CN116837009A

CN116837009A - Nucleic acid construct and related preparation method

Info

Publication number: CN116837009A
Application number: CN202211736942.7A
Authority: CN
Inventors: 郭敏; 胡鼎楷; 张俊; 徐丽琼; 于雪
Original assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Current assignee: Kangma Healthcode Shanghai Biotech Co Ltd
Priority date: 2021-12-31
Filing date: 2022-12-31
Publication date: 2023-10-03
Also published as: CN114214345A

Abstract

The invention provides a nucleic acid construct, a vector, a cell, a strain, a culture residue, a related application and a related biological preparation method, which can adapt to the extracellular secretion expression of small polypeptides, can practically and accurately cut without destroying the N-terminal functionality of a peptide chain and maintain a high cutting positive rate, wherein the nucleic acid construct has a structure shown in the following formula one from 5 'to 3': formula one: a-B-C, wherein a is a first nucleotide sequence encoding a promoter element; b is a second nucleotide sequence encoding a signal peptide; c is a third nucleotide sequence encoding a small molecule short peptide, each "-" is independently a bond or a nucleotide linking sequence.

Description

Nucleic acid construct and related preparation method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid construct, a related vector, cells, a strain, culture residues, related applications and a preparation method of related biological products.

Background

At present, accurate N-terminal expression of peptide chains is always a hard injury for application of small molecule functional peptide chains (small molecule short chain polypeptides), particularly clinical application hard injury, and the reason is that the cleavage modification of proteins in cells cannot be truly simulated in academic at present, so that peptide chain cleavage of small molecule short chain polypeptides still only ends in the prediction stage of in vitro experiments, even if successful prediction is carried out, various proteases in cells still add a plurality of variables to the secondary structure of peptide chains, so that expression of heterologous proteins still stays only in intracellular expression and extraction processing, and the situation can lead people to limit and threshold for production or use of various proteins.

Disclosure of Invention

The invention provides a nucleic acid construct, a vector, a cell, a strain, a culture residue and a biological production method, which can adapt to the extracellular secretion expression of small polypeptides, can practice accurate cleavage without damaging the N-terminal functionality of a peptide chain, and maintain high cleavage positive rate.

For this purpose, the invention provides the following technical scheme:

the present invention provides a nucleic acid construct having a structure represented by formula one from 5 'to 3':

formula one: A-B-C (I)

Wherein a is a first nucleotide sequence encoding a promoter element;

b is a second nucleotide sequence encoding a signal peptide;

c is a third nucleotide sequence encoding a small molecule short peptide,

each "-" is independently a bond or a nucleotide linking sequence.

The nucleic acid construct provided by the invention further has the following characteristics: wherein said A, B is derived from yeast, further wherein said yeast cell is selected from the group consisting of:

in another preferred embodiment, the yeast of the genus Kluyveromyces is selected from the group consisting of: kluyveromyces lactis, kluyveromyces marxianus, and a combination of one or more of kluyveromyces polybuyveromyces lactis.

The nucleic acid construct provided by the invention further has the following characteristics: wherein B and C are linked by a fourth nucleotide sequence encoding linkage segment D,

the linkage segment D comprises an amino acid sequence as shown in any one of SEQ ID NOs 4, 33-35 or having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similarity to any one of SEQ ID NOs 4, 33-35; or the fourth nucleotide sequence comprises a nucleotide sequence as set forth in any one of SEQ ID NOS.38-41 or having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similarity to any one of SEQ ID NOS.38-41.

The nucleic acid construct provided by the invention further has the following characteristics: wherein the signal peptide comprises an amino acid sequence as shown in SEQ ID NO. 2 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 2.

The nucleic acid construct provided by the invention further has the following characteristics: wherein the first nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO. 1 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 1, and/or

The second nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO. 36 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 36.

The nucleic acid construct provided by the invention further has the following characteristics: wherein the size range of the small molecule short peptide is any one of 15kDa, 1kDa to 15kDa,5kDa-10kDa and 2kDa to 7 kDa.

The nucleic acid construct provided by the invention further has the following characteristics: wherein the small molecule short peptide is a peptide having wide-area antibacterial and antiviral activity, preferably the small molecule short peptide has an antibacterial and antiviral activity against gram-negative bacteria, more preferably gram-positive bacteria are E.coli, pseudomonas aeruginosa, proteus, bacillus dysenteriae, bacillus pneumoniae, bacillus brucei, influenza (haemophilus), parainfluenza (haemophilus), moraxella catarrhalis, acinetobacter, yersinia, legionella pneumophila, pertussis, bordetella parapertussis, shigella, pasteurella, vibrio cholerae, parhaemolyticus or Shi He Bilin monad; still more preferably, it is resistant to gram-positive bacteria, and even more preferably, gram-positive bacteria are Staphylococcus (Staphylococcus), staphylococcus aureus, streptococcus (Streptococcus), diplococcus pneumoniae, bacillus anthracis, bacillus diphtheriae, bacillus tetani, etc.; common gram-negative bacteria are shigella dysenteriae, typhoid bacillus, proteus or vibrio cholerae; another preferred small molecule short peptide is a peptide having antiviral activity.

The nucleic acid construct provided by the invention further has the following characteristics: the small molecule short peptide contains an amino acid sequence as shown in SEQ ID NO. 3 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 3.

The nucleic acid construct provided by the invention further has the following characteristics: wherein the third nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO. 37 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 37.

The invention also provides a carrier, characterized by comprising: the aforementioned nucleic acid construct.

The invention also provides a cell characterized in that: the nucleic acid construct described above is integrated into the genome of the cell, or the vector described above is contained.

The cell provided by the invention also has the following characteristics: wherein the cell is selected from the group consisting of yeast cells, further wherein the yeast cells are selected from the group consisting of: in another preferred embodiment, the yeast of the genus Kluyveromyces is selected from the group consisting of: kluyveromyces lactis, kluyveromyces marxianus, and a combination of one or more of kluyveromyces polybuyveromyces lactis.

The cell provided by the invention also has the following characteristics: wherein the cell is selected from kluyveromyces lactis, and the locus of the integrated nucleic acid construct in the genome of the cell is: located on the D chromosome, the gene number is KLLA0D11660g.

The invention also provides a strain, which is characterized in that: the cells were cultured to obtain the above-mentioned cells.

The invention also provides a culture residue, which is characterized in that: the culture residue is obtained after the strain is cultured in a culture medium to obtain corresponding cells: the remainder after the sediment including the above cells is substantially removed, wherein the strain is the strain described above, and the cells are the cells described above.

The present invention also provides a use of the aforementioned nucleic acid construct, the aforementioned vector, the aforementioned cell, the aforementioned strain or the aforementioned cell culture residue, characterized in that: is used for the production of cell-free in vitro protein synthesis and/or the production of small molecule short peptides.

The invention also provides a preparation method of the biological product, which is characterized by comprising the following steps:

culturing a target strain by using a culture medium, then harvesting to obtain corresponding cells, and/or obtaining culture residues, wherein the obtained cells are used for cell-free in-vitro protein synthesis, the cell culture residues are used for obtaining a small molecule short peptide product, the target strain is the strain,

the cells are the aforementioned cells; the culture residue was the aforementioned culture residue.

The actions and effects of the invention

(1) The invention discovers that the nucleic acid construct which is a formula I is obtained by constructing the coding sequences of the promoter, the signal peptide and the small molecule short chain peptide, and the small molecule short chain polypeptide can be cut with high yield and high precision, thereby improving the extracellular secretion expression of the intracellular small polypeptide;

(2) The second nucleotide sequence of the coding signal peptide is connected with the third nucleotide sequence of the coding small molecule short chain peptide through the specific connecting segment D, so that the accurate cutting and extracellular position transportation of the small molecule short chain polypeptide can be ensured, and the secretion expression of the small polypeptide reaching the extracellular is improved;

(3) The yield of the small molecule short-chain polypeptide can be improved through a specific promoter, the small molecule short-chain polypeptide can be expressed forcefully in the early stage compared with other products, and the later expression can be inferior to part of other products;

(4) Through specific signal peptide, positive cleavage of small molecule short chain polypeptide can be improved, thereby improving extracellular secretion expression of small polypeptide.

Thus, on one hand, the nucleic acid construct provided by the invention can adapt to the extracellular secretion expression of small polypeptides with the sizes of 15kDa, 1kDa to 15kDa,5kDa to 10kDa or 2kDa to 7kDa, can practically and accurately cut without damaging the N-terminal functionality of a peptide chain, and can maintain a high cutting positive rate, so that a culture system of small molecule short chain polypeptides expressed by cells can produce the small molecule short chain polypeptides with high yield and high precision without extraction or additional processing, thereby reducing the limitation and threshold caused by the production or use of various proteins and enlarging the clinical application of the small molecule short chain polypeptides; furthermore, when the yeast cells with the nucleic acid constructs are used for expressing small molecule short chain polypeptides, the removal process of toxin increase generated by an escherichia coli system can be avoided, and the difficulties of gene expression and transformation can meet the current market with faster expression requirements for various different proteins;

on the other hand, in the preparation process of the biological product for cell-free in-vitro protein synthesis, the nucleic acid construct provided by the invention can be used for obtaining cells of cell extracts for in-vitro protein synthesis in the stage of culturing strains, and the small molecular short-chain antibacterial peptide which is expressed in a large quantity and precisely can be forcefully expressed in the early stage, so that the cultured strains can quickly obtain dominant growth, and the later stage does not compete for the whole resources of the cells, and the whole resources expressed by the cell growth can be better utilized, and meanwhile, the culture residues can be used for obtaining small molecular short-chain products due to the production of high-yield small molecular short-chain polypeptides.

Drawings

FIG. 1 shows the results of an antibacterial test against E.coli in example 1;

FIG. 2 shows the results of the antibacterial test against Lactobacillus acidophilus in example 1.

Detailed Description

Specific embodiments of the present invention are described below with reference to the accompanying drawings. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.

The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

The nucleic acid construct of the present invention is directed to a nucleic acid construct having a structure represented by formula one from 5 'to 3', specifically as follows:

formula one: A-B-C

Wherein a is a first nucleotide sequence encoding a promoter element;

b is a second nucleotide sequence encoding a signal peptide;

c is a third nucleotide sequence encoding a small short chain peptide.

The nucleic acid construct realizes accurate expression of small molecule short-chain peptide at N-terminal.

In the present invention, there is also provided a vector comprising the nucleic acid construct of formula one.

The nucleic acid construct described above, or a cell containing the vector described above, is integrated into the genome of the present invention, and the cell is derived from a prokaryotic cell or a eukaryotic cell.

The expression "in vitro cell-free protein synthesis system" in the present invention has the same meaning as the expression of "in vitro expression system", "in vitro protein synthesis reaction system", "cell-free protein synthesis system", etc., and may be described as a protein in vitro synthesis system, an in vitro protein synthesis system, a cell-free protein synthesis system, an in vitro cell-free synthesis system, a CFS system (cell-free system), a CFPS system (cell-free protein synthesis system), etc. Including in vitro translation systems, in vitro transcription translation systems (IVTT systems, IVTT reaction systems), and the like.

In vitro protein synthesis reaction refers to a reaction for synthesizing a protein in an in vitro cell-free synthesis system, and at least comprises a translation process. Including but not limited to the IVTT reaction (in vitro transcription translation reaction). In the present invention, the IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of in vitro transcription and translation of DNA into Protein, so we also refer to such in vitro Protein synthesis system as D2P system, D-to-P system, D_to_P system, DNAto-Protein system; corresponding in vitro Protein synthesis methods, also known as D2P method, D-to-P method, D_to_P method, DNA-to-Protein method.

In the in vitro cell-free synthesis method of the present invention, the in vitro protein synthesis system, the template, the plasmid, the target protein, the in vitro protein synthesis reaction (incubation reaction), various production methods, various detection methods, and other technical elements of the present invention may be selected, independently of each other, from the following documents, such as, but not limited to, CN111484998A, CN106978349A, CN108535489A, CN108690139A, CN 108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, CN110938649A, CN110964736 a. Unless otherwise contradicted by purpose of the present invention, these documents and their cited documents are incorporated by reference in their entirety for all purposes.

The above-mentioned prokaryotic cells include E.coli cells.

The eukaryotic cells include yeast cells, rabbit reticulocytes, wheat germ cells, insect cells, humanized cells, and the like. The eukaryotic in vitro biosynthesis system has the advantages of being capable of synthesizing RNA or protein with complex structure, post-translational modification of protein and the like.

In the present invention, the in vitro foreign protein synthesis system is not particularly limited, and a preferred in vitro foreign protein synthesis system includes a yeast in vitro biosynthesis system, preferably a yeast in vitro protein synthesis system, preferably a Kluyveromyces lactis expression system, more preferably a Kluyveromyces lactis expression system.

The term "medium" as used herein refers to a substance used for culturing the above-mentioned prokaryotic or eukaryotic cells, and the term "culture remainder" refers to a substance obtained by culturing a strain in a medium to obtain the corresponding cells: the remainder after substantial removal of the precipitate, where the precipitate includes the cells described above.

The small molecule short peptide is also called nano collagen, and the oligopeptide, the micro peptide and the short peptide are also called small molecule peptide. The small molecular peptide is generally formed by combining 2-3 amino acids, and has a smaller average molecular weight of about 300 daltons. The size range of the expressed small molecule short peptide is any one of 15kDa, 1kDa to 15kDa,5kDa-10kDa and 2kDa to 7 kDa.

The expressed small molecule short peptide is a peptide with wide-area antibacterial and antiviral activity, namely, can be used for resisting bacteria and inactivating viruses.

Preferably, the small molecule peptide expressed in the present invention has an anti-gram-negative bacterium, more preferably, gram-positive bacterium is escherichia coli, pseudomonas aeruginosa, proteus, bacillus dysenteriae, bacillus pneumoniae, bacillus buchneri, influenza (haemophilus), parainfluenza (haemophilus), catarrhalis (moraxella), acinetobacter, yersinia, legionella pneumophila, pertussis, b.parapertussis, shigella, bassinense, vibrio cholerae, haemolyticus or shi He Bilin monad; still more preferably, it is resistant to gram-positive bacteria, and even more preferably, gram-positive bacteria are Staphylococcus (Staphylococcus), staphylococcus aureus, streptococcus (Streptococcus), diplococcus pneumoniae, bacillus anthracis, bacillus diphtheriae, bacillus tetani, etc.; common gram-negative bacteria are shigella dysenteriae, typhoid bacillus, proteus or vibrio cholerae; another preferred small molecule short peptide is a peptide having antiviral activity.

The biological product of the invention refers to all products or products produced and prepared by adopting biological correlation as raw materials.

Example 1

This example illustrates the expression of an antimicrobial small molecule short chain peptide.

The strain used in this experiment (experimental strain) for expressing the small short-chain polypeptide was Kluyveromyces lactis (strain NRRL Y-1140), a yeast of Candida globosa, and crabtree negative type.

The experiment uses wide-area antibacterial peptide (small molecule short chain polypeptide) with the coding sequence of SEQ ID NO. 4 as a reporter gene, and the expressed intensity of the wide-area antibacterial peptide is detected by testing antibacterial broth of gram-negative strains (test strains) and gram-positive strains (test strains).

Test strain used in this experiment: the gram-positive strain is Lactobacillus acidophilus (Lactobacillus Acidophilus) and the gram-negative strain is Escherichia coli (Escherichia coli).

(1) Construction of the nucleic acid construct of interest:

gene editing was performed using CRISPR-Cas9 technology.

By means of gene sequence comparison, it is obtained that in the K.lactis genome, the insertion site is located on D chromosome, the gene number is KLLA0D11660g (gene name: CTA 1), and the coding sequence is referred to SEQ ID NO. 5. Three gRNAs were selected for the KLLA0D11660g knockdown to create strand break.

The three gRNAs sequences are gRNA1: AGTATTAGTAGGATGGCCCA (SEQ ID NO: 6), gRNA2: CTTGAGAGTTTGTGACCACA (SEQ ID NO: 7) and gRNA3: CGTGTGGTCACAAACTCTCA (SEQ ID NO: 8).

We constructed gRNA1, gRNA2, gRNA3 and, respectively, on the pCas9pKMCas9 plasmid using the following primers (PF: 5'-3', PR:3 '-5'):

primer pair for gRNA1:

PF，SEQ ID NO:9：

CAGTATTAGTAGGATGGCCCAGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGC；

PR，SEQ ID NO:10：

CTGGGCCATCCTACTAATACTGATTCGAACTGCCGAGAAAG TAACTTTTTTTTATTTG。

primer pair for gRNA2:

PF，SEQ ID NO:11：

CCTTGAGAGTTTGTGACCACAGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGC；

PR，SEQ ID NO:12：

CTGTGGTCACAAACTCTCAAGGATTCGAACTGCCGAGAAA GTAACTTTTTTTTATTTG。

primer pair for gRNA3:

PF，SEQ ID NO:13：

CCGTGTGGTCACAAACTCTCAGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGC；

PR，SEQ ID NO:14：

CTGAGAGTTTGTGACCACACGGATTCGAACTGCCGAGAAA GTAACTTTTTTTTATTTG。

meanwhile, the donor DNA (donor DNA) used was constructed on the pKMD1 plasmid, and the donor DNA contained left and right homology arms HR1 and HR2, promoter (promoter a), signal peptide (signal peptide B), linker (linker D), reporter gene (reporter, small short-chain peptide C), and terminator. HR1 and HR2 were amplified from the K.lactis genome, and other portions of the pKMD1 vector fragment (donor DNA) were also amplified by primers of the following sequences:

promoter-PF，SEQ ID NO:15：

GCAAAATTTTTTTCTAACCTGTCTTTTTCTTTTTTTGCGGTC ACCCCCATGTG；

promoter-PR，SEQ ID NO:16：

GAAAGACAACAAGAACAAGAAAATATAGAAAATATTCATT TTTGATAAGTATTTAAGCG。

signal peptide-PF，SEQ ID NO:17：

GCTTAAATACTTATCAAAAATGAATATTTTCTATATTTTCTTG TTCTTGTTGTCTTTCG；

signal peptide-PR，SEQ ID NO:18：

CTCTTCTATGAGTATGTTCCAAACCTTGAACGAAAGACAAC AAGAACAAGAAAATATAG。

linker-PF，SEQ ID NO:19：

CTTGTTCTTGTTGTCTTTCGTTCAAGGTTTGGAACATACTCA TAGAAGAGGTTCTTTGG；

linker-PR，SEQ ID NO:20：

CTTCTTGAAAACCTTCCACTTTCTTTTATCCAAAGAACCTCT TCTATGAGTATGTTCC。

reporter gene-PF，SEQ ID NO:21：

CTCATAGAAGAGGTTCTTTGGATAAAAGAAAGTGGAAGGT TTTCAAGAAGATTGAAAAG；

reporter gene-PR，SEQ ID NO:22：

GATTAAAATAGAACAACTACAATATAAAAAAAttaAGACAAA ATAGCCTTAGCTTCGCC。

terminator-PF，SEQ ID NO:23：

CTATTTTGTCTtaaTTTTTTTATATTGTAGTTGTTCTATTTTAAT CAAATGTTAGCGTG；

terminator-PR，SEQ ID NO:24：

GAGAGAGAGAAAGAAAGCACAGCTAATTCTCTCAGTATAG CGACCAGCATTCACATAC。

the specific sequences of the obtained parts are respectively corresponding to:

promter (initiation element, first nucleotide sequence): SEQ ID NO. 1;

signal peptide: SEQ ID NO. 2;

reporter gene (reporter gene, small short-chain peptide C): SEQ ID NO. 3;

linker (connection segment D): SEQ ID NO. 4;

terminator：SEQ ID NO:25；

homology arm HR1: SEQ ID NO. 26;

homology arm HR2: SEQ ID NO. 27.

Then Gibson from Transgene Inc. was usedThe Master Mix seamlessly splices the parts. The correctly sequenced fragment was amplified to give a linear donor fragment (nucleic acid construct of interest 1, SEQ ID NO: 28), which was electrotransformed into yeast competent cells together with gRNA1 and gRNA2, screened for G418 resistance, and then screened for P1 (SEQ ID NOID No. 29) and P2 (SEQ ID No. 30) were subjected to PCR identification, and then the full-length sequences of the sites were amplified with P3 (SEQ ID No. 31) and P4 (SEQ ID No. 32) for sequencing, and clones with correct sequencing were subjected to subsequent experiments.

P1：CGAGTCGTATTCTCCCATTATTGTCTGC

P2：CACAGGTTTAAGGGTACAGGGTACG

P3：GCGGCCATTTCCTTTTGATATTGGAGATTAC

P4：CTTCGCATCTGGGCAGATGATG。

Referring to the above construction procedure, a nucleic acid construct of interest 2 was additionally constructed: the nucleic acid construct 2 differs from the nucleic acid construct of interest 1 in that the expressed reporter gene is a nonfunctional peptide chain.

(2) Bacteriostasis test of broth

Culturing and obtaining culture residues: the test selects the strain which is correspondingly treated with the target nucleic acid construct, uses YPD to cultivate for 40 hours, when the OD600 of the bacterial liquid reaches more than 13, the bacterial liquid is centrifugated for obtaining the supernatant by 4000g and 30 minutes, and then the precipitate is removed by a 0.22 micron filter membrane to obtain the sterile supernatant, namely the culture residue;

lactobacillus acidophilus (30 ℃) and E.coli (37 ℃) were treated for 24 hours with the following groups, each with 15 ml centrifuge tubes:

the first group, designated 84, is the nucleic acid construct of interest 1 added using the present test strain;

the second group, 86, is the nucleic acid construct of interest 2 added using the current test strain;

a third group, control NC, which does not contain any modified wild type experimental strain;

and the fourth group, YPD, pure YPD medium.

FIG. 1 shows the results of an antibacterial test against E.coli in example 1;

The results are shown in fig. 1 and 2.

As can be seen from fig. 1 and 2:

(1) During the bacteriostasis for 24 hours, the 86 group, the control NC group and the YPD group have a great trend of increasing gram negative and gram positive along with time change, while the 84 group keeps the bacteriostasis of the negative gram bacteria and the gram positive gram bacteria at a stable level along with time, and the effect reaches 100 percent and nearly 80 percent respectively;

(2) The bacteriostasis effect of the culture medium is eliminated by comparing the 84 and YPD results;

(3) The results caused by the error expression are eliminated by comparing the results of the groups 84 and 86, so that the fact that the small molecule short chain peptide can be accurately expressed after the nucleic acid construct of the invention, namely the target nucleic acid construct 1 is added is also shown;

(4) The result comparison of 84 and wild NC controls eliminates the bacteriostasis effect existing in the non-reconstruction;

(5) The results of this test 84 set demonstrate that the aforementioned nucleic acid construct 1 can achieve precise cleavage of the N-terminus and precise delivery of the position of the small molecule short chain polypeptide.

Therefore, the nucleic acid construct with the formula I provided by the invention has good gram antibacterial effect on negative and positive gram bacteria, and the antibacterial effect is up to 100% and 80% respectively, which indicates that the nucleic acid construct with the formula I provided by the invention can be used for carrying out large-scale and accurate expression and position transportation on target small molecule short-chain polypeptides for a long time.

Example 2

In this example, cells were cultured with K lactis strain in the medium, RNA content was detected and sequenced in the cells of the strain cultured for different periods of time to determine the transcription strength of the gene, and the results are shown in Table 1.

In Table 1, the sequence number 1 is a transcription initiated by the initiation element A according to the present invention. As can be seen from table 1, there is a trend within 24 hours as follows: within 12 hours, it can be seen that the gene of SEQ ID NO. 1 is strongly transcribed compared to the transcription of other genes, indicating that the promoter corresponding to this gene (i.e., the promoter element A involved in the examples of the present invention) is able to strongly transcribe the corresponding gene in the early stage, whereas the transcription intensity of SEQ ID NO. 1 for the gene at 24 hours is less varied than the transcription intensity shown at 12 hours, unlike the 24-hour transcription intensity shown at SEQ ID NO. 2 which is greatly increased compared to 12 hours, and the transcription intensity of SEQ ID NO. 1 has become inferior to that of SEQ ID NO. 2 at 24 hours.

Such a trend in performance as described above brings about several advantages:

(1) The initiation element A can be used for initially (within 12 hours) transcribing the target product, so that colony growth advantages of the strain can be realized: for example, when the small short-chain polypeptide secreted by the expression of the promoter A of the present invention is an antibacterial peptide (target product) and is broad-spectrum antibacterial but harmless to the cultured strain, the strong transcription of the antibacterial peptide can cause the antibacterial peptide to be forcefully expressed and filled in the whole culture medium at the initial stage of the culture, thereby allowing the cultured strain to grow rapidly to obtain population growth advantages;

(2) Compared with other transcription, the transcription of the target product by the starting element A in the later stage (after 12 hours) is not particularly strong any more, and in the preparation process of the biological product outside the target product, the main target can be ensured, and the whole resource in the cell can be prevented from being wasted: for example, when the desired product is the above-mentioned antibacterial peptide, on the one hand, since the cultured strain has obtained population growth advantage, secretion of such antibacterial peptide is not already required, and if the advantageous growth is continued, the above-mentioned whole resource is wasted; on the other hand, if the dominant growth is continued, the above-mentioned overall resources will be contended with the main target product, so that the production of the main target product will be affected.

(3) Thus, when the expression system cells required for producing cell-free protein synthesis are taken as main targets, the expression of the antibacterial peptide is carried out by adopting the promoter such as the promoter element A, so that the production of the expression system cells can be ensured, and the antibacterial peptide with high yield can be obtained, thereby being more beneficial to the use of culture residues for obtaining the antibacterial peptide.

The sequences mentioned previously herein are summarized in Table 1.

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Claims

1. A nucleic acid construct having a structure represented by formula one from 5 'to 3':

formula one: A-B-C, wherein the group consisting of A, B and C,

wherein a is a first nucleotide sequence encoding a promoter element;

b is a second nucleotide sequence encoding a signal peptide;

c is a third nucleotide sequence encoding a small molecule short peptide,

each "-" is independently a bond or a nucleotide linking sequence.

2. The nucleic acid construct of claim 1, wherein:

wherein said A, B is derived from yeast, further wherein said yeast cell is selected from the group consisting of: in another preferred embodiment, the yeast of the genus Kluyveromyces is selected from the group consisting of: kluyveromyces lactis, kluyveromyces marxianus, and a combination of one or more of kluyveromyces polybuyveromyces lactis.

3. The nucleic acid construct of claim 1, wherein:

wherein B and C are linked by a fourth nucleotide sequence encoding a linker segment D comprising an amino acid sequence as shown in any one of SEQ ID NOS.4, 33-35 or having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similarity to any one of SEQ ID NOS.4, 33-35.

4. The nucleic acid construct of claim 1 or 2, wherein:

wherein the signal peptide contains a polypeptide shown as SEQ ID NO. 2 or has a sequence identical to SEQ ID

Amino acid sequence of at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similarity to NO 2.

5. The nucleic acid construct of any one of claims 1-3, wherein: wherein the first nucleotide sequence comprises a nucleotide sequence as set forth in SEQ ID NO. 1 or having at least 50%, 60%, 70%, 80%, 90% or 95% similarity to SEQ ID NO. 1. .

6. The nucleic acid construct of any one of claims 1-4, wherein: wherein the size range of the small molecule short peptide is any one of 15kDa, 1kDa to 15kDa,5kDa-10kDa and 2kDa to 7 kDa.

7. The nucleic acid construct of any one of claims 1-5, wherein: wherein the small molecule short peptide is a peptide with wide-area antibacterial and antiviral activity,

preferably, the small-fraction short peptide has an anti-gram-negative bacterium, more preferably, the gram-positive bacterium is escherichia coli, pseudomonas aeruginosa, proteus, bacillus dysenteriae, pneumobacillus, bacillus, influenza bacillus, parainfluenza bacillus, catarrhalis, acinetobacter, yersinia, legionella pneumophila, pertussis bacillus, parapertussis bacillus, shigella, pasteurella, vibrio cholerae, parahaemolyticus, or shi He Bilin monad;

still more preferably, it is resistant to gram-positive bacteria, and even more preferably, the gram-positive bacteria are staphylococci, staphylococcus aureus, streptococcus, diplococcus pneumoniae, bacillus anthracis, diphtheria bacillus, tetanus bacillus, etc.; common gram-negative bacteria are shigella dysenteriae, typhoid bacillus, proteus or vibrio cholerae;

another preferred small molecule short peptide is a peptide having antiviral activity.

8. The nucleic acid construct of claim 6, wherein:

the small molecule short peptide contains an amino acid sequence as shown in SEQ ID NO. 3 or having at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similarity to SEQ ID NO. 3.

9. A carrier, comprising:

the nucleic acid construct of any one of claims 1-8.

10. A cell, characterized in that:

the nucleic acid construct of any one of claims 1 to 8, or the vector of claim 9, is integrated into the genome of the cell.

11. The cell of claim 10, wherein:

wherein the cell is selected from kluyveromyces lactis, and the locus of integration of the nucleic acid construct in the genome of the cell is: located on the D chromosome, the gene number is KLLA0D11660g.

12. A strain, characterized in that:

for culturing to give a cell according to any one of claims 10-11.

13. A culture residue, characterized in that:

the culture residues are obtained after corresponding cells are obtained by culturing the strain in a culture medium: the remainder after the sediment including the above-mentioned cells is substantially removed,

wherein the strain is the strain of claim 12,

the cell is a cell according to any one of claims 10 to 11.

14. Use of the nucleic acid construct according to any one of claims 1 to 8, the vector according to claim 9, the cell according to any one of claims 10 to 11, the strain according to claim 12 or the cell culture residue according to claim 13, characterized in that:

is used for the production of cell-free in vitro protein synthesis and/or the production of small molecule short peptides.

15. A method of making a biologic comprising:

culturing target strain with culture medium, harvesting to obtain corresponding cells, and/or obtaining culture residue,

wherein the cells obtained are used for cell-free in vitro protein synthesis, the cell culture residues are used for obtaining small molecule short peptide products,

wherein the strain of interest is the strain of claim 12,

the cell is a cell according to any one of claims 9 to 10;

the culture residue is the culture residue according to claim 13.