Non-replicating recombinant bacteriophage packaging plasmid and application thereof

Abstract

The invention provides a non-replicating recombinant bacteriophage packaging plasmid and application thereof, and particularly relates to the non-replicating recombinant bacteriophage packaging plasmid and a method for preparing ssDNA and mcDNA, the plasmid comprises a starting sequence from a bacteriophage replication origin, a target sequence and a terminating sequence from the bacteriophage replication origin, and the target sequence and the terminating sequence are in sequence from 5'to 3 '. The initial sequence at least comprises a sequence as shown in the x-v site in SEQ ID NO: 1, the termination sequence at least comprises sequences as shown in the 1-y site and the z-w site in SEQ ID NO: 2, x is smaller than or equal to 291, v is larger than or equal to 329 and smaller than or equal to 381, and 67 is smaller than or equal to result; z < = 279, 312 < = w < = 330. Compared with the original full-length sequence, the non-replicating recombinant bacteriophage packaging plasmid has at least one segment of deletion in the initial sequence and / or the termination sequence of the replication origin, and can still generate a circular single-stranded DNA containing a target sequence and a deleted bacteriophage replication origin sequence; therefore, a safer single-stranded DNA donor containing fewer non-target sequences is provided for downstream applications such as gene editing, ssDNA and mcDNA preparation and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a non-replicating recombinant phage packaging plasmid and an application method for preparing ssDNA and mcDNA based on the non-replicating recombinant phage. Background Art

[0002] Single-stranded DNA is considered as a biomaterial with great application potential in many biological reactions and has a wide range of applications in DNA nanotechnology. However, due to the limitations of chemical synthesis methods, the synthesis of long single-stranded DNA is difficult to ensure yield, output and satisfactory cost-effectiveness. Therefore, long single-stranded DNA needs to be prepared with the help of biological enzymes in vivo or in vitro and some auxiliary denaturation methods. Currently, the commonly used methods for preparing long single-stranded DNA mainly include reverse transcription, enzyme degradation, denaturing high-performance liquid chromatography (HPLC), magnetic bead biotin modification, asymmetric PCR, RCA, etc. However, in practical applications, these methods have problems such as low yield and high cost.

[0003] The helper phage method is a relatively new method for preparing single-stranded DNA. The basic principle is to construct a plasmid containing the M13 replication origin (M13 ori) or the f1 replication origin (f1 ori), transfer it into a host cell containing the F factor, and then infect it with a defective helper phage. The helper phage can help the plasmid form single-stranded DNA and encapsulate it into a phage, which is secreted out of the host cell. This method has low cost and high yield, but the single-stranded DNA it forms contains a necessary 2-3 nt M13ori / f1ori conserved sequence.

[0004] It has been reported that pUC18 can be converted into a recombinant phage packaging plasmid for producing custom single-stranded DNA by adding four components: wild-type M13 ori as the start sequence (M13 ori start), restriction sites, M13 PS for phage particle output, and mutant M13 ori as the termination sequence (M13 ori terminator). The resulting single-stranded circular DNA still contains 381 bases of the wild-type M13 ori conservative sequence (Nafisi, Parsa M.; Aksel, Tural; Douglas, Shawn M. Synthetic Biology (Oxford, United Kingdom) (2018), 3(1), ysy015). However, how to further shorten the length of the conservative sequence remains an urgent problem to be solved.

[0005] All the above-mentioned documents are fully introduced into this paper. Summary of the invention

[0006] The present inventors have found that after certain deletions of the starting sequence (M13 ori start) and the terminating sequence (M13 ori terminator) on the recombinant phage packaging plasmid, circular single-stranded DNA containing the target sequence and the phage origin of replication (ori) sequence can still be generated. This can provide a safer single-stranded DNA donor with fewer non-target sequences for downstream applications such as gene editing and the preparation of minicircle DNA (mcDNA). Moreover, the length of the target sequence can reach several thousand nt. The resulting circular single-stranded DNA has a shorter conserved sequence than the traditional full-length phage origin of replication sequence, which can reduce the potential impact of the phage origin of replication as a backbone sequence on the target sequence. Specifically, the present invention includes the following: On the one hand, the present invention provides a recombinant phage packaging plasmid, which contains a starting sequence (M13ori start), a target sequence (GOI), and a terminating sequence (M13 ori terminator). In the order from 5' to 3', the starting sequence at least contains the sequence shown at positions x-v in SEQ ID NO:1, and the terminating sequence at least contains the sequences shown at positions 1-y and z-w in SEQ ID NO:2, where x ≤ 291, 329 ≤ v ≤ 381, 67 ≤ y < z ≤ 279, 312 ≤ w ≤ 330, x, y, z, v, w are all positive integers, and the starting sequence is not SEQ ID NO:1 and / or the terminating sequence is not SEQ ID NO:2. The sequence shown in SEQ ID NO:1 is the wild-type M13 ori sequence with a length of 381 nt, and the sequence shown in SEQ ID NO:2 is the mutant M13 ori sequence with a length of 330 nt. Moreover, the sequence shown at positions 1-319 in SEQ ID NO:2 is identical to the sequence shown at positions 1-319 in SEQ ID NO:1. The starting sequence and the terminating sequence provided by the present invention, at least one of which has a sequence inconsistent with SEQ ID NO:1 or SEQ ID NO:2, that is, at least one has a deletion compared to the full-length starting sequence or terminating sequence, but can still generate circular single-stranded DNA containing the target sequence.

[0007] In some embodiments, the start site and / or the stop site of the recombinant phage packaging plasmid is deleted, and the M13 ori sequence on the generated circular single-stranded DNA containing the target sequence is also deleted compared with the sequence shown in the wild-type SEQ ID NO: 1. In some embodiments, the start site and / or the stop site of the recombinant phage packaging plasmid is deleted, and the M13 ori sequence on the generated circular single-stranded DNA containing the target sequence is consistent with the sequence shown in the wild-type SEQ ID NO: 1, and there is no deletion.

[0008] Unless otherwise specified, all integers involved in the present invention are positive integers greater than 0. Unless otherwise specified, nucleic acid sequences mentioned herein are from 5' to 3' from left to right.

[0009] In some embodiments, x is an integer less than 292; in some embodiments, x is an integer less than 290; in some embodiments, x is an integer less than 280; in some embodiments, x is an integer less than 270; in some embodiments, x is an integer less than 260; in some embodiments, x is an integer less than 250; in some embodiments, x is an integer less than 240; in some embodiments, x is an integer less than 230; in some embodiments, x is an integer less than 220; in some embodiments, x is an integer less than 210; in some embodiments, x is an integer less than 200; in some embodiments, x is an integer less than 190; in some embodiments, x is an integer less than 180; in some embodiments, x is an integer less than 170; in some embodiments In some embodiments, x is an integer less than 160; in some embodiments, x is an integer less than 150; in some embodiments, x is an integer less than 140; in some embodiments, x is an integer less than 130; in some embodiments, x is an integer less than 120; in some embodiments, x is an integer less than 100; in some embodiments, x is an integer less than 90; in some embodiments, x is an integer less than 80; in some embodiments, x is an integer less than 70; in some embodiments, x is an integer less than 60; in some embodiments, x is an integer less than 50; in some embodiments, x is an integer less than 40; in some embodiments, x is an integer less than 30; in some embodiments, x is an integer less than 20; in some embodiments, x is an integer less than 10.

[0010] In some embodiments, the starting sequence of the recombinant phage packaging plasmid is the sequence shown in positions x-329 in SEQ ID NO:1 in order from 5' to 3', wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 291.

[0011] In some embodiments, the starting sequence of the recombinant phage packaging plasmid is the sequence shown at position x-381 in SEQ ID NO: 1 in order from 5' to 3'.

[0012] In some embodiments, the starting sequence is the sequence shown in SEQ ID NO: 1. In some embodiments, the starting sequence is the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 19.

[0013] In some embodiments, the recombinant phage packaging plasmid comprises an initiation sequence, a target sequence and a termination sequence, and in order from 5' to 3', the initiation sequence comprises at least the sequence shown at positions 291-381 in SEQ ID NO:1, and the termination sequence comprises at least the sequence shown at positions 1-67 and 279-312 in SEQ ID NO:2.

[0014] In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-y and zw in SEQ ID NO: 2 in order from 5' to 3'. In some embodiments, y is an integer greater than 66; in some embodiments, y is an integer greater than 70; in some embodiments, y is an integer greater than 80; in some embodiments, y is an integer greater than 90; in some embodiments, y is an integer greater than 100; in some embodiments, y is an integer greater than 110; in some embodiments, y is an integer greater than 120; in some embodiments, y is an integer greater than 130; in some embodiments, y is an integer greater than 140; in some embodiments, y is an integer greater than 150; in some embodiments, y is an integer greater than 160; in one In some embodiments, y is an integer greater than 170; in some embodiments, y is an integer greater than 180; in some embodiments, y is an integer greater than 190; in some embodiments, y is an integer greater than 200; in some embodiments, y is an integer greater than 210; in some embodiments, y is an integer greater than 220; in some embodiments, y is an integer greater than 230; in some embodiments, y is an integer greater than 240; in some embodiments, y is an integer greater than 250; in some embodiments, y is an integer greater than 260; in some embodiments, y is an integer greater than 270.

[0015] In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequences shown in positions 1-y and zw of SEQ ID NO:2 in order from 5' to 3', wherein y is 67, 68, 69, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300; z is 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 278.

[0016] In some embodiments, w is 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, or 330.

[0017] In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-67 and positions 279-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-70 and positions 260-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-80 and positions 250-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-90 and positions 240-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-100 and positions 230-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-110 and positions 220-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-120 and positions 210-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-130 and positions 200-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-140 and positions 190-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-150 and positions 180-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown in positions 1-160 and positions 170-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant phage packaging plasmid comprises at least the sequence shown at positions 1-150 and 161-312 in SEQ ID NO:2.

[0018] In some embodiments, the termination sequence is selected from one of the sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:21.

[0019] Furthermore, the recombinant phage packaging plasmid further comprises at least one deoxyribozyme cleavage site or restriction endonuclease cleavage site. In some embodiments, a deoxyribozyme cleavage site or restriction endonuclease cleavage site is also provided to obtain linear single-stranded DNA. In some embodiments, there is 1 deoxyribozyme cleavage site or restriction endonuclease cleavage site. In some embodiments, there are 2 deoxyribozyme cleavage sites or restriction endonuclease cleavage sites, which are respectively arranged at both ends of the target sequence.

[0020] On the other hand, the present invention also provides a method for preparing phage circular single-stranded DNA, including designing and constructing a recombinant phage packaging plasmid, which is any of the above recombinant phage packaging plasmids, transforming a host cell with the recombinant phage packaging plasmid and an auxiliary plasmid. Specifically, it can be a competent cell, replicating, and extracting phage circular single-stranded DNA.

[0021] On the other hand, the present invention also provides a phage circular single-stranded DNA, which comprises a target sequence and a deleted M13 ori sequence. The deleted M13 ori sequence at least comprises the sequences shown at positions 1-y and z-329 in SEQ ID NO:1, wherein 67 ≤ y < z ≤ 279, and z is an integer at least 2 greater than y; in some embodiments, z is an integer at least 6 greater than y; in some embodiments, z is an integer at least 11 greater than y; in some embodiments, z is an integer at least 21 greater than y; in some embodiments, z is an integer at least 31 greater than y; in some embodiments, z is an integer at least 41 greater than y; in some embodiments, z is an integer at least 51 greater than y; in some embodiments, z is an integer at least 61 greater than y; in some embodiments, z is an integer at least 71 greater than y; in some embodiments, z is an integer at least 81 greater than y; in some embodiments, z is an integer at least 91 greater than y; in some embodiments, z is an integer at least 101 greater than y; in some embodiments, z is an integer at least 113 greater than y; in some embodiments, z is an integer at least 150 greater than y; in some embodiments, z is an integer at least 188 greater than y; in some embodiments, z is an integer at least 196 greater than y; in some embodiments, z is an integer at least 204 greater than y; in some embodiments, z is an integer at least 212 greater than y. In some embodiments, the deleted M13 ori sequence at least comprises the sequences shown at positions 1-67 and 279-381 in SEQ ID NO:1, and is not the sequence shown in SEQ ID NO:1.

[0022] For reference on the method for preparing circular single-stranded DNA Figure 1, the wild-type M13 ori is a full-length 381-nt sequence as shown in SEQ ID NO:1. When the starting sequence of the recombinant phage packaging plasmid adopts the full-length sequence of the wild-type M13 ori (SEQ ID NO:1) and the terminating sequence adopts the full-length sequence of the mutant M13 ori (SEQ ID NO:2), the circular single-stranded DNA generated by the combination of the recombinant phage packaging plasmid and the helper plasmid has the full-length sequence of the wild-type M13 ori as its M13 ori. When the recombinant phage packaging plasmid provides the x - v position of the full-length sequence of the wild-type M13 ori shown in SEQ ID NO:1 at its starting sequence and the 1 - y position and the z - w position of the full-length sequence of the mutant M13 ori shown in SEQ ID NO:2 at its terminating sequence, since 67 ≤ y < z ≤ 279 and z is an integer at least 2 greater than y, there must be a deletion in the middle section of the terminating sequence. In some embodiments, and when x is also an integer at least 2 greater than y, the deleted part of the terminating sequence cannot be fully complemented by the starting sequence, and thus the circular single-stranded DNA packaged has a certain deletion in the middle section of its M13 ori sequence compared with the wild-type full-length sequence shown in SEQ ID NO:1. When 329 ≤ v < 381, there is also a deletion at its end, that is, the circular single-stranded DNA can be deleted two sequences relative to the wild-type M13 ori full-length sequence, which are the sequences between the y position and the z position or the x position and the sequences shown from z to 381; when x ≤ y, the deleted part of the terminating sequence can be fully complemented by the starting sequence, and thus there is no deletion in the middle section of the circular single-stranded DNA packaged.

[0023] For ease of understanding, the present invention provides Figure 1 the preparation method of the circular single-stranded DNA shown, but it is not used to limit the present invention. Any circular single-stranded DNA, when its M13 ori is an M13 ori that is at least deleted for the sequences at positions 1 - 67 and 279 - 381 shown in SEQ ID NO:1, is within the protection scope of the present invention.

[0024] Compared to the full-length fixed sequence shown in SEQ ID NO: 1, in some embodiments, the fixed sequence lacks 10 consecutive bases; in some embodiments, the fixed sequence lacks 20 consecutive bases; in some embodiments, the fixed sequence lacks 30 consecutive bases; in some embodiments, the fixed sequence lacks 40 consecutive bases; in some embodiments, the fixed sequence lacks 50 consecutive bases; in some embodiments, the fixed sequence lacks 60 consecutive bases; in some embodiments, the fixed sequence lacks 70 consecutive bases; in some embodiments, the fixed sequence lacks 80 consecutive bases; in some embodiments, the fixed sequence lacks 90 consecutive bases; in some embodiments, the fixed sequence lacks 100 consecutive bases; in some embodiments, the fixed sequence lacks 110 consecutive bases; in some embodiments, the fixed sequence lacks 120 consecutive bases; in some embodiments, the fixed sequence lacks In some embodiments, the fixed sequence is missing 130 consecutive bases; in some embodiments, the fixed sequence is missing 140 consecutive bases; in some embodiments, the fixed sequence is missing 150 consecutive bases; in some embodiments, the fixed sequence is missing 160 consecutive bases; in some embodiments, the fixed sequence is missing 170 consecutive bases; in some embodiments, the fixed sequence is missing 180 consecutive bases; in some embodiments, the fixed sequence is missing 190 consecutive bases; in some embodiments, the fixed sequence is missing 200 consecutive bases; in some embodiments, the fixed sequence is missing 210 consecutive bases; in some embodiments, the fixed sequence is missing 220 consecutive bases; in some embodiments, the fixed sequence is missing 230 consecutive bases; in some embodiments, the fixed sequence is missing 240 consecutive bases; in some embodiments, the fixed sequence is missing 250 consecutive bases.

[0025] Further, the phage circular single-stranded DNA also contains at least one deoxyribozyme cleavage site or a restriction endonuclease cleavage site. In some embodiments, the phage circular single-stranded DNA contains one deoxyribozyme cleavage site or a restriction endonuclease cleavage site, and linear single-stranded DNA can be obtained by linear cleavage of the deoxyribozyme or restriction endonuclease. In some embodiments, the phage circular single-stranded DNA contains two deoxyribozyme cleavage sites or restriction endonuclease cleavage sites, and the two cleavage sites are respectively located on both sides of the target sequence, and linear single-stranded DNA of the target sequence can be obtained by linear cleavage of the deoxyribozyme or restriction endonuclease.

[0026] On the other hand, the present invention also provides a method for preparing minicircle DNA, which includes preparing the above-mentioned circular single-stranded DNA, then performing in vitro complementation on the circular single-stranded DNA, and finally obtaining minicircle DNA. In some embodiments, minicircle DNA is obtained by asymmetric amplification of single-stranded DNA.

[0027] In some embodiments, one of the single strands of minicircle DNA consists of the sequences at positions 1-67 and 279-329 shown in SEQ ID NO:1 and the target sequence.

[0028] The traditional method for preparing minicircle DNA is that the parental plasmid (PP) undergoes site-specific recombination under the action of LR clonase and is transformed into two circular DNAs. One contains a large amount of bacterial backbone sequences and is called a miniplasmid (MP); the other is a eukaryotic expression framework containing the target gene, that is, minicircle DNA (MC). However, this method has the following disadvantages: 1. The LR clonase is expensive and the cost is high; 2. The minicircle DNA is obtained through gel recovery and purification, resulting in large losses and small yields; 3. The steps are cumbersome. After the LR reaction, restriction enzyme digestion is also required to destroy the microvector, causing difficulties in the subsequent purification and recovery of minicircle DNA. The method for minicircle DNA provided by the present invention is simpler and more convenient, and the cost is also lower.

[0029] The present invention provides a recombinant phage packaging plasmid, which contains a starting sequence, a target sequence, and a termination sequence. In the order from 5' to 3', the starting sequence contains at least a segment of sequence shown by positions x-v in SEQ ID NO:1, the termination sequence contains at least two segments of sequence shown by positions 1-y and z-w in SEQ ID NO:2, and x ≤ 291, 329 ≤ v ≤ 381, 67 ≤ y < z ≤ 279, 312 ≤ w ≤ 330. The circular single-stranded DNA prepared by the recombinant phage packaging plasmid provided by the present invention has a deletion in the M13 ori sequence and a shorter length, which can reduce the influence of the M13 ori as a backbone sequence on the target sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to better understand the present invention and more clearly show how to implement the present invention, the features of the embodiments according to the present invention are described by way of examples and with reference to the drawings, where: Figure 1 : Schematic diagrams of wild-type M13 ori, deleted starting sequence, deleted termination sequence, and deleted M13 ori structures.

[0031] Figure 2: Schematic diagram of the sequence structure of the recombinant phage packaging plasmid and the corresponding circular single-stranded DNA, where A is a schematic diagram of pMPdID2C-css DNA; B is a schematic diagram of pMT2d5C9MRdM75-css DNA; C is a schematic diagram of pMT2d5C9MRdM83R16-css DNA, and the dotted part is the missing sequence.

[0032] Figure 3 :css-DNA agarose gel electrophoresis detection diagram. (A) Lane M: standard sample; Lane 1: pMPdID2C-cssDNA, Lane 2: pMT2d5C9-cssDNA; (B) Lane M: standard sample; Lane 1: NA; Lane 2: NA; Lane 3: NA; Lane 4: pMT2d5C9M10-cssDNA; Lane 5: pMT2d5C9R10-cssDNA; (C) Lane M: standard sample; Lane 1: pMT2d5C9MRd-cssDNA clone 1; Lane 2: pMT2d5C9MRd-cssDNA clone 2; (D) Lane M: standard sample; Lane 1: NA; Lane 2: NA; Lane 3: pMT2d5C9MRdM75-cssDNA clone 1; Lane 4: pMT2d5C9MRdM75-cssDNA clone 2; (E) Lane M: standard sample; Lane 1: pMT2d5C9MRdM75R8-cssDNA clone 1; Lane 2: pMT2d5C9MRdM75R8-cssDNA clone 2; Lane 3: pMT2d5C9MRdM75R16-cssDNA clone 1; Lane 4: pMT2d5C9MRdM75R16-cssDNA clone 2; (F) Lane M: standard sample; Lane 1: pMT2d5C9MRdM83R16-cssDNA.

[0033] Figure 4 : Agarose gel electrophoresis of pMT2d5C9MRdM75R16dS-cssDNA. Lane M: standard sample; Lane 1: pMT2d5C9MRdM75R16dS-cssDNA, where the white horizontal line marks the expected site of the target product.

[0034] Figure 5 : Condensation electrophoresis of pMT2d5C9-cssDNA, pMT2d5C9-ssDNA and pMT2d5C9-ssDNA digestion products. Lane M: standard sample; Lane 1: pMT2d5C9-cssDNA; Lane 2: pMT2d5C9-ssDNA; Lane 3: pMT2d5C9-ssDNA exonuclease digestion identification.

[0035] Figure 6:Sequencing map of pMT2d5C9MRdM83R16-cssDNA ori.

[0036] Figure 7 :css-DNA agarose gel electrophoresis detection diagram, in which the white horizontal line marks the expected site of the target product. (A) Electrophoresis of pMT1d3C2 packaging product; (B) Electrophoresis of pMT1d5C1 packaging product; (C) Electrophoresis of pMT2d5C10 packaging product; (D) Electrophoresis of pMT2d3C1 packaging product; (E) Electrophoresis of pMT2d5C10 packaging product; (F) Electrophoresis of pMT2d5C9MRdR25 packaging product; (G) Electrophoresis of pMT2d5C9MRdM90R16 packaging product.

[0037] Figure 8 : pMPdID2C-cssDNA was used as a template, and the mcDNA produced after 2, 3, and 4 cycles of amplification was digested and purified by column. Gel electrophoresis detection of the obtained products. DETAILED DESCRIPTION

[0038] definition In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

[0039] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" may mean "one", but it is also known with the meanings of "one or more", "at least one" and "one or more than one". Similarly, the word "another" may mean at least a second or many.

[0040] As used in this specification and claims, the words "comprising" (and any forms of comprising, such as "including" and "comprising"), "having" (and any forms of having, "having", "including" and "containing") are inclusive and open-ended, and do not exclude additional, unrecited elements or processing steps.

[0041] As used herein, "recombinant phage packaging plasmid" or "Phagemid" are used interchangeably and are a special type of artificially constructed vector containing a single-stranded phage packaging sequence, a replicon and a plasmid replicon, a cloning site, and a marker gene.

[0042] As used herein, "minicircle DNA", "mcDNA", "miniCircle" and "miniCircle DNA" are used interchangeably. Minicircle DNA is a novel small supercoiled expression cassette that lacks bacterial backbone sequences such as resistance marker genes, which enhances its safety in clinical applications. Compared with viral vectors and plasmid vectors, minicircle DNA reduces the possibility of inflammation and gene silencing, has a longer expression cycle, and enhances the intensity of gene expression by 10-1000 times, whether for gene expression in vivo or in vitro.

[0043] Examples: The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way.

[0044] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0045] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

[0046] The experimental methods not specifically described in the present invention are all carried out according to the specific methods in the book "Molecular Cloning: A Laboratory Manual (Fourth Edition)" by Joseph. Sambrook, or according to the instructions of relevant products. When used in this article, unless otherwise specified, all terms in the present invention should be understood according to their common meanings known in the art. The biological reagents used in the present invention can be obtained from commercial channels unless otherwise specified. The brand information of the main experimental materials is shown in Table 1 below.

[0047] Table 1 Main materials

[0048] Example 1: pMPdID2C generates circular single-stranded DNA 1) The recombinant phage packaging plasmid pMPdID2C was obtained by PCR subcloning, which was composed of a start sequence, a target sequence and a stop sequence in order. The start sequence was the full-length start sequence shown in SEQ ID NO:1, with a sequence length of 381 nt, the stop sequence was the full-length stop sequence shown in SEQ ID NO:2, with a sequence length of 330 nt, and the target sequence was shown in SEQ ID NO:4.

[0049] 2) Recombinant phage packaging plasmid and helper plasmid transformation 2.1. Take out the competent cells DH5α (NoviZan) from -80 ℃ and quickly thaw them on ice.

[0050] 2.2. Add 25 ng of Phagemid pMPdID2C and 50 ng of helper plasmid to be transformed into 100 μL competent cells, flick the tube wall to mix, and place on ice for 30 min.

[0051] 2.3. After heat shock in a 42℃ water bath for 45 seconds, quickly place on ice for 2 minutes.

[0052] 2.4. Add + 900 μL LB or SOC liquid medium (without antibiotics) to the centrifuge tube, mix well, and place at 30°C and shake at 220 rpm for 1 h.

[0053] 2.5. Take 100 μL of bacterial solution and spread it evenly on an LB solid culture medium plate containing chloramphenicol and kanamycin antibiotics.

[0054] 2.6. Invert the plate and incubate overnight.

[0055] 2.7. Mark the bacterial batch number and clone number.

[0056] 2.8. Pick single clones from the plate into 10 mL 2 x YT (containing antibiotics) liquid culture medium and incubate at 30°C, 270 rpm for 16-24 h.

[0057] 3) Extraction of bacteriophage single-stranded DNA 3.1. Bacterial culture medium at 4000 rcf, 4 ℃ for 15 min.

[0058] 3.2. Weigh PEG8000 4 g / 100 mL and NaCl 3 g / 100 mL in a new centrifuge tube and add to the supernatant after centrifugation to avoid disturbing the precipitate below. Mix at room temperature until the solid is completely dissolved.

[0059] 3.3. Incubate on ice for 30 min.

[0060] 3. Centrifuge at 45000 rcf, 4°C for 30 min and discard the supernatant.

[0061] 3.5. Resuspend the pellet with 133 μL / TE.

[0062] 3.6. Add 2 times the volume of P2 in the plasmid extraction kit and mix gently.

[0063] 3.7. Add 1.5 times the volume of P3 in the plasmid extraction kit and mix gently.

[0064] 3.8. Add RNase A, incubate at room temperature for 10 min, then place on ice for 10 min, and centrifuge at 16,000 rcf, 4°C for 30 min.

[0065] 3.9. Aspirate the supernatant and add an equal volume of -20 ℃ precooled anhydrous ethanol, and precipitate at -20 ℃ overnight.

[0066] 3.10.16000 rcf, 4 ℃, centrifuge for 30 min, and discard the supernatant.

[0067] 3.11. Washing: Add 70% ethanol, centrifuge at 16000 rcf, 4 ℃ for 30 min, and discard the supernatant.

[0068] 3.12. Repeat the previous step.

[0069] 3.13. Perform agarose gel electrophoresis and sequencing on the extracted M13 phage genome.

[0070] The total length of pMPdID2C-cssDNA is 4968 nt, of which the target sequence is 4587 nt. The generated M13 ori sequence is the full-length sequence of the wild-type M13 ori as shown in SEQ ID NO: 1, with a sequence length of 381 nt. Figure 3 .

[0071] Example 2: pMT2d5C9 generates circular single-stranded DNA and linear single-stranded DNA 1) Circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9 was obtained by PCR subcloning, which was composed of a starting sequence, a ribozyme cleavage sequence, a target sequence and a termination sequence in order. The starting sequence is the deleted starting sequence as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 position sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 position sequence; the termination sequence is the deleted termination as shown in SEQ ID NO:6, and the sequence length is 312 nt. The termination sequence shown in SEQ ID NO:6 lacks the 313-330 position sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-312 position sequence. The ribozyme cleavage sequence is shown in SEQ ID NO:3, and the target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0072] The total length of pMT2d5C9-cssDNA is 5058 nt, of which the ribozyme cleavage sequence is 82 nt and the target sequence is 4595 nt. M13ori is still a full-length sequence, as shown in SEQ ID NO:5, and the sequence length is 381 nt. Agarose gel electrophoresis shows Figure 3 .

[0073] 2) Linear single-stranded DNA pMT2d5C9-cssDNA was further cleaved by ribozyme. The reaction system is shown in Table 2. After preparation, it was fully mixed and centrifuged briefly in a mini centrifuge to eliminate bubbles in the system. The reaction was carried out at 37°C for 30 min.

[0074] Table 2 Enzyme digestion reaction system

[0075] The single-stranded product of the enzyme cleavage reaction was verified by directly hydrolyzing the reaction solution of the reaction system with exonuclease Exo V, and then detecting it by gel electrophoresis. The detection results are shown in Figure 5 .Depend on Figure 5 It can be seen that the product after enzyme digestion can be hydrolyzed by exonuclease, that is, pMT2d5C9-ssDNA is successfully prepared.

[0076] Example 3: pMT2d5C9M10 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9M10 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is a starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is a termination sequence deleted as shown in SEQ ID NO:7, and the sequence length is 302nt. The termination sequence shown in SEQ ID NO:7 lacks the 151-160 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-150 and 161-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0077] The total length of pMT2d5C9M10-cssDNA is 4966 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO:8. Compared with the wild-type M13 ori, 10 nucleotides at positions 151-160 are deleted, and the length is 371 nt. Agarose gel electrophoresis shows Figure 3 .

[0078] Example 4: pMT2d5C9R10 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9R10 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO:9, and the sequence length is 302 nt. The termination sequence shown in SEQ ID NO:9 lacks the 253-262 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-252, 263-312 sequence, and the target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0079] The total length of pMT2d5C9R10-cssDNA is 4966 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO: 10. Compared with the wild-type M13 ori, 10 nucleotides at positions 253-262 are deleted, and the length is 371 nt. Agarose gel electrophoresis shows Figure 3 .

[0080] Example 5: pMT2d5C9MRd generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRd was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO:11, and the sequence length is 200nt. The termination sequence shown in SEQ ID NO:11 lacks the 151-262 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-150 and 263-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0081] The total length of pMT2d5C9MRd-cssDNA is 4864 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO: 12. Compared with the wild-type M13 ori, 112 nucleotides from positions 151 to 262 are deleted, and the length is 269 nt. Agarose gel electrophoresis shows Figure 3 .

[0082] Example 6: pMT2d5C9MRdM75 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRdM75 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO:13, and the sequence length is 125nt. The termination sequence shown in SEQ ID NO:13 lacks the 76-232 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-75 and 233-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0083] The total length of pMT2d5C9MRdM75-cssDNA is 4789 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO:14. Compared with the wild-type M13 ori, 187 nucleotides from positions 76 to 232 are deleted, and the length is 194 nt. Agarose gel electrophoresis shows Figure 3 .

[0084] Example 7: pMT2d5C9MRdM75R8 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRdM75R8 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO:15, and the sequence length is 117 nt. The termination sequence shown in SEQ ID NO:15 lacks the 76-270 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-75 and 271-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0085] The total length of pMT2d5C9MRdM75R8-cssDNA is 4781 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO:16. Compared with the wild-type M13 ori, 195 nucleotides from positions 76 to 270 are deleted, and the length is 186 nt. The results of agarose gel electrophoresis show Figure 3 .

[0086] Example 8: pMT2d5C9MRdM75R16 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRdM75R16 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO:17, and the sequence length is 109 nt. The termination sequence shown in SEQ ID NO:17 lacks the 76-278 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-75 and 279-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0087] The total length of pMT2d5C9MRdM75R16-cssDNA is 4773 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO:18. Compared with the wild-type M13 ori, 203 nucleotides at positions 76-278 are deleted, and the length is 178 nt. The results of agarose gel electrophoresis show Figure 3 .

[0088] Example 9: pMT2d5C9MRdM75R16dS generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRdM75R16dS was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the starting sequence deleted as shown in SEQ ID NO: 19, and the sequence length is 39 nt. The starting sequence shown in SEQ ID NO: 18 lacks the 1-290 sequence and the 330-381 sequence of the full-length starting sequence shown in SEQ ID NO: 1, and is consistent with the 291-329 sequence; the termination sequence is the termination sequence deleted as shown in SEQ ID NO: 17, and the sequence length is 109 nt. The termination sequence shown in SEQ ID NO: 17 lacks the 76-278 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO: 2, and is consistent with the 1-75 and 279-312 sequences. The target sequence is shown in SEQ ID NO: 4. For other steps, refer to Example 1.

[0089] The total length of pMT2d5C9MRdM75R16dS-cssDNA is 4704 nt, of which the target sequence is 4595 nt. The deleted M13ori is shown in SEQ ID NO:20. Compared with the wild-type M13 ori, 203 nucleotides at positions 76-278 and 69 sequences at positions 313-381 are deleted, with a length of 109 nt. Agarose gel electrophoresis shows Figure 4 .

[0090] Example 10: pMT2d5C9MRdM83R16 generates circular single-stranded DNA The recombinant phage packaging plasmid pMT2d5C9MRdM83R16 was obtained by PCR subcloning, which was composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is a starting sequence deleted as shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is a termination sequence deleted as shown in SEQ ID NO:21, and the sequence length is 94 nt. The termination sequence shown in SEQ ID NO:21 lacks the 68-278 sequence and the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-67 and 279-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1.

[0091] The total length of pMT2d5C9MRdM83R16-cssDNA is 4765 nt, of which the target sequence is 4595 nt. The deleted M13 ori is shown in SEQ ID NO:22. Compared with the wild-type M13 ori, 211 nucleotides at positions 68-278 are deleted, and the length is 170 nt. Agarose gel electrophoresis shows Figure 3 , Sequencing map see Figure 6 .

[0092] Example 11: pMT1d3C2 The recombinant phage packaging plasmid pMT1d3C2 was obtained by PCR subcloning, which is composed of a start sequence, a target sequence and a termination sequence in order. The start sequence is the complete start sequence shown in SEQ ID NO:1, and the sequence length is 381 nt; the termination sequence is the deleted termination sequence shown in SEQ ID NO:23, and the sequence length is 262 nt. The termination sequence shown in SEQ ID NO:23 lacks the 263-330 position sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-262 position sequence. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1. Agarose gel electrophoresis is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C10 did not successfully package circular single-stranded DNA.

[0093] Example 12: pMT1d5C1 The recombinant phage packaging plasmid pMT1d5C1 was obtained by PCR subcloning, which is composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the complete starting sequence shown in SEQ ID NO:1, and the sequence length is 381 nt; the termination sequence is the missing termination sequence shown in SEQ ID NO:24, and the sequence length is 280 nt. The termination sequence shown in SEQ ID NO:24 lacks the 281-330 position sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-280 position sequence. The target sequence is shown in SEQ ID NO:4. The other steps refer to Example 1, and the agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT1d5C1 did not successfully package circular single-stranded DNA.

[0094] Example 13: pMT2d5C10 The recombinant phage packaging plasmid pMT2d5C10 was obtained by PCR subcloning, which consists of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the missing starting sequence shown in SEQ ID NO:25, and the sequence length is 81 nt. The starting sequence shown in SEQ ID NO:25 lacks the 1-300 position sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 301-381 position sequence; the termination sequence is the missing termination shown in SEQ ID NO:6, and the sequence length is 312 nt. The termination sequence shown in SEQ ID NO:6 lacks the 313-330 position sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-312 position sequence. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1. Agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C10 did not successfully package circular single-stranded DNA.

[0095] Example 14: pMT2d3C1 The recombinant phage packaging plasmid pMT2d3C1 was obtained by PCR subcloning, which consists of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the missing starting sequence shown in SEQ ID NO:26, and the sequence length is 331 nt. The starting sequence shown in SEQ ID NO:26 lacks the 332-381 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 1-331 sequence; the termination sequence is the missing termination sequence shown in SEQ ID NO:6, and the sequence length is 312nt. The termination sequence shown in SEQ ID NO:6 lacks the 313-330 sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-312 sequence. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1. Agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C10 did not successfully package circular single-stranded DNA.

[0096] Example 15: pMT2d5C9F10 The recombinant phage packaging plasmid pMT2d5C9F10 was obtained by PCR subcloning, which is composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the missing starting sequence shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 position sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 position sequence; the termination sequence is the missing termination shown in SEQ ID NO:27, and the sequence length is 302 nt. The termination sequence shown in SEQ ID NO:27 lacks the 51-60 position and 312-330 position sequence of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-50 and 61-312 position sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1, and the agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C9F10 did not successfully package circular single-stranded DNA.

[0097] Example 16: pMT2d5C9MRdR25 The recombinant phage packaging plasmid pMT2d5C9MRdR25 was obtained by PCR subcloning, which is composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the missing starting sequence shown in SEQ ID NO:5, and the sequence length is 91nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the missing termination shown in SEQ ID NO:28, and the sequence length is 175nt. The termination sequence shown in SEQ ID NO:28 lacks the 151-287 and 312-330 sequences of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-150 and 288-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1. Agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C9MRdR25 did not successfully package circular single-stranded DNA.

[0098] Example 17: pMT2d5C9MRdM90R16 The recombinant phage packaging plasmid pMT2d5C9MRdM90R16 was obtained by PCR subcloning, which is composed of a starting sequence, a target sequence and a termination sequence in order. The starting sequence is the missing starting sequence shown in SEQ ID NO:5, and the sequence length is 91 nt. The starting sequence shown in SEQ ID NO:5 lacks the 1-290 sequence of the full-length starting sequence shown in SEQ ID NO:1, and is consistent with the 291-381 sequence; the termination sequence is the missing termination shown in SEQ ID NO:29, and the sequence length is 94 nt. The termination sequence shown in SEQ ID NO:29 lacks the 61-278 and 312-330 sequences of the full-length termination sequence shown in SEQ ID NO:2, and is consistent with the 1-60 and 179-312 sequences. The target sequence is shown in SEQ ID NO:4. For other steps, refer to Example 1. Agarose gel electrophoresis of the final product is shown in Figure 7 .Depend on Figure 7 It can be seen that the phage packaging plasmid pMT2d5C9MRdM90R16 did not successfully package circular single-stranded DNA.

[0099] Example 18: Preparation of minicircle DNA Using circular single-stranded DNA (pMPdID2C-cssDNA) as a template, a single-primer PCR method was used to perform asymmetric PCR amplification of css DNA. Then, exonuclease V (RecBCD) was used to digest the double-stranded DNA and single-stranded DNA in the product, retaining the circular (with nicked) cdsDNA. Finally, the product was purified and quantified using column purification.

[0100] 1) Asymmetric amplification of cssDNA 1.1 Perform asymmetric PCR amplification on pMPdID2C-cssDNA. The amplification system is shown in Table 3. After preparation, mix thoroughly and centrifuge briefly in a mini centrifuge to eliminate bubbles in the system. The PCR amplification program is shown in Table 4.

[0101] Table 3 cssDNA asymmetric PCR amplification system

[0102] Table 4 PCR amplification program

[0103] 2) Digestion reaction solution The reaction solution obtained in the previous step was digested. The digestion system is shown in Table 5. Centrifuge clockwise for 5 seconds and place in a 37°C constant temperature incubator for digestion for 30 minutes.

[0104] Table 5 Digestion system

[0105] 3) Purification of reaction solution The reaction solution was purified using the Tiangen brand common DNA product purification kit. Before use, anhydrous ethanol was added to the rinse solution PW, and the added volume was referred to the label on the bottle.

[0106] 3.1 Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0107] 3.2 Add 5 times the volume of PB to the reaction solution and mix thoroughly.

[0108] 3.3 Add the solution obtained in the previous step to the adsorption column CB2 (the adsorption column is placed in the collection tube), leave it at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube.

[0109] 3.4 Add 600 μL of rinse solution PW to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube.

[0110] 3.5 Repeat step 3.4.

[0111] 3.6 Place the adsorption column CB2 back into the collection tube and centrifuge at 12,000 rpm for 2 min. Then place the adsorption column CB2 at room temperature for several minutes to dry thoroughly.

[0112] 3.7 Add a certain amount of enzyme-free water (about 30-60 μl) to the adsorption column for elution, let it stand for 2 minutes, and then centrifuge at 12,000 rpm for 2 minutes.

[0113] 3.8 The products after centrifugation were detected by agarose gel electrophoresis.

[0114] After the above experimental process, Figure 8 The agarose gel electrophoresis results shown in the figure show that there is only one bright band after digestion and purification, that is, the circular single-stranded DNA changes from single-stranded to double-stranded.

[0115] The present invention finds that even if there are certain deletions in the starting sequence and the termination sequence of M13ori, it can be successfully packaged as a recombinant phage packaging plasmid for further packaging, and finally generate single-stranded DNA. The recombinant phage packaging plasmid provided by the present invention has a starting sequence that can be shortened to the sequence shown in positions 291-329 of SEQ ID NO:1, but its termination sequence must at least contain two sequences shown in positions 1-67 and 279-312 of SEQ ID NO:2 to successfully package circular single-stranded DNA. The circular single-stranded DNA prepared by the recombinant phage packaging plasmid provided by the present invention has a shorter length of its fixed phase sequence compared to the traditional full-length M13ori sequence, which can reduce the impact of M13 ori as a backbone sequence on the target sequence, and the preparation of mini-circular DNA by the circular single-stranded DNA provided by the present invention is also simpler and more convenient than the traditional method, and has lower cost.

[0116] The above-described embodiments only express several embodiments of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that a person skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

[0117] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

Claims

1. A recombinant phage packaging plasmid, characterized in that: The recombinant phage packaging plasmid comprises a starting sequence, a target sequence and a termination sequence, in order from 5' to 3', the starting sequence comprises at least the xv position sequence in the sequence shown in SEQ ID NO:1, the termination sequence comprises at least the 1-y position and zw position sequence in the sequence shown in SEQ ID NO:2, and x≤ 291, 329 ≤ v ≤ 381, 67 ≤ y < z ≤ 279, 312 ≤ w ≤ 330, wherein x, y, z, v, w are all positive integers, and the starting sequence is not the sequence shown in SEQ ID NO:1 and / or the termination sequence is not the sequence shown in SEQ ID NO:

2.

2. The recombinant phage packaging plasmid according to claim 1, characterized in that The recombinant phage packaging plasmid further comprises at least one DNAzyme cleavage site or a restriction endonuclease cleavage site.

3. The recombinant phage packaging plasmid according to claim 1, characterized in that The x=291, the v=329, and the starting sequence at least includes the sequence from positions 291 to 329 in the sequence shown in SEQ ID NO:

1.

4. The recombinant phage packaging plasmid according to claim 1, characterized in that The y=67, the z=279, the w=312, and the termination sequence at least comprises the sequence of positions 1-67 and positions 279-312 in the sequence shown in SEQ ID NO:

2.

5. The recombinant phage packaging plasmid according to claim 1, characterized in that The starting sequence shown is the sequence shown in SEQ ID NO:5 or SEQ ID NO:

19.

6. The recombinant phage packaging plasmid according to claim 1, characterized in that: The termination sequence is selected from any one of the sequences shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:

21.

7. A method for preparing circular single-stranded DNA, characterized in that: The preparation method comprises designing and constructing the recombinant phage packaging plasmid according to any one of claims 1 to 6, transforming host cells with the recombinant phage packaging plasmid and a helper plasmid for replication, and extracting the circular single-stranded DNA generated in the host cells.

8. A circular single-stranded DNA, characterized in that The circular single-stranded DNA comprises a target sequence and a deleted M13 ori sequence, wherein the deleted M13 ori sequence comprises at least the sequences shown at positions 1-y and z-329 in SEQ ID NO:1, wherein 67 ≤ y < z ≤ 279, and z is an integer at least 2 greater than y.

9. The circular single-stranded DNA according to claim 8, characterized in that The circular single-stranded DNA further comprises at least one deoxyribozyme cleavage site or a restriction endonuclease cleavage site.

10. A method for preparing linear single-stranded DNA, characterized in that: The circular single-stranded DNA according to claim 9 is digested with a deoxyribozyme or a restriction endonuclease to obtain a linear single-stranded DNA.

11. A method for preparing minicircle DNA, characterized in that: The preparation method comprises preparing the circular single-stranded DNA according to claim 8 or 9, and performing in vitro complementation on the circular single-stranded DNA to obtain minicircular DNA.

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