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Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein

A technology of fusion protein and protein sequence, which is applied in the field of genetic engineering and bioengineering, can solve the problems of complex renaturation process, high production cost, and inactivity of inclusion bodies, so as to increase the overall immune regulation effect, increase the time of effect, and reduce the The effect of production costs

Active Publication Date: 2013-05-08
江苏晶红生物医药科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former method uses animal cells as raw materials, the production process is complicated, the production cost is high, and the product has the risk of carrying exogenous viruses and other pathogenic microorganisms
In contrast, the E. coli heterologous expression method has the advantages of large yield, high purity, suitable for large-scale production, and easy control of product quality, but its production process is relatively cumbersome
Moreover, due to the lack of protein post-translational processing system in the E. coli system, the expression products are mostly produced in the form of inactive inclusion bodies
However, the renaturation process of inclusion bodies is complex and the renaturation rate is low, resulting in low protein specific activity.
In addition, the purity of the product of the E. coli expression system is not high, and it contains other miscellaneous proteins, so it is difficult to separate and purify

Method used

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  • Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein
  • Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein
  • Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the cDNA gene design of porcine IFNγ-Fc fusion protein

[0044] According to the cDNA sequence of Sus scrofa breed Fengjing interferon gamma that GenBank has published (GenBank sequence accession number: EU249804.1), the Fc fragment tag is added before the stop codon at the 3' end of the sequence, and the gene of the Fc fragment is provided by Sus scrofa The IgG heavy chain (GenBank accession number: NM_213828.1) consists of the cDNA sequences of the hinge region, CH2 region and CH3 region, and the obtained sequence is recorded as the original gene sequence of porcine IFNγ-Fc. According to the characteristics of the silkworm expression system, the gene sequence was optimized as a whole, and the optimized gene sequence was recorded as the first optimized gene sequence of pig IFNγ-Fc ， As shown in SEQ ID NO:1.

[0045] The optimized first optimized gene sequence of porcine IFNγ-Fc is more suitable for the silkworm expression system, which is mainly reflec...

Embodiment 2

[0055] Example 2. Construction of recombinant transfer vector pFastBac-(IFNγ-Fc)

[0056] 1. PCR amplification of the second optimized gene product of porcine IFNγ-Fc

[0057] Using the artificially synthesized pVL1393-(IFNγ-Fc) plasmid as a template, carry out PCR amplification to remove the 23 amino acid signal peptide of IFNγ itself, and add the system gp64 signal peptide suitable for the insect expression system. The obtained gene sequence is marked as porcine IFNγ- The second optimized Fc gene, the gene sequence is shown in SEQ ID NO:2. details as follows:

[0058] Use primers as follows:

[0059] Forward primer 1: 5'-

[0060] CGC GGATCC ATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCGCAAGCCCCTTTCTTCAAAGA-3',

[0061] Reverse primer 1: 5'-CCC AAGCTT TTATTTTTCCTTGGGTCTTGCT-3'.

[0062] The upstream primer contains a BamH I restriction site and the gp64 signal peptide sequence, and the downstream primer contains a Hind III restriction site.

[0063]...

Embodiment 3

[0075] Example 3, Construction of Baculovirus BmN-(IFNγ-Fc)

[0076] 1. Construction of transfer plasmid Bacmid-(IFNγ-Fc)

[0077] Add 5 μL of pFastBac-(IFNγ-Fc) plasmid into 100 μL of DH10Bac competent cells, heat shock in a water bath at 42°C for 90 seconds after 30 minutes on ice. After the heat shock, the product was quickly cooled on ice for 3 minutes, and then added to 900mL LB medium that had been incubated to 37°C, and revived at 37°C with shaking at 220rpm for 1h. Take 100 μL of recovered bacterial solution and spread it on the LB solid plate containing 50 μg / mL kanamycin, 7 μg / mL gentamycin, 10 μg / mL tetracycline, 100 μg / mL X-gal and 40 μg / mL IPTG at 37 Cultivate in an upside-down incubator. After 48 hours, a single white colony was picked from the LB solid plate and added to LB liquid medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline for overnight expansion. After extracting Bacmid genomic DNA, PCR verification was performed us...

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Abstract

The invention provides a pig IFN (interferon) gamma-Fc fusion protein optimized according to a silkworm reaction system and a coding gene of the pig IFNgamma-Fc fusion protein and a method of expressing pig IFNgamma-Fc fusion protein by using a silkworm bioreactor, and belongs to the field of a biological genetic engineering. The pig interferon gamma serving as a non-special broad spectrum antiviral biological agent has a wide medicinal prospect in a veterinary drug field; but like most of genetic engineering veterinary drugs, the pig interferon gamma also has the problems such as insufficient output, expensive price, irregular quality, short acting time and the like. A target protein with a high expression efficiency and strong activity is obtained by expressing the pig IFNgamma-Fc fusion protein optimized by using a silkworm expression system. In addition, the acting time of the pig interferon gamma is prolonged by addition of an Fc segment, so that the overall immune regulation effect is enhanced, and purification at a later stage is facilitated, thus a pig IFNgamma-Fc fusion protein preparation produced by using a genetic engineering method is possible, and a condition is also created for developing a novel feed containing the fusion protein.

Description

technical field [0001] The invention relates to genetic engineering and bioengineering technology, in particular to a porcine IFNγ-Fc fusion protein and its encoding gene and a method for expressing the porcine IFNγ-Fc fusion protein using a silkworm bioreactor. Background technique [0002] Interferon (Interferon, IFN) is a group of active proteins (mainly glycoproteins) with multiple functions, and is a cytokine produced by monocytes and lymphocytes. They have broad-spectrum anti-virus, affect cell growth, differentiation, regulation of immune function and other biological activities on the same kind of cells. According to the source of IFN, that is, animal species, cell type, the nature of the inducer and the induction conditions, it can be divided into three types: α, β, and γ. Among them, interferon-γ (Interferon-gamma, IFN-γ) is a cytokine with anti-virus, anti-tumor and immunoregulatory effects, mainly produced by activated T cells and NK cells, and plays an importan...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N7/01A61K38/21A61K47/48A61P31/12A61P37/02A61P35/00A23K1/16
Inventor 马永钱林徐春林陈晨王耀方
Owner 江苏晶红生物医药科技股份有限公司
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