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Recombinant glucanase capable of being expressed and secreted in animal cell as well as recombination method and application of recombinant glucanase

A technology of glucanase and animal cells, which is applied in the field of recombinant glucanase and its recombination, can solve the problems of deviation from the optimum action temperature, uneven trypsin ability, and high heat resistance of the enzyme, and achieve a wide range Effects of pH adaptation and tolerance

Inactive Publication Date: 2014-08-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After these genes are cloned, they are fermented by Escherichia coli or yeast to produce enzymes. According to reports, the optimal pH of most enzyme genes is narrow, and there is only one peak in different pH buffers, which is difficult to adapt to the pH of the gastrointestinal tract of animals from 1.0 to 7.0 In the constantly changing acid-base environment, in the gastrointestinal tract of animals, it can only perform hydrolysis in the stomach or intestine, and the action time is short. The normal body temperature of animals is mostly distributed between 40-70°C
Most of the feed pelleting temperature requires 80-95°C. In its practical application, the heat resistance of the enzyme is required to be high. Affected by many factors, it is difficult to choose an ideal enzyme for production.

Method used

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  • Recombinant glucanase capable of being expressed and secreted in animal cell as well as recombination method and application of recombinant glucanase
  • Recombinant glucanase capable of being expressed and secreted in animal cell as well as recombination method and application of recombinant glucanase
  • Recombinant glucanase capable of being expressed and secreted in animal cell as well as recombination method and application of recombinant glucanase

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 can express the recombinant method of the secreted recombinant glucanase in animal cell

[0031] Include the following steps:

[0032] 1. Candidate gene screening

[0033]According to literature reports, the present invention screened 4 bacterial and fungal glucanase genes from NCBI, and obtained the CelA4 gene derived from Alicyclobacillus sp.A4 (Bai et al., 2010), and the PsBg16A gene derived from Paecilomyces sp.FLH30 (Hua et al.,2011), Bg17 gene from Bisporasp.MEY-1 (Luo et al.,2010), eg1314 gene from B.licheniformis EGW039 (Teng et al.,2006), the complete sequence of these genes in High activity in bacteria and fungi.

[0034] 2. Optimization and transformation of candidate genes

[0035] Using Signal P3.0 online software ( http: / / www.cbs.dtu.dk / services / SignalP / ), predict the mature peptide region and signal peptide region of the four genes respectively, and then according to the pig codon preference, use the Optimum Gene TM Gene Design system s...

Embodiment 2 Embodiment 1

[0050] Example 2 The stability test of the recombinant β-glucanase protein obtained in Example 1 to pH

[0051] Transfect porcine kidney pK15 cells with pCD-pBgA3pEG and pCD-pBg2ApEG, and collect the cell culture fluid 3 days later as the initial enzyme solution. Refer to the national standard to determine the activity of pBgA3pEG and pBg2ApEGβ-glucanase in different pH buffers. Buffer: 0.1M KCl-HCl (pH10-2.0), 0.1M Na 2 HPO 4 - Citrate (pH2.6-7.6) and 0.1M Tris-HCl (pH8.0-9.0).

[0052] After incubating pBgA3pEG and pBg2ApEG and their monomeric enzymes in different pH buffers for 2 hours, the remaining enzyme activities were determined under the optimal pH conditions. see results Figure 6 ,from Figure 6 It can be seen that compared with the monomeric enzyme, the recombinant enzyme has a significantly wider range of action on pH (more than 50% of the enzyme activity can be maintained at pH 1.0-7.0), while the working pH of the monomeric enzyme pBgA is 1.0-5.0, When the ...

Embodiment 3 Embodiment 1

[0053] Example 3 Enzyme Activity Comparison of Two Recombinant β-Glucanase Proteins Obtained in Example 1

[0054] Transfect porcine kidney pK15 cells with pCD-pBgA3pEG and pCD-pBg2ApEG, and collect the cell culture fluid 3 days later as the initial enzyme solution. Refer to the national standard to determine the activity of pBgA3pEG and pBg2ApEGβ-glucanase in different pH buffers. The buffer is 0.1M Na 2 HPO 4 – Citrate (pH 2.6-7.6).

[0055]After pBgA3pEG and pBg2ApEG were incubated in the buffer for 2 hours, the remaining enzyme activities were determined under the conditions of pH 4.0 and pH 5.6. see results Figure 7 ,from Figure 7 It can be seen that the recombinase pBgA3pEG exhibited higher enzyme activity than pBg2ApEG.

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Abstract

The invention discloses a recombinant glucanase capable of being expressed and secreted in an animal cell as well as a recombination method and application of recombinant glucanase. The nucleotide sequence of the recombinant glucanase capable of being expressed and secreted in an animal cell is SEQ ID No:1 or SEQ ID No:3. The recombinant glucanase is obtained through the steps as follows: a microbe source gene is subjected to codon transformation and signal peptide substitution to screen out two glucanase genes suitable for being expressed and secreted in an animal cell; the two genes to recombined and transformed to obtain two new recombinases capable of being expressed and secreted in an animal cell. According to the invention, the obtained new recombinant beta-glucanase pBgA3pEG and the new recombinant beta-glucanase pBg2ApEG can be expressed and secreted in an animal cell, have wider pH adaptability and tolerance, can be used for the development of animal feed enzymic preparations, and can be used for the transgenic animal production.

Description

technical field [0001] The technology of the invention belongs to the field of biotechnology, and more specifically, the invention relates to a recombinant glucanase that can be expressed and secreted in animal cells and its recombinant method and application. Background technique [0002] β-glucan belongs to the structural non-starch polysaccharide in the plant cell wall. It is a D-glucose polymer connected by β-1,3 and β-1,4 glycosidic bonds. In the cell walls of higher plants, the content is high in the endosperm cell walls of barley and oats, and the content of β-glucan in barley is about 4%-10%. It also has certain content in wheat, rye, corn, sorghum, millet and other cereal crops. According to its solubility, β-glucan is divided into two types: water-soluble and water-insoluble, and its soluble part can be formed in the gastrointestinal tract of animals. Viscous chyme affects the diffusion of endogenous digestive enzymes in animals, thereby affecting the digestion of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/10C12N15/85C12N5/10A01K67/027
CPCC12N9/244C12Y302/01006
Inventor 吴珍芳张献伟李紫聪刘德武贺晓燕
Owner SOUTH CHINA AGRI UNIV