A kind of method for improving the expression of Bacillus subtilis ovalbumin

A Bacillus subtilis, protein expression technology, applied in the field of Bacillus subtilis metabolic engineering and genetic engineering, can solve the problems of egg price uncertainty, egg price rise, etc., to achieve growth rate and ovalbumin expression, ratio Increased growth rate and shortened production time

Active Publication Date: 2022-07-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, an outbreak of poultry-borne diseases in chicken farms will lead to an increase in the price of eggs, that is, the price of eggs is uncertain, and there is also a risk of disease transmission from chickens in farms to humans

Method used

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  • A kind of method for improving the expression of Bacillus subtilis ovalbumin
  • A kind of method for improving the expression of Bacillus subtilis ovalbumin
  • A kind of method for improving the expression of Bacillus subtilis ovalbumin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of the oppD knockout integration cassette and acquisition of the knockout strain BS168ΔoppD

[0051] Knockout of ATP-binding protein gene oppD (shown in SEQ ID NO. 2):

[0052] Design primers rh_oppD(S)_1F / rh_oppD(S)_1R, carry out colony PCR on wild-type Bacillus subtilis to obtain the left arm of the integration frame; design primers rh_oppD(S)_2F / rh_oppD(S)_2R, which are resistant to plasmid p7S6 (such as SEQ ID NO.7) to carry out PCR to obtain the resistance amplification fragment; design primers rh_oppD(S)_3F / rh_oppD(S)_3R, carry out colony PCR on wild-type Bacillus subtilis, and obtain the right arm of the integration frame; finally by fusion By PCR, the three fragments were fused and amplified to construct an integration frame for knocking out oppD.

[0053] The constructed integration cassette was transformed into wild-type Bacillus subtilis strain 168. Using yz-oppD-750-F: 5'-gaatcaggagtatgtgcttgcttca-3' and yz-750-R: 5'-ttcaaatatcctcct...

Embodiment 2

[0054] Example 2: Construction of flgD gene knockout integration cassette and acquisition of gene knockout strain BS168ΔflgD

[0055] The flagellar hook-cap component protein gene flgD (shown as SQE ID NO. 4) was knocked out according to the same strategy in Example 1. The specific steps are: design primers rh_flgD(423+)_1F / rh_flgD(423+)_1R, perform colony PCR on wild-type Bacillus subtilis to obtain the left arm of the integration frame; design primers rh_flgD(423+)_2F / rh_flgD(423+) _2R, carry out PCR to the resistance plasmid p7S6 (as shown in SEQ ID NO.7), obtain the resistance amplification fragment; PCR to obtain the right arm of the integration frame; finally, by fusion PCR, the three fragments were fused and amplified to construct an integration frame knocking out flgD.

[0056] The constructed integration cassette was transformed into wild-type Bacillus subtilis strain 168. Using yz-flgD-750-F: 5'-cctcagctgaagcaatcattgccgaatatgg-3' and yz-750-R: 5'-ttcaaatatatcctcctc...

Embodiment 3

[0057] Example 3: Construction of hag knockout integration cassette and acquisition of knockout strain BS168Δhag

[0058] The flagellin gene hag (shown in SEQ ID NO. 6) was knocked out according to the same strategy as in Example 1 or 2, and the specific steps were: using primers rh_hag(915+)_1F / rh_hag(915+)_1R to carry out wild-type Bacillus subtilis Colony PCR was performed to obtain the left arm of the integration frame; primers rh_hag(915+)_2F / rh_hag(915+)_2R were used to perform PCR on the resistant plasmid p7S6 (as shown in SEQ ID NO. 7) to obtain a resistant amplified fragment; The primers rh_hag(915+)_3F / rh_hag(915+)_3R were used to perform colony PCR on wild-type Bacillus subtilis to obtain the right arm of the integration frame; finally, by fusion PCR, the three fragments were fused and amplified to construct a knockout hag integration box. The constructed integration cassette was transformed into wild-type Bacillus subtilis strain 168. Using primers yz-hag-750-F: ...

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Abstract

The invention discloses a method for increasing the ovalbumin expression of Bacillus subtilis, and belongs to the field of genetic engineering. The invention improves the ovalbumin expression of Bacillus subtilis by increasing the specific growth rate of Bacillus subtilis. In the present invention, the wild-type 168 is used as the starting strain, and a plurality of recombinant strains with increased specific growth rate can be obtained by using the method of knockout of growth-related genes, adaptive evolution (Adaptive Laboratory Evolution, ALE) and the combination of the two. The specific growth rate of Bacillus to increase the expression of Bacillus subtilis ovalbumin provides a new idea and method for increasing the expression of Bacillus subtilis ovalbumin.

Description

technical field [0001] The invention relates to a method for increasing the ovalbumin expression of Bacillus subtilis, and belongs to the technical field of Bacillus subtilis metabolic engineering and genetic engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a typical representative of Gram-positive bacteria. It does not produce cell wall endotoxin, and the products it produces are certified as food-grade safe by the U.S. Food and Drug Administration (FDA). In addition, as a non-pathogenic microorganism, Bacillus subtilis has the advantages of fast growth rate (20min proliferation for one generation), short culture time, low culture requirements, clear genetic background for transformation, and strong purine synthesis ability. Therefore, it is widely used in scientific research. The application of Bacillus subtilis in industrial production is very extensive. Ovalbumin, referred to as OVA, is the main protein component in egg white, accounti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/77C12N15/75C12N1/21C12R1/125
CPCC07K14/77C12N15/75C07K14/32
Inventor 刘延峰刘龙宿安琪堵国成李江华陈坚
Owner JIANGNAN UNIV
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