N15/66C12N15/74C12N15/75C12N2800/101
Inventor 万小平
Owner 成都虎标行生物科技有限公司
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