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Antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, bacillus subtilis expressing fragment, preparation and application

A technology of Bacillus subtilis and porcine hemoglobin, applied in Bacillus subtilis and its application fields, can solve the problems of poor stability of porcine hemoglobin peptide structure, physical and chemical properties and functional properties, poor antibacterial and antiviral performance, and reduced activity, and achieve good secretion , good non-pathogenicity, growth-inhibiting effect

Active Publication Date: 2021-11-09
山东仙普爱瑞科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the preparation methods of porcine hemoglobin peptide are all using pig blood as raw material, and the porcine hemoglobin peptide prepared by the prior art has the following disadvantages: the antibacterial and antiviral performance of porcine hemoglobin peptide is relatively poor; the yield is small ; Porcine hemoglobin peptide will reduce its activity when it is prepared into porcine hemoglobin peptide preparation; the structure, physical and chemical properties and functional properties of porcine hemoglobin peptide have poor stability

Method used

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  • Antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, bacillus subtilis expressing fragment, preparation and application
  • Antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, bacillus subtilis expressing fragment, preparation and application
  • Antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, bacillus subtilis expressing fragment, preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Determination of the C-terminal Fragment of Porcine Hemoglobin β Chain with Antibacterial and Antiviral Dual Activities

[0037] Using porcine hemoglobin β chain as template, from N- end to C- The end is gradually decomposed into fragments with a size of 15-17 amino acids. The two connected fragments overlap by 5 amino acids. A total of 14 polypeptide fragments are designed. See the design intention figure 1 . Changzhou Xiangtai Biotechnology Co., Ltd. was commissioned to use automated peptide synthesis equipment to efficiently and quickly synthesize the designed 14 peptide fragments, which were recorded as fragment 1 to fragment 14. See Table 1.

[0038] Table 1 Sequences of 14 polypeptide fragments

[0039]

[0040] 1.1 Determination of antiviral activity of polypeptide fragments according to cytopathic inhibition method

[0041] Use the PEDV / Vero system to detect the biological activity of the polypeptide fragments. The Vero cell suspension digeste...

Embodiment 2

[0055] Example 2 High expression of porcine hemoglobin β chain C-terminal fragment in Bacillus subtilis

[0056] 2.1 Construction of pHT01-P43-HBB expression vector

[0057] Respectively according to the restriction site BamH I nucleic acid artificial sequence (sequence 1), Bacillus subtilis P43 constitutive promoter nucleic acid artificial sequence (sequence 2), porcine hemoglobin β chain C-terminal fragment nucleic acid artificial sequence [abbreviation: HBB] (sequence 3 ), the enzyme cutting site Sma I nucleic acid artificial sequence (sequence 4) entrusted Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize its entire expression cassette (sequence 5) and insert it into the Bacillus subtilis expression vector pHT01 to obtain pHT01-P43- HBB recombinant vector, see Figure 4 .

[0058] The above sequences 1, 2, 3, 4 are sequentially connected in series.

[0059] 2.2 Competent preparation of Bacillus subtilis

[0060] Pick a single colony of B. subtilis WB800n on the ...

Embodiment example 3

[0070] Implementation Case 3 Production of high-density fermentation of Bacillus subtilis recombinant bacteria producing porcine hemoglobin β chain C-terminal fragment

[0071]The recombinant strain of Bacillus subtilis producing the C-terminal fragment of the porcine hemoglobin β chain was streaked on an LB plate containing 30 µg / mL of chloramphenicol resistance, and a single colony was selected and inoculated into a 1L Erlenmeyer flask containing 250 mL of LB liquid medium. Cultivate in a shaker at 37°C and 200rpm until OD600=0.6-0.8; after microscopic examination without bacteria, it is used as the first-class seed for later use. A total of 1 L of 4 bottles of first-grade seed liquid was added to 100 L of sterilized fermentation medium. During the fermentation process, the temperature was maintained at 37 °C and the pH value was maintained at 8.0 (the pH value was maintained by dropping 10% ammonia water), and The dissolved oxygen (dissolved oxygen, DO) was maintained at 20...

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Abstract

The invention provides an antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment. The amino acid sequence of the fragment is shown in a sequence table SEQ ID NO. 6. The invention further provides bacillus subtilis expressing the antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, a preparation containing the porcine hemoglobin beta chain C-terminal fragment and application. The prepared porcine hemoglobin beta chain C-terminal fragment is formed by determining the optimal polypeptide fragment according to the activity change and connecting the optimal polypeptide fragment in series. The porcine hemoglobin beta chain C-terminal fragment has the antiviral activity of 8.16 * 10<4>U / ml and the antibacterial activity that the diameter of an inhibition zone is 13.47 + / -0.67mm. The bacillus subtilis spray-dried powder containing the porcine hemoglobin beta chain C-terminal fragment has a remarkable treatment effect on porcine viral diarrhea and bacterial diarrhea.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to an antibacterial and antiviral porcine hemoglobin beta chain C-terminal fragment, bacillus subtilis expressing the fragment and application thereof. Background technique [0002] In the development of the national economy, the pig breeding industry occupies an increasingly important position. It is not only related to people's daily life, but also plays a certain role in promoting the economic development of the place where the breeding farm is located. In recent years, pig farm epidemics have become more and more complicated. There are mainly multi-pathogen mixed infections such as porcine blue ear disease, porcine circovirus disease, suis streptococcosis and Haemophilus parasuis disease, with high incidence and low survival rate. , causing serious economic losses. [0003] Hemoglobin is a protein that is responsible for transporting oxygen in the blood vessels of most a...

Claims

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Application Information

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IPC IPC(8): C07K14/805C07K14/47C12N1/21C12N15/12C12N15/75A61K38/42A61K35/742A61K9/14A61K47/26A61K47/24A61K47/36A61K47/10A61P31/04A61P31/14A61P1/12C12R1/125
CPCC07K14/805C07K14/47C12N15/75A61K38/42A61K35/742A61K9/145A61K9/146A61P31/04A61P31/14A61P1/12A61K38/00A61K2300/00Y02A50/30
Inventor 王兴业韩国英李琦王晓冉乔雪刘刚郭海岩
Owner 山东仙普爱瑞科技股份有限公司
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