CN-121975023-A - Recombinant bovine lactoferrin peptide capable of reducing feedback inhibition to host bacteria and construction method

CN121975023ACN 121975023 ACN121975023 ACN 121975023ACN-121975023-A

Abstract

In order to overcome the practical application difficulty of the current genetic engineering method in the process of producing the bovine lactoferrin peptide, the invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition to host bacteria and a construction method thereof. The invention adopts bovine lactoferrin peptide, calmodulin and enhanced green fluorescent protein to form bovine lactoferrin peptide fusion protein in series, constructs pichia pastoris recombinant strain and realizes high-level expression of bovine lactoferrin peptide. The method is based on the neutralization of positive charges of bovine lactoferrin peptide by calmodulin, reduces the toxicity of the antibacterial peptide to a host, and the serial enhanced green fluorescent protein can emit green fluorescence under 488 nm excitation light so as to realize the real-time analysis of the expression level of bovine lactoferrin peptide fusion protein. The method overcomes the toxicity of the antibacterial peptide to a host when the antibacterial peptide is produced by a genetic engineering method by adopting a fusion expression technology, thereby improving the expression quantity and laying a foundation for high-density fermentation production of the bovine lactoferrin peptide.

Inventors

WANG TAO
WU HAODONG
HE KUN
WANG LIN
WANG LIANG

Assignees

济南亿民动物药业有限公司

Dates

Publication Date: 20260505
Application Date: 20260211

Claims (6)

1. A recombinant bovine lactoferrin peptide capable of reducing feedback inhibition to host bacteria is characterized in that the recombinant bovine lactoferrin peptide fusion protein is obtained by sequentially connecting enhanced green fluorescent protein, calmodulin and bovine lactoferrin peptide in series from N end to C end, wherein the calmodulin can weaken or shield toxicity of the bovine lactoferrin peptide to a host and enhance stability of the fusion protein to protease, the enhanced green fluorescent protein can emit green fluorescence under 488 nm excitation light so as to realize real-time quantitative analysis of expression level of the fusion protein, the amino acid sequence of the calmodulin is shown as SEQ ID No.1, the amino acid sequence of the bovine lactoferrin peptide is shown as SEQ ID No.2, and the amino acid sequence of the enhanced green fluorescent protein is shown as SEQ ID No. 3.
2. The recombinant bovine lactoferrin peptide of claim 1, wherein in the bovine lactoferrin peptide fusion protein, an enterokinase cleavage site is introduced between calmodulin and bovine lactoferrin peptide for separation and purification of bovine lactoferrin peptide, and the amino acid sequence of the enterokinase cleavage site is shown as SEQ ID No. 4.
3. The recombinant bovine lactoferrin peptide of claim 2, wherein a6 xhis tag is provided at the C-terminus of the bovine lactoferrin peptide fusion protein and the amino acid sequence is HHHHHH, such that the bovine lactoferrin peptide fusion protein carrying the 6 xhis tag can be purified by Ni-NTA agarose purification resin.
4. The recombinant bovine lactoferrin peptide of claim 2, wherein the nucleotide sequence of the enhanced green fluorescent protein is shown in SEQ ID No.6, the nucleotide sequence of the calmodulin is shown in SEQ ID No.7, the nucleotide sequence of the bovine lactoferrin peptide is shown in SEQ ID No.8, and the nucleotide sequence of the enterokinase cleavage site is shown in SEQ ID No. 9.
5. The method for constructing the recombinant bovine lactoferrin peptide vector according to claim 4, comprising the steps of inserting a gene fragment CaM-LfcinB shown in SEQ ID No.5 into an expression vector plasmid pPIC9K-EGFP-His to construct a recombinant expression vector pPIC9K-EGFP-CaM-LfcinB-His, then converting the constructed recombinant expression vector pPIC9K-EGFP-CaM-LfcinB-His into Pichia pastoris GS115 to construct a recombinant strain pPIC9K-EGFP-CaM-LfcinB-His-GS115, and performing methanol induction fermentation to obtain a fermentation broth of the recombinant strain bovine lactoferrin peptide fusion protein.
6. A method for separating and purifying bovine lactoferrin peptide is characterized by comprising the steps of collecting fermentation liquor of the recombinant strain bovine lactoferrin peptide fusion protein of claim 5, centrifuging and collecting supernatant, purifying by using Ni-NTA agarose purification resin to obtain bovine lactoferrin peptide fusion protein, and then performing enterokinase digestion and purifying by using Ni-NTA agarose purification resin to obtain bovine lactoferrin peptide.

Description

Recombinant bovine lactoferrin peptide capable of reducing feedback inhibition to host bacteria and construction method Technical Field The invention relates to a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition on host bacteria and a construction method thereof, belonging to the technical field of antibacterial peptides. Background At present, the problem of antibiotic abuse is increasingly severe, so that the curative effect of the traditional antibiotics is gradually weakened or even is invalid, and the public health safety is seriously influenced, so that the search for a feed antibiotic substitute is used for promoting the green production of feed to be an urgent requirement for the development of current enterprises. Bovine lactoferrin peptide is a novel antimicrobial peptide. Bovine lactoferrin peptide (LfcinB, bovine Lactoferricin) is located at position 17-41 of bovine lactoferrin and consists of 25 amino acid residues, the amino acid sequence being FKCRRWQWRMKKLGAPSITCVRRAF. In the amino acid sequence of bovine lactoferrin peptide, two cysteines are connected to each other by disulfide bonds to form an eighteen-membered ring structure. Bovine lactoferrin peptide has broad-spectrum antibacterial activity, has inhibition effect on bacteria, fungi and viruses, can induce death of cancer cells, and has great potential in the fields of animal feed, functional food, clinical medicine and the like. Bovine lactoferrin peptide, as a novel antibacterial peptide, has broad-spectrum antibacterial activity and does not cause the problem of bacterial resistance as in the case of conventional antibiotics. However, due to the broad-spectrum antibacterial activity of the bovine lactoferrin peptide, the growth of host cells can be inhibited and even dead in the process of continuously expressing the bovine lactoferrin peptide by engineering strains such as pichia pastoris, and the fermentation process is finished in advance, so that the yield and the production efficiency of the bovine lactoferrin peptide are obviously reduced. Disclosure of Invention In order to overcome the practical application difficulty of the current genetic engineering method in the process of producing the bovine lactoferrin peptide, the invention provides a recombinant bovine lactoferrin peptide capable of reducing feedback inhibition to host bacteria and a construction method thereof. The invention adopts bovine lactoferrin peptide (LfcinB), calmodulin (CaM) and Enhanced Green Fluorescent Protein (EGFP) to form bovine lactoferrin peptide fusion protein in series, constructs pichia pastoris recombinant strain and realizes high-level expression of bovine lactoferrin peptide. The method is based on the neutralization of positive charges of bovine lactoferrin peptide by calmodulin, reduces the toxicity of the antibacterial peptide to a host, and the serial enhanced green fluorescent protein can emit green fluorescence under 488 nm excitation light so as to realize the real-time analysis of the expression level of bovine lactoferrin peptide fusion protein. The method overcomes the toxicity of the antibacterial peptide to a host when the antibacterial peptide is produced by a genetic engineering method by adopting a fusion expression technology, thereby improving the expression quantity and laying a foundation for high-density fermentation production of the bovine lactoferrin peptide. The first object of the invention is to provide a recombinant bovine lactoferrin peptide (bovine lactoferrin peptide fusion protein) capable of reducing feedback inhibition to host bacteria, which is characterized in that the recombinant bovine lactoferrin peptide (bovine lactoferrin peptide fusion protein) is serially connected with Enhanced Green Fluorescent Protein (EGFP), calmodulin (CaM) and bovine lactoferrin peptide (LfcinB) from the N end to the C end in sequence, wherein the calmodulin can weaken or shield toxicity of the bovine lactoferrin peptide to the host and enhance the stability of the fusion protein to protease, and the enhanced green fluorescent protein can emit green fluorescence under 488 nm excitation light, so that real-time quantitative analysis of the expression level of the fusion protein is realized. Furthermore, in the bovine lactoferrin peptide fusion protein, an enterokinase cleavage site is introduced between calmodulin and bovine lactoferrin peptide for separation and purification of bovine lactoferrin peptide. Furthermore, in order to facilitate subsequent purification, the C-terminal end of the bovine lactoferrin peptide fusion protein is provided with a 6 XHis tag, and the amino acid sequence is HHHHHH, so that the bovine lactoferrin peptide fusion protein carrying the 6 XHis tag can be purified by Ni-NTA agarose purification resin. The amino acid sequence of the calmodulin is shown as SEQ ID No.1, the amino acid sequence of the bovine lactoferrin peptide is shown as SEQ ID No.2, the amino acid sequen