Soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as preparation method and application of soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein

A GM-CSF and fusion protein technology, applied in the field of biogenetic engineering, can solve the problems of affecting curative effect, high cost of interferon, and increased number of treatments, and achieve strong anti-VSV virus activity, simple purification steps, and low production costs.

Inactive Publication Date: 2020-06-19
WUHU TIANMING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This brings a lot of inconvenience to clinical treatment, such as the increase in the number of treatments, the corresponding time cost and economic cost increase, which also affects the curative effect and increases the probability of adverse reactions in the body
Although polyethylene glycol-modified interferon can prolong the half-life, the cost of polyethylene glycol-modified interferon is very high, and it is difficult to be applied in clinical treatment of poultry

Method used

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  • Soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as preparation method and application of soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein
  • Soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as preparation method and application of soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein
  • Soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as preparation method and application of soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A fusion protein composed of recombinant chicken GM-CSF and recombinant chicken IFN-α, the preparation method of which is as follows:

[0047] a. Acquisition of chicken GM-CSF and chicken IFN-α target genes and design of amplification primers:

[0048] Synthetic primers were designed according to the target gene sequence reported in Genebank, BamHI restriction site and Linker sequence were introduced into the upstream and downstream primers of chicken GM-CSF respectively, and the upstream and downstream primers of chicken IFN-α were respectively introduced into Linker sequence and HindⅢ restriction site, as shown in Table 1:

[0049] Table 1 PCR amplification primers

[0050]

[0051] RT-PCR to obtain the target gene, as shown in Table 2:

[0052] RNA was extracted from chicken liver tissue, and the target genes of chicken GM-CSF and chicken IFN-α were obtained by reverse transcription. The gene sequences of the two are shown in the sequence table SEQ ID NO.1 and S...

Embodiment 2

[0074] A rChGM-CSF-IFNα protein is prepared by freeze-drying after mixing the fusion protein in Example 1 and a freeze-drying protective agent. The lyoprotectants are glycerol, mannitol and sucrose, and 10mmol / L PBS is used as the buffer solution. The final concentrations of the three are glycerol 100mL / L, mannitol 0.12g / mL and sucrose 0.025g / mL.

Embodiment 3

[0076] Identification of rChGM-CSF-IFNα protein:

[0077] a. Protein quantitative detection:

[0078] The Lowry method was used to determine the standard protein provided by the China Institute for Food, Drug and Biological Products Control.

[0079] b. SDS-PAGE electrophoresis detection:

[0080] Compared with the empty vector bacterium protein, the target protein expressed by the recombinant bacteria showed the expected wide and thick band of the target protein at about 55kD, which was basically consistent with the theoretical value.

[0081] c. Western Blot results:

[0082] The mouse anti-chicken GM-CSF polyclonal antibody from Bio-Rad was used as the primary antibody (1:2000 dilution), and the HRP-labeled goat anti-mouse IgG from Zhongshan Jinqiao Company was used as the secondary antibody (1:50000 dilution). rChGM-CSF-IFNα protein can specifically react with chicken GM-CSF polyclonal antibody, and a specific band appears at about 55kD, see Figure 5 .

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Abstract

The invention discloses a soluble efficiently-expressed rChGM-CSF-IFNalpha fusion protein as well as a preparation method and application of the soluble efficiently-expressed rChGM-CSF-IFNalpha fusionprotein. The preparation method comprises the following steps: (1) respectively extracting and amplifying chicken GM-CSF genes and chicken IFN-alpha genes; (2) linking the amplified chicken GM-CSF genes with the amplified chicken IFN-alpha genes, so as to obtain a fusion protein gene; (3) constructing a recombinant clone vector and a recombinant expression vector, and carrying out screening, so as to obtain gene engineering strains; (4) carrying out soluble efficient expression on a fusion protein; and (5) purifying the fusion protein. By virtue of ChGM-CSF-IFNalpha fusion expression, the antiviral activity of pure ChIFNalpha is improved, and the in-vivo drug half-life period of the pure ChIFNalpha is prolonged; and by virtue of the soluble efficient expression of a ChGM-CSF-IFNalpha pronucleus, the application cost problems of the chicken GM-CSF genes and the chicken IFN-alpha genes in the poultry breeding industry are effectively solved.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a soluble highly expressed rChGM-CSF-IFNα fusion protein and a preparation method and application thereof. Background technique [0002] With the rapid development of the global livestock and poultry breeding industry in recent years, my country's chicken industry has developed from a sideline business in rural households into a major social industry. However, viral infectious diseases of chickens, such as Newcastle disease, infectious bursal disease, Marek's disease, infectious bronchitis and avian influenza, break out in different seasons every year. Once the chicken flock is infected with the disease, it will cause a large area of ​​chickens to die, causing huge economic losses to the breeding industry. At present, the prevention and treatment of chicken viral infectious diseases mainly adopts vaccine immunization and drug treatment. Because the se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/21A61K47/64A61P31/04
CPCC07K14/535C07K14/56C12N15/70A61K47/64A61P31/04C07K2319/00A61K38/00
Inventor 王明丽何志远赵俊张俊玲
Owner WUHU TIANMING BIOTECH CO LTD
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