阅读说明：本技术 一种能胞外分泌抗菌肽的衣藻的构建方法及其应用 (Construction method and application of chlamydomonas capable of extracellularly secreting antibacterial peptide ) 是由 王潮岗 李安国 胡章立 于 2019-10-30 设计创作，主要内容包括：本发明属于衣藻基因工程技术领域,尤其涉及一种能胞外分泌抗菌肽的衣藻的构建方法及其应用。本发明将莱茵衣藻碳酸酐酶信号肽和抗菌肽在莱茵衣藻中进行融合表达,获得的转基因衣藻能够胞外分泌抗菌肽,并且分泌的抗菌肽能够有效地抑制海水微生物的生长,减少抗生素的使用,显示其在水产养殖方面具有潜在的应用前景。衣藻构建过程包括：构建质粒载体并提取质粒DNA,所述质粒载体的构建主要为将碳酸酐酶信号肽的编码序列和抗菌肽ALFPm3/ALFPm11基因融合并克隆到载体质粒中；酶切质粒DNA；将酶切后的DNA分子转入基因受体,培养,得到遗传转化子。(The invention belongs to the technical field of chlamydomonas genetic engineering, and particularly relates to a construction method and application of chlamydomonas capable of extracellularly secreting antibacterial peptide. According to the invention, the Chlamydomonas reinhardtii carbonic anhydrase signal peptide and the antibacterial peptide are subjected to fusion expression in Chlamydomonas reinhardtii, the obtained transgenic Chlamydomonas reinhardtii can secrete the antibacterial peptide extracellularly, and the secreted antibacterial peptide can effectively inhibit the growth of seawater microorganisms, reduce the use of antibiotics, and show that the transgenic Chlamydomonas reinhardtii has a potential application prospect in the aspect of aquaculture. The construction process of the chlamydomonas comprises the following steps: constructing a plasmid vector and extracting plasmid DNA, wherein the construction of the plasmid vector mainly comprises fusing and cloning a coding sequence of carbonic anhydrase signal peptide and antibacterial peptide ALFPm3/ALFPm11 genes into a vector plasmid; carrying out enzyme digestion on plasmid DNA; transferring the DNA molecule after enzyme digestion into a gene receptor, and culturing to obtain a genetic transformant.)

1. A construction method of Chlamydomonas capable of extracellularly secreting antibacterial peptide is characterized by comprising the following steps:

step 1: constructing a signal peptide-antibacterial peptide chlamydomonas expression vector, connecting the signal peptide with an antibacterial peptide sequence, synthesizing and then connecting the signal peptide and the antibacterial peptide sequence to the vector to obtain an expression vector;

step 2: transforming the plasmid DNA of the expression vector to a transgenic receptor strain to obtain a genetic transformant;

and step 3: and detecting the genome DNA of the genetic transformant, and analyzing the transcription activity of the antibacterial peptide to obtain the transgenic alga capable of stably inheriting.

2. The method of claim 1, wherein the method comprises the steps of: the amino acid sequence of the signal peptide in the step 1 is shown in SEQ ID NO.1, and the gene sequence of the signal peptide expressed in Chlamydomonas reinhardtii is shown in SEQ ID NO. 2.

3. The method of claim 1, wherein the method comprises the steps of: in the step 1, the antibacterial peptide is an anti-lipopolysaccharide factor.

4. The method of claim 3, wherein the method comprises the steps of: the amino acid sequence of the anti-lipopolysaccharide factor is shown in SEQ ID NO.3 or SEQ ID NO. 8.

5. The method of claim 1, wherein the method comprises the steps of: the vectors involved in the construction of the expression vector in step 1 include the pH105 vector and the pSP124 vector.

6. The method of claim 1, wherein the method comprises the steps of: and (3) the transgenic receptor algae strain in the step 2 is cell wall-deficient Chlamydomonas reinhardtii.

7. The method of claim 1, wherein the method comprises the steps of: and 2, transforming the plasmid DNA of the expression vector to a transgenic receptor strain by adopting a bead milling method.

8. Use of a transgenic chlamydomonas constructed by the method for constructing a chlamydomonas capable of extracellular secretion of an antimicrobial peptide according to any one of claims 1 to 7 for inhibiting the growth of a microorganism in seawater.

9. The use of a chlamydomonas capable of extracellular secretion of an antimicrobial peptide according to claim 8 for inhibiting the growth of a marine microorganism, wherein: the application shows an inhibition effect on bacillus alginolyticus and/or vibrio parahaemolyticus.

Technical Field

The invention belongs to the technical field of microalgae genetic engineering, and particularly relates to a construction method and application of chlamydomonas capable of extracellularly secreting antibacterial peptide.

Background

The antibacterial peptide is a general name of peptides and small molecular proteins which are widely present in various organisms and have the function of inhibiting or killing pathogenic microorganisms. Researches show that the antibacterial peptide can effectively kill pathogenic microorganisms, viruses, pathogenic protozoa and the like. In recent years, the abuse of antibiotics causes the appearance of superbacteria, and due to the characteristics of small molecular weight, wide antibacterial spectrum, brand new antibacterial mechanism and the like, the antibacterial peptide increasingly becomes a new compound which is expected to replace antibiotics. Among them, anti-lipopolysaccharide factors (ALFs) are an antimicrobial peptide in crustaceans, which were first identified from two kinds of limulus hemolymphocytes, and generally consist of 98-123 amino acids, including a signal peptide sequence and a lipopolysaccharide binding domain, and secondary structures consisting of a plurality of α -helices and β -sheets. The LBD domain is a functional region of ALF, consisting of 22 amino acid residues, containing a pair of conserved cysteines forming a disulfide bond. ALF is an antibacterial peptide with broad-spectrum antiviral, antibacterial and antifungal activities, and has important application potential in industries such as aquaculture and the like. However, the content of the antibacterial peptide in the nature is low, the extraction is difficult, and the synthesis by the genetic engineering technology is a feasible method.

Currently, the heterologous expression of antibacterial peptides is mainly an escherichia coli expression system and a yeast expression system.

The colibacillus expression system is the expression system established for expressing heterologous genes at the earliest time and has the advantages of high expression level, simple operation, low cost and the like. Currently, antibacterial peptides are mainly obtained by recombinant expression of prokaryotic escherichia coli systems. The molecular weight of the antibacterial peptide is small, and the antibacterial peptide can be heterologously expressed by cloning the antibacterial peptide coding gene onto an expression vector and converting the antibacterial peptide coding gene into escherichia coli. Escherichia coli grows rapidly and is easy to operate, and only IPTG (isopropyl-beta-thiogalactoside) is needed to be added for induction, so that the escherichia coli is widely used as a receptor cell for heterologous expression of antibacterial peptides. However, Escherichia coli contains a large amount of endotoxin capable of causing immune response, and the endotoxin can affect the separation and purification of antibacterial peptide; and the Escherichia coli is a prokaryotic expression system, lacks biological processes of folding, modifying and processing eukaryotic protein, and is easy to form inclusion bodies; since antimicrobial peptides may have a killing effect on E.coli, many antimicrobial peptides cannot be expressed by E.coli. Therefore, the recombinant antibacterial peptide obtained by the prokaryotic expression system is low in activity and cytotoxic. Yeast belongs to eukaryotic microorganisms, and is one of the microorganisms that have been used for production by humans for the first time. It has enzyme and organelle needed by protein post-translational modification, and makes up the shortage that the protein can not be modified in the colibacillus expression system. Thus, yeast is also frequently used as a host cell for the heterologous production of antimicrobial peptides. Research shows that the codon optimized fungal defensin gene is cloned to a corresponding expression vector and electrically transformed to a pichia pastoris cell, and intracellular expression, secretion expression and fusion expression of the protein in yeast can be realized. The 96-well plate bacteriostasis experiment proves that the expressed antibacterial peptide has the capacity of resisting staphylococcus aureus (gram positive bacteria), escherichia coli and vibrio parahaemolyticus (gram negative bacteria). However, yeast expression systems have a significant problem in that they are prone to excessive glycosylation of proteins, which can have a major effect on the activity of the antimicrobial peptides; the yeast expression system needs to solve another problem that dissolved oxygen is insufficient due to mass growth of thalli during high-density fermentation, so that proliferation is inhibited, the expression level of the antibacterial peptide is reduced, difficulty is caused in separation and purification, and the separated antibacterial peptide lacks biological activity; meanwhile, the yeast expression level is low, and the subsequent separation and purification cost is high, which is not beneficial to industrial application. The results of the prior studies also suggest that: both escherichia coli expression systems and yeast expression systems need heterotrophic fermentation, consume a large amount of culture medium, oxygen and electric energy, and cause environmental pollution.

Meanwhile, Chlamydomonas reinhardtii, as a unicellular eukaryotic green alga, is the only eukaryotic expression system that can perform transformation of cell nucleus, mitochondria and chloroplast and express heterologous proteins.Three genomes of the mutant strain are sequenced, hundreds of thousands of mutant strains exist, and the genetic background is clear. Chlamydomonas reinhardtii has post-translational processing of eukaryotic proteins, can correctly express heterologous proteins, has glycosylation close to that of human, and has no endotoxin. The Chlamydomonas reinhardtii has high growth speed, can perform photosynthesis, and can absorb CO₂And the product is synthesized, the culture cost is low and the environment is protected. The chlamydomonas reinhardtii can also be fermented and cultured by utilizing an optical bioreactor, the culture technology is mature, no farmland is occupied, and the cost is low. Compared with an escherichia coli and yeast expression system, the chlamydomonas reinhardtii can also be used as bait for aquatic organisms such as fish, shrimps and the like, can also be cultured in a closed culture pond, and has important application potential in the aquaculture industry.

Genetic transformation of chlamydomonas reinhardtii chloroplasts, mitochondria and nuclei has been accomplished today. Various proteins have been successfully expressed in chlamydomonas chloroplasts as reported in the literature, including fusion antigens, monoclonal antibodies, cytokines, vaccines, and the like. Mitochondrial expression systems have not been reported to utilize mitochondria to produce pharmaceutical proteins due to the problems of heterogeneity of genetic transformants, problems with screening methods, problems with expression regulation, and the like. The nuclear genome is the most used and technically mature system in the chlamydomonas expression system. Rouholah Barahimipour et al expressed the HIVP24 gene in Chlamydomonas, and the expression level of the recombinant protein was 0.25% of the total protein. The expression of erythropoietin in Chlamydomonas nuclei is up to 100. mu.g/L by Alke Eichler-Stahlberg et al. The plum-Zhefang gene and the like connect 3 copies of the same antibacterial peptide gene Bacteriocin LS2 in series, realize intracellular expression in Chlamydomonas reinhardtii, the recombinant Bacteriocin LS2 protein accounts for 0.29% of soluble protein, and show strong antibacterial activity on salmonella, Escherichia coli (gram-negative bacteria), Bacillus subtilis and Listeria monocytogenes (gram-positive bacteria) through antibacterial activity analysis. In conclusion, the chlamydomonas reinhardtii can solve the problems of low activity, endotoxin and the like existing in escherichia coli expression, and avoid the problems of excessive glycosylation, high cost and the like existing in yeast expression. However, Chlamydomonas reinhardtii expression also has its own problems, such as complicated process for separating and purifying the expression product.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a construction method and application of chlamydomonas capable of extracellularly secreting antibacterial peptide. The invention adopts a secretion type expression system to express the antibacterial peptide outside cells, thereby avoiding product inhibition; an inducible promoter is adopted to improve the expression quantity of the product; target protein is recovered through the culture medium, and the protein can be used for production through simple purification, so that the cost is low; the pathogenic bacteria are killed by the culture in the closed culture pond, and a healthy environment is provided for aquaculture.

The invention is realized in such a way that the construction method of the chlamydomonas capable of extracellularly secreting the antibacterial peptide comprises the following steps:

step 1: constructing a signal peptide-antibacterial peptide chlamydomonas expression vector, connecting the signal peptide with an antibacterial peptide sequence, synthesizing and then connecting the signal peptide and the antibacterial peptide sequence to the vector to obtain an expression vector;

step 2: transforming the plasmid DNA of the expression vector to a transgenic receptor strain to obtain a genetic transformant;

and step 3: and detecting the genome DNA of the genetic transformant, and analyzing the transcription activity of the antibacterial peptide to obtain the transgenic alga capable of stably inheriting.

The invention can secrete the antibacterial peptide expressed in cells to the outside of the cells, and relates to a signal peptide (S-peptide) expressed by extracellular secretion, wherein the amino acid sequence of the signal peptide is as follows: MARTGALLLVALALAGCAQA, see SEQ ID NO.1, the gene expression sequence in Chlamydomonas reinhardtii is: atggcgcgtactggcgctctactcctggtcgcgctggcgcttgcgggctgcgcgcaggct, see SEQ ID NO. 2.

The invention constructs a plasmid vector, which is mainly characterized in that a coding sequence of a signal peptide (S-peptide) and a coding sequence of an antibacterial peptide gene are fused, a fusion sequence of the signal peptide and the antibacterial peptide is obtained by a gene synthesis technology, and then the fusion sequence is cloned into a vector plasmid. Wherein, the antibacterial peptide demonstrated by the research is the prawn pelagic acid shrimp antibacterial peptide ALFPm3, the amino acid sequence is shown in SEQ ID NO.3, and the gene optimization sequence expressed in the chlamydomonas is SEQ ID NO. 4; obtaining a sequence of a signal peptide-antibacterial peptide fusion gene by double enzyme digestion vector plasmids, inserting the sequence into a Chlamydomonas expression vector pH124 with a light induction or heat shock induction promoter, and successfully constructing a Chlamydomonas expression vector; the expression vector with the signal peptide-antibacterial peptide fusion gene is introduced into the chlamydomonas genome for expression, and genetic transformation is carried out by a bead milling method.

Further, the antibacterial peptide in the step 1 is an anti-lipopolysaccharide factor.

Further, vectors involved in the construction of the expression vector in step 1 include the pH105 vector and the pSP124 vector, which are purchased from the center of Chlamydomonas USA (https:// www.chlamycollection.org).

Further, the transgenic recipient strain in step 2 was cell wall-deficient Chlamydomonas reinhardtii, purchased from the American Chlamydomonas center (https:// www.chlamycollection.org).

Further, primers used for PCR amplification detection of the genomic DNA of the genetic transformant in the step 3 are CAH-F and All-R respectively, and the sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively.

The application of the transgenic chlamydomonas constructed by the construction method of the chlamydomonas capable of extracellularly secreting the antibacterial peptide in inhibiting the growth of seawater microorganisms.

Further, the application shows an inhibitory effect on Bacillus alginolyticus and Vibrio parahaemolyticus.

Further, step 3 is followed by the addition of: detecting the expression of the antibacterial peptide, culturing the transgenic algae to a logarithmic phase, applying strong light or heat shock to increase the expression quantity of the antibacterial peptide, concentrating a culture medium to obtain the antibacterial peptide, and identifying the expression quantity of the antibacterial peptide through protein hybridization; the antibacterial peptide is secreted into a culture medium and is obtained by centrifugation and freeze drying.

Further, the expression of antibacterial peptide ALFPm3 or ALFPm11 is induced by a secondary heat shock induction method.

In summary, the advantages and positive effects of the invention are:

the invention fuses the coding sequence of the carbonic anhydrase signal peptide and the ALFPm3 or ALFPm11 gene and clones the signal peptide to the pH124 vector according to the codon preference of Chlamydomonas reinhardtii. The Chlamydomonas reinhardtii cell wall defective strain CC-849 is transformed by a bead mill method and screened by Zeocin. Then, the ALFPm3 or ALFPm11 protein is induced and expressed by a secondary heat shock induction method. The crude extract containing ALFPm3 or ALFPm11 protein can be obtained by centrifuging and separating algae cells, and freeze drying. The transgenic chlamydomonas constructed by the invention can secrete the antibacterial peptide into a culture medium, namely the obtained antibacterial peptide is extracellularly secreted antibacterial peptide, is convenient to separate and purify and has higher purity. And the secreted antibacterial peptide can effectively inhibit the growth of seawater microorganisms, reduce the use of antibiotics and show that the antibacterial peptide has potential application prospects in the aspect of aquaculture.

The method for producing the antibacterial peptide by using the chlamydomonas as the host cell has the main advantages that ① genetic transformation is convenient to operate, various mutant strains are obtained for molecular biology research, ② has enzyme and organelle which can modify the antibacterial peptide, and the antibacterial peptide can be secreted into a culture medium, the separation and purification steps are simplified, ③ the chlamydomonas is convenient to culture, not only can be photoautotrophic, but also can be chemoheterotrophic, and the culture medium is cheap and easy to obtain.

Drawings

FIG. 1 is a schematic diagram of the pH-S-ALF3 plasmid structure;

FIG. 2 is CAH transformants on TAP plates;

FIG. 3 is the identification of transformants by PCR;

FIG. 4 is a transcriptional activity analysis of CAH transformants;

FIG. 5 is a Western Blot analysis of antimicrobial peptides, 1: negative control; 2: a positive control; 3: CAH-3; 3: CAH-2; 3: CAH-1;

fig. 6 is a graph of the inhibition of bacterial growth by antimicrobial peptide ALFPm3, wherein a-SIV: ampicillin kills vibrio parahaemolyticus; coli: ampicillin kills escherichia coli; algicola: ampicillin kills algae-inhabiting bacillus; pm 3-SIV: ALFPm3 kills Vibrio parahaemolyticus; pm 3-E.coli: ALFPm3 can kill Escherichia coli; pm3-B. algicola: ALFPm3 Bacillus algicidal;

fig. 7 is the inhibition of bacterial growth by antimicrobial peptide ALFPm11, where Amp: ampicillin; pm 11: ALFPm 11; PBS: phosphate buffer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses a construction method and application of chlamydomonas capable of extracellularly secreting antibacterial peptide, which are shown in the following embodiments.