阅读说明：本技术 一种诱导细菌变形的方法及其应用 (Method for inducing bacterial deformation and application thereof ) 是由 陈力 王蕾 邹琳 于 2018-07-25 设计创作，主要内容包括：本发明属生物技术领域,涉及改变细菌细胞形态的方法,具体涉及一种恒间隔短回文重复序列相关蛋白2(简称Cas2序列表达蛋白)序列诱导细菌细胞形变的方法及其在用于提高细菌适应性等方面的应用。本发明包括,取编码Cas2蛋白的序列连接到载体上得到重组载体；取重组载体,转化细胞,进行蛋白表达,进而促使杆状细胞发生丝状形变。本发明方法利用源自于细菌的Cas2序列改变了细菌的形态,并且发现Cas2序列能有效增加细菌的适应性,为药物筛选提供了新的分子、条件和方法,具有应用前景。(The invention belongs to the technical field of biology, relates to a method for changing the morphology of bacterial cells, and particularly relates to a method for inducing bacterial cell deformation by a constant interval short palindromic repeat (Cas2) related protein 2 (Cas2 sequence expression protein for short) sequence and application thereof in aspects of improving bacterial adaptability and the like. The method comprises the steps of connecting a Cas2 protein coding sequence to a vector to obtain a recombinant vector; and (3) taking the recombinant vector, transforming the cell, and expressing the protein, thereby promoting the rod-shaped cell to generate filamentous deformation. The method changes the form of the bacteria by using the Cas2 sequence derived from the bacteria, and finds that the Cas2 sequence can effectively increase the adaptability of the bacteria, provides new molecules, conditions and methods for drug screening, and has application prospect.)

The application of the Cas2 protein and derivative polypeptide thereof in preparing preparations for changing the cell morphology of bacteria, wherein the derivative protein is a polypeptide sequence of the amino acid sequence of the Cas2 protein shown as SEQ ID NO.1 through substitution, deletion or addition of one or more amino acids.

2. The use of claim 1, wherein the Cas2 protein causes bacterial cells to transform rod-like into filamentous.

3. The use of claim 1, wherein the Cas2 protein is an elizasa meningitidis Cas2 protein (Cas2Em), and the Cas gene sequence is isolated from the elizasa meningitidis genome.

4. The use according to claim 1, wherein the bacterium is e.

5. The use of claim 1, wherein the Cas2Em protein induces increased bacterial adaptation.

6. The use of claim 3, wherein the Cas2Em protein is any polypeptide between 30-101 amino acids and derivative sequences.

7. A method of altering the morphology of a bacterial cell comprising the steps of:

step 1), connecting a DNA sequence encoding Cas2Em protein to a vector to obtain a recombinant vector;

and 2) taking the obtained recombinant vector, transfecting cells, and carrying out protein expression so as to change the morphology of the cells.

8. The method of claim 7, wherein the recombinant vector comprises a 30 amino acid functional domain sequence encoding the C-terminus of Cas2Em protein.

9. The method of claim 7, wherein the recombinant vector is constructed by ligating additional sequences N-terminal or C-terminal to the sequence encoding Cas2 protein.

10. The method of claim 8, wherein the sequence of the domain in the recombinant vector is a sequence encoding a domain of Cas2Em protein 30-101 aa.

11. A protein or fusion protein expressed from the recombinant vector of claim 8.

12. Use of the method of claim 7 for increasing the adaptation of a cell.

Technical Field

The invention belongs to the technical field of biology, relates to a method for changing the morphology of bacterial cells, and particularly relates to a method for inducing bacterial cell deformation by a constant-spacing Short palindromic repeat (CRISPR) associated protein 2 (Cas)2, which is called as Cas2 sequence expression protein for Short) sequence and application thereof in the aspects of improving bacterial adaptability and the like.

Background

The literature states that the basic morphology of bacterial cells is stable, and that the basic morphology of bacteria is a central feature for classification and characterization of bacteria. Studies have shown that the basic morphology of bacteria is altered in the presence of certain molecules, which are capable of inducing changes in the basic morphology of bacterial cells, and may be referred to as cell deformers. Research reports that the bacterial cell deformation agent has good application value, wherein a part of the bacterial cell deformation agent becomes a lead compound for drug development, for example, Rosenberg and the like found in 1967 show that cisplatin in an electric field can induce rod-shaped bacteria to generate filamentous deformation and is the bacterial cell deformation agent, and the finding lays a foundation for the development of cisplatin as an anti-tumor drug; it has also been reported that bacterial deformation of treated cells occurs when the cells are treated with antibiotic drugs acting on different targets under specific conditions [ Bos, j., et al, where antibiotic resistance from multinuclear bacterial membranes. proc Natl Acadsi U S A,2015.112(1): p. 178-83 ].

Polypeptide deformers were first reported in Delumen et al 1999. The research team found that expression of the plant polypeptide Lunasin in bacteria induces filamentous deformation of rod-shaped bacteria [ Galvez, A.F. and B.O. de Lumen, Asoybean cDNA encoding a chromium-binding peptide inhibitors biology of mammalian cells. Nat Biotechnol,1999.17(5): p.495-500 ], which has potential anti-tumor effect.

Cas2 protein is a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated protein, and the CRISPR-Cas system is a system which has been found in recent years to have close relationship in controlling bacterial lateral gene Transfer and can effectively defend against the horizontal Transfer of genes [ Barrangou, R., et al., CRISPR precursors acquired resistance genes in prokaryotes.science,2007.315(5819): p.1709-12. Marraffini, L.A.and E.J.Sonthheimer, CRITnterground Limits horizotalGene Transfer staphylococci by Targeting DNA science,2008.322(5909): p.1843. 1845 ]. The system is widely existed in archaea and bacterial genomes, provides a mechanism for resisting exogenous DNA invasion for bacterial cells, and researches show that Cas2 plays an important role in the immune acquisition process.

The elizabetha meningitidis (Em) is a clinical conditional pathogen, can cause symptoms such as neonatal meningitis, pneumonia, septicemia and the like, is clinically manifested as multi-drug resistance, and has been researched to discover a II-C type CRISPR-Cas system in a genome sequence of the bacteria standard, wherein Cas2Em protein is derived from the CRISPR-Cas system of the Em bacteria. Clinical studies have found that bacteria undergo changes in their basic morphology in vivo to resist attack by the immune system and drugs.

disclosure of Invention

The invention aims to provide a novel method for changing the cell morphology of bacteria aiming at the limitations of the bacterial deformation technology in the prior art, and particularly relates to a method for inducing bacterial deformation by a Cas2 sequence and application thereof. The Cas2 sequence disclosed by the invention not only induces bacterial deformation, but also can improve the strain capacity of bacteria, and has a wide application prospect.

Based on the deformation observation of bacteria without and after antibiotics in clinical research and the correlation between deformation and pathogenicity and drug resistance of the bacteria, as an extension of the experimental thought, Cas2 protein expression bacteria are used as experimental objects, a screening scheme aiming at deformation bacteria antibiotics is screened in a view, and inhibitors are replaced by candidate drugs of novel antibiotics;

experiments show that the Cas2 protein and the derivative short peptide expressed in bacteria can induce the bacteria to deform and improve the adaptability of the bacteria. Compared with the plant polypeptide deforming agent, the bacterial deforming agent has species and structure difference, and can be used for preparing a novel polypeptide bacterial deforming agent. The invention utilizes the bacterial cell deforming agent Cas2Em to change the form of cells and improve the adaptability of the cells, provides a new way for the research and development of novel drug targets and novel drug-resistant bacteria drugs, and has wide practicability.

The invention is realized by the following technical scheme:

connecting a sequence encoding Cas2 protein to a vector to obtain a recombinant vector; and (3) taking the recombinant vector, transforming the cell, and expressing the protein, thereby promoting the rod-shaped cell to generate filamentous deformation. The invention changes the form of bacteria by using the Cas2 sequence derived from the bacteria, and finds that the Cas2 sequence can effectively increase the adaptability of the bacteria, provides new molecules, conditions and methods for drug screening, and has application prospect.

Specifically, the invention provides a method for changing the cell morphology of bacteria, in particular to a method for inducing the change of the bacterial morphology by a Cas2 sequence, which comprises the following steps:

step 1), connecting a DNA sequence encoding Cas2Em protein to a vector to obtain a recombinant vector;

and 2) taking the obtained recombinant vector, transfecting cells, and carrying out protein expression so as to change the morphology of the cells.

Preferably, the sequence encoding the Cas2 protein is isolated in the genome of elizabetha meningitidis.

Preferably, the vector is pET-28a (+).

Preferably, the bacterium used to express the recombinant plasmid is e.

Preferably, the bacteria used to express Cas2Em protein are e.colibl21(DE3) and e.colibl21-AI.

In the invention, the recombinant vector comprises a 30 amino acid functional domain sequence capable of encoding the C end of the Cas2Em protein; the functional domain sequence in the recombinant vector is a sequence encoding the functional domain of Cas2Em protein 30-101 aa.

In the invention, the recombinant vector is constructed by connecting other sequences to the N end or the C end of the sequence for encoding the Cas2 protein.

The Cas2 protein described in the present invention causes bacterial cells to transform from rod-like to filamentous.

The Cas2 protein is an Elizabalanopsis meningitidis Cas2 protein (Cas2Em), and the Cas gene sequence is obtained by separating from an Elizabeth meningitidis genome.

The bacteria of the invention are Escherichia coli E.coli BL21(DE3), E.coli BL21-AI and the like.

Preferably, the bacterium is e.

The invention also provides a protein expressed by the recombinant vector in the method and a derivative protein or a fusion protein.

The invention provides a Cas2 protein and application of derivative polypeptide thereof in preparing a preparation for changing bacterial cell morphology, wherein the derivative protein is a polypeptide sequence of a Cas2 protein amino acid sequence shown as SEQ ID NO.1 through substitution, deletion or addition of one or more amino acids.

Preferably, the use is of Cas2 protein in inducing normal bacterial deformation.

Preferably, the Cas2 protein is a Cas2Em protein.

In the present invention, the Cas2Em protein includes any polypeptide between 30-101 amino acids and derivative sequences.

In the invention, the Cas2Em protein induces increased bacterial adaptation.

Experiments show that after Cas2Em is induced and expressed in escherichia coli BL21(DE3) and BL21-AI, the bacterial morphology is changed from short rod shape to long wire shape, and after the analysis of a fluorescence microscope, the expression of Cas2Em in bacteria does not influence the replication and division of nucleic acid, but can block the normal division of cell membranes; analysis of the functional domain of Cas2Em showed that filamentous deformation could be mediated by a short 30aa peptide at the carboxy terminus (C-segment); analysis of Cas2 proteins derived from different types of CRISPR-Cas systems shows that there is some conservation in the filamentous deformation mediated by Cas2, which is unique to Cas2 protein of type ii CRISPR.

According to the invention, the Cas2 sequence exists in prokaryotic cells, the form of bacteria is changed, the adaptability of the bacteria is effectively increased, and the Cas2 sequence is prompted to have application prospects in the fields of industry, environment, research and development of novel antibiotics and antitumor drugs. Compared with the prior art, the invention has the following characteristics: according to the invention, the Cas2 sequence existing in the elizabeth meningitis and septicemia is utilized to change the cell morphology of the bacteria; the method can improve the adaptability of bacteria, and has wide application prospect in the fields of industrial and environmental microorganism and drug research and development.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1: PET28a-Cas2Em expression mediates the production of filamentous deformations of e.coli BL21(DE3) bacterial cells;

wherein, the result of an optical microscope expressing no-load (control) and Cas2Em protein (Cas2) is IPTG induction, BL21(DE3) host bacteria scale is 25 μm;

FIG. 2: PET28a-Cas2Em expression mediates the growth of filamentous shape change of e.coli BL21-AI bacterial cells;

wherein, the result of the optical microscope expressing no-load (control) and Cas2Em protein (Cas2) is IPTG induction, BL21-AI host bacteria scale is 25 μm;

FIG. 3: the result of the truncated mutant of the Cas2Em protein mediating the generation of long-filamentous deformation of the Escherichia coli cells;

a summary of the phenotypic results of 5N-terminal truncation mutations of Cas2Em protein is shown;

FIG. 4: the result of the Cas2 protein from different CRISPR types mediating the generation of long-thread shape change of escherichia coli cells;

among these, the phenotypic results for different types of Cas2 proteins: neisseria meningitidis (Nm: Neisseria meningitidis) Type II, Elizabethiia meningitidis (Em: Elizabethikingia meningitidis) Type II, Escherichia coli (Ec: Escherichia coli) Type I, and Mycobacterium tuberculosis (Tb: Mycobacterium tuberculosis) Type III);

as shown in fig. 1 and 2, PET28a-Cas2Em expression mediates filamentous deformation of e.coli BL21(DE3) and e.coli BL21-AI bacterial cells; the truncation mutation experiment shown in fig. 3 shows that a short peptide of 30 amino acids at the C-terminal end of the Cas2 protein is sufficient to mediate the bacterial cell to generate long filamentous deformation; the results shown in fig. 4 demonstrate that the function of inducing bacterial filamentous deformation is unique to Cas2 protein of type ii CRISPR-Cas system.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.