Non-replicative recombinant bacteriophage packaging plasmid and use thereof

Abstract

The application provides a non-replicative recombinant bacteriophage packaging plasmid and application thereof, and particularly relates to a non-replicative recombinant bacteriophage packaging plasmid and a method for preparing ssDNA and mcDNA, wherein the plasmid comprises a starting sequence derived from a bacteriophage replication origin, a target sequence and a termination sequence derived from the bacteriophage replication origin, and the sequences are arranged in order from 5' to 3', the starting sequence comprises at least a sequence shown in x-v positions in SEQ ID NO: 1, the termination sequence comprises at least a sequence shown in 1-y positions and z-w positions in SEQ ID NO: 2, and x≤291, 329≤v≤381, 67≤y<z≤279, 312≤w≤330. The non-replicative recombinant bacteriophage packaging plasmid of the application has at least one deletion in the starting sequence and / or the termination sequence of the replication origin compared with the original full-length sequence, and can still generate a circular single-stranded DNA containing the target sequence and the deletion of the bacteriophage replication origin sequence, thereby providing a safer single-stranded DNA donor containing less non-target sequences for downstream applications such as gene editing, ssDNA and mcDNA preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a non-replicative recombinant bacteriophage packaging plasmid and an application method for preparing ssDNA and mcDNA based on the non-replicative recombinant bacteriophage. BACKGROUND

[0002] Single-stranded DNA is considered as a biological material, which has great application potential in many biological reactions and has a wide range of applications in DNA nanotechnology. However, due to the limitation of chemical synthesis method, the synthesis of long single-stranded DNA is difficult to ensure yield, output and satisfactory cost performance, so the long single-stranded DNA needs to be assisted by in vivo or in vitro action of biological enzymes and some auxiliary denaturation means. At present, the commonly used preparation methods of long single-stranded DNA mainly include reverse transcription method, enzyme degradation method, denaturing high performance liquid chromatography (HPLC) method, magnetic bead biotin (Biotin) modification method, asymmetric PCR method, RCA method, etc. However, in practical application, these methods all have problems of 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 M13 replication initiation point (M13 ori) or f1 replication initiation point (f1 ori), which is transferred into a host cell containing F factor, and then infected with a defective helper phage. The helper phage can help the plasmid to form single-stranded DNA and wrap into phage, and then secreted out of the host cell. This method has low cost and high yield, but the single-stranded DNA formed contains a 2-3 nt M13 ori / f1 ori conserved sequence.

[0004] It has been reported that pUC18 can be converted into a recombinant bacteriophage packaging plasmid for producing custom single-stranded DNA by adding four components: wild-type M13 ori for start sequence (M13 ori start), restriction site, M13 PS for phage particle export, and mutated M13 ori as terminator sequence (M13 ori terminator). The final single-stranded circular DNA still contains a 381-base wild-type M13 ori conserved 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 conserved sequence is still a problem to be solved.

[0005] The above-mentioned documents are incorporated herein in their entirety. SUMMARY

[0006] The present inventors found that the start sequence (M13 ori start) and the termination sequence (M13 ori terminator) on the recombinant phage packaging plasmid can still generate circular single-stranded DNA containing target sequences and phage replication origin (ori) sequences after certain deletions, thereby providing safer single-stranded DNA donors with fewer non-target sequences for downstream applications such as gene editing, minicircle DNA (mcDNA) preparation, etc., and the length of the target sequence can reach several thousand nt. The conserved sequence of the resulting circular single-stranded DNA is shorter than the traditional full-length phage replication origin sequence, which can reduce the potential impact of the phage replication origin as a backbone sequence on the target sequence. Specifically, the present application includes the following contents:

[0007] In one aspect, the present application provides a recombinant phage packaging plasmid, which comprises a start sequence (M13 ori start), a target sequence (GOI) and a termination sequence (M13 ori terminator), in order from 5' to 3', the start sequence comprises at least the sequence shown as x-v in SEQ ID NO: 1, and the termination sequence comprises at least the sequence shown as 1-y and z-w in SEQ ID NO: 2, wherein x ≤ 291, 329 ≤ v ≤ 381, 67 ≤ y<z ≤ 279, 312 ≤w ≤ 330, x, y, z, v, w are all positive integers, and the start sequence is not SEQ ID NO: 1 and / or the termination sequence is not SEQ ID NO: 2. The sequence shown in SEQ ID NO: 1 is the wild-type M13 ori sequence, which is 381 nt in length, and the sequence shown in SEQ ID NO: 2 is the mutant M13 ori sequence, which is 330 nt in length, and the sequence shown as 1-319 in SEQ ID NO: 2 sequence is consistent with the sequence shown as 1-319 in SEQ ID NO: 1 sequence. At least one of the start sequence and the termination sequence provided in the present application is inconsistent with SEQ ID NO: 1 or SEQ ID NO: 2, i.e. at least one of the start sequence or the termination sequence has a deletion compared to the full-length sequence, but still generates circular single-stranded DNA containing target sequences.

[0008] In some embodiments, the initiation site and / or the termination site of the recombinant phage packaging plasmid is deleted, and the M13 ori sequence on the resulting circular single-stranded DNA containing the sequence of interest also has a deletion compared to the sequence set forth in wild-type SEQ ID NO: 1. In some embodiments, the initiation site and / or the termination site of the recombinant phage packaging plasmid is deleted, and the M13 ori sequence on the resulting circular single-stranded DNA containing the sequence of interest is identical to the sequence set forth in wild-type SEQ ID NO: 1, and does not have a deletion.

[0009] As used herein, and unless otherwise indicated, the term "integer" refers to a positive integer greater than zero. As used herein, and unless otherwise indicated, reference to a nucleic acid sequence herein is from left to right in the 5' to 3' direction.

[0010] 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, 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.

[0011] In some embodiments, the initiation sequence of the recombinant bacteriophage packaging plasmid is the sequence set forth in SEQ ID NO: 1 at position x-329, where 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, in order from 5' to 3'.

[0012] In some embodiments, the initiation sequence of the recombinant bacteriophage packaging plasmid is the sequence set forth in SEQ ID NO: 1 at position x-381, in order from 5' to 3'.

[0013] In some embodiments, the initiation sequence is the sequence set forth in SEQ ID NO: 1. In some embodiments, the initiation sequence is the sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 19.

[0014] In some embodiments, the recombinant bacteriophage packaging plasmid comprises an initiation sequence, a target sequence, and a termination sequence, in order from 5' to 3', the initiation sequence comprises at least the sequence set forth in SEQ ID NO: 1 at positions 291-381, and the termination sequence comprises at least the sequence set forth in SEQ ID NO: 2 at positions 1-67 and 279-312.

[0015] In some embodiments, the stop sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequences set forth as positions 1-y and z-w 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 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.

[0016] In some embodiments, the stop sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequences set forth as positions 1-y and z-w in 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; and 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.

[0017] 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.

[0018] In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-67 and 279-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-70 and 260-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-80 and 250-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-90 and 240-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-100 and 230-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-110 and 220-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-120 and 210-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-130 and 200-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-140 and 190-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-150 and 180-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-160 and 170-312 of SEQ ID NO: 2. In some embodiments, the termination sequence of the recombinant bacteriophage packaging plasmid comprises at least the sequence set forth in positions 1-150 and 161-312 of SEQ ID NO: 2.

[0019] In some embodiments, the termination sequence is selected from one of the sequences set forth 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, SEQ ID NO: 21.

[0020] Further, the recombinant phage packaging plasmid further comprises at least one deoxyribonuclease cleavage site or restriction endonuclease cleavage site. In some embodiments, in order to obtain linear single-stranded DNA, the deoxyribonuclease cleavage site or restriction endonuclease cleavage site is further provided. In some embodiments, the deoxyribonuclease cleavage site or restriction endonuclease cleavage site is 1. In some embodiments, the deoxyribonuclease cleavage site or restriction endonuclease cleavage site is 2, and is respectively provided at both ends of the target sequence.

[0021] In another aspect, the present application also provides a method for preparing a phage circular single-stranded DNA, comprising designing and constructing a recombinant phage packaging plasmid, the recombinant phage packaging plasmid being any of the above-mentioned recombinant phage packaging plasmids, transforming a host cell, in particular a competent cell, with the recombinant phage packaging plasmid and a helper plasmid, and extracting the phage circular single-stranded DNA.

[0022] In another aspect, the present application also provides a phage circular single-stranded DNA, the circular single-stranded DNA comprising a target sequence and a deleted M13 ori sequence, the deleted M13 ori sequence comprising at least the sequence shown in positions 1-y and z-329 of 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 comprises at least the sequence shown in positions 1-67 and 279-381 of SEQ ID NO: 1, and is not the sequence shown in SEQ ID NO: 1.

[0023] The method for preparing the circular single-stranded DNA is described in reference 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 termination sequence adopts the full-length sequence of the mutant M13 ori (SEQ ID NO: 2), the recombinant phage-packaging plasmid and the helper plasmid generate a circular single-stranded DNA with a M13 ori of the full-length sequence of the wild-type M13 ori. When the recombinant phage-packaging plasmid has a starting sequence providing x-v of the full-length sequence of the wild-type M13 ori as shown in SEQ ID NO: 1 and a termination sequence providing 1-y and z-w of the full-length sequence of the mutant M13 ori as shown in SEQ ID NO: 2, since 67≤y<z≤279 and z is an integer at least 2 greater than y, there must be a deletion in the middle of the termination sequence. In some embodiments, and when x is also an integer at least 2 greater than y, the portion of the termination sequence deleted cannot be fully complemented by the starting sequence, and thus the circular single-stranded DNA packaged has a M13 ori sequence with a deletion in the middle compared to the full-length sequence of the wild-type M13 ori as shown in SEQ ID NO: 1, and has a deletion at the end when 329≤v<381, i.e. the circular single-stranded DNA can have two deletions in the sequence relative to the full-length sequence of the wild-type M13 ori, one between y and z or x and the other between z and 381 as shown in the sequence; when x≤y, the portion of the termination sequence deleted can be fully complemented by the starting sequence, and thus the circular single-stranded DNA packaged has no deletion in the middle.

[0024] For ease of understanding, the present application provides Figure 1 The preparation method of the circular single-stranded DNA as shown is not intended to limit the present application. Any circular single-stranded DNA, when its M13 ori is a M13 ori with a deletion of at least the sequence of 1-67 and 279-381 as shown in SEQ ID NO: 1, is within the scope of the present application.

[0025] In some embodiments, the fixed sequence is missing 10 consecutive bases compared to the full-length fixed sequence set forth in SEQ ID NO: 1; in some embodiments, the fixed sequence is missing 20 consecutive bases; in some embodiments, the fixed sequence is missing 30 consecutive bases; in some embodiments, the fixed sequence is missing 40 consecutive bases; in some embodiments, the fixed sequence is missing 50 consecutive bases; in some embodiments, the fixed sequence is missing 60 consecutive bases; in some embodiments, the fixed sequence is missing 70 consecutive bases; in some embodiments, the fixed sequence is missing 80 consecutive bases; in some embodiments, the fixed sequence is missing 90 consecutive bases; in some embodiments, the fixed sequence is missing 100 consecutive bases; in some embodiments, the fixed sequence is missing 110 consecutive bases; in some embodiments, the fixed sequence is missing 120 consecutive bases; 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.

[0026] Further, the phage circular single-stranded DNA further comprises at least one deoxyribonuclease cleavage site or restriction endonuclease cleavage site. In some embodiments, the phage circular single-stranded DNA comprises one deoxyribonuclease cleavage site or restriction endonuclease cleavage site, and linear single-stranded DNA can be obtained by linear cleavage of the deoxyribonuclease or the restriction endonuclease. In some embodiments, the phage circular single-stranded DNA comprises two deoxyribonuclease 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 deoxyribonuclease or the restriction endonuclease.

[0027] In another aspect, the present application also provides a method for preparing a minicircle DNA, which comprises preparing any of the above-mentioned circular single-stranded DNA, and then performing in vitro complementation on the circular single-stranded DNA to obtain the minicircle DNA. In some embodiments, the minicircle DNA is obtained by asymmetric amplification of the single-stranded DNA.

[0028] In some embodiments, one of the single strands of the minicircle DNA is composed of the sequences at positions 1-67 and 279-329 of SEQ ID NO: 1 and the target sequence.

[0029] The conventional method for preparing a minicircle DNA is to perform site-specific recombination on a parental plasmid (PP) under the action of an LR clonase to convert the parental plasmid into two circular DNAs, one of which is a miniplasmid (MP) containing a large amount of bacterial backbone sequences, and the other of which is a eukaryotic expression framework containing a target gene, i.e., a minicircle (MC). However, this method has the following disadvantages: 1. The LR clonase is expensive and has a high cost; 2. The minicircle DNA is obtained by gel recovery and purification, resulting in a large loss and a small amount of obtained product; and 3. The steps are complicated, and the minicarriers need to be destroyed by restriction enzyme digestion after the LR reaction, which causes difficulties in subsequent purification and recovery of the minicircle DNA. The minicircle DNA provided in the present application has a simpler and more convenient method and a lower cost.

[0030] The present application provides a recombinant bacteriophage packaging plasmid, which comprises a start sequence, a target sequence and a stop sequence, in order from 5' to 3', the start sequence at least comprises a sequence shown as positions x-v in SEQ ID NO: 1, the stop sequence at least comprises two sequences shown as 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 bacteriophage packaging plasmid provided in the present application has a missing 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 DRAWINGS

[0031] In order to better understand the present application and more clearly show how to implement the present application, the features of the embodiments according to the present application are described by way of examples and with reference to the accompanying drawings, in which:

[0032] Figure 1 : Schematic diagram of wild-type M13 ori, missing start sequence, missing stop sequence and missing M13 ori structure.

[0033] Figure 2: Schematic diagram of the sequence structure of the recombinant phage packaging plasmid and the corresponding circular single-stranded DNA, wherein 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 deleted sequence.

[0034] Figure 3 : Agarose gel electrophoresis detection diagram of css-DNA. (A) Lane M: marker; Lane 1: pMPdID2C-css DNA, Lane 2: pMT2d5C9-css DNA; (B) Lane M: marker; Lane 1: NA; Lane 2: NA; Lane 3: NA; Lane 4: pMT2d5C9M10-css DNA; Lane 5: pMT2d5C9R10-css DNA; (C) Lane M: marker; Lane 1: pMT2d5C9MRd-css DNA clone 1; Lane 2: pMT2d5C9MRd-css DNA clone 2; (D) Lane M: marker; Lane 1: NA; Lane 2: NA; Lane 3: pMT2d5C9MRdM75-css DNA clone 1; Lane 4: pMT2d5C9MRdM75-css DNA clone 2; (E) Lane M: marker; Lane 1: pMT2d5C9MRdM75R8-css DNA clone 1; Lane 2: pMT2d5C9MRdM75R8-css DNA clone 2; Lane 3: pMT2d5C9MRdM75R16-css DNA clone 1; Lane 4: pMT2d5C9MRdM75R16-css DNA clone 2; (F) Lane M: marker; Lane 1: pMT2d5C9MRdM83R16-css DNA.

[0035] Figure 4 : Agarose gel electrophoresis diagram of pMT2d5C9MRdM75R16dS-css DNA. Lane M: marker; Lane 1: pMT2d5C9MRdM75R16dS-css DNA, wherein the white horizontal line marked interval is the expected site of the target product.

[0036] Figure 5 : Agarose gel electrophoresis diagram of pMT2d5C9-css DNA, pMT2d5C9-ssDNA and pMT2d5C9-ssDNA enzyme digestion product. Lane M: marker; Lane 1: pMT2d5C9-css DNA; Lane 2: pMT2d5C9-ssDNA; Lane 3: pMT2d5C9-ssDNA exonuclease digestion identification.

[0037] Figure 6: Sequencing map of pMT2d5C9MRdM83R16-cssDNA ori.

[0038] Figure 7 : Agarose gel electrophoresis detection map of css-DNA, where the white horizontal line marks the expected location of the target product. (A) pMT1d3C2 packaging product electrophoresis map; (B) pMT1d5C1 packaging product electrophoresis map; (C) pMT2d5C10 packaging product electrophoresis map; (D) pMT2d3C1 packaging product electrophoresis map; (E) pMT2d5C10 packaging product electrophoresis map; (F) pMT2d5C9MRdR25 packaging product electrophoresis map; (G) pMT2d5C9MRdM90R16 packaging product electrophoresis map.

[0039] Figure 8 : Gel electrophoresis detection map of the products obtained after digestion and column purification of the mcDNA produced after 2, 3, 4 cycles of amplification using pMPdID2C-cssDNA as template. DETAILED DESCRIPTION

[0040] DEFINITIONS

[0041] In order to provide a clear and consistent understanding of the terminology used in the description of the present application, a number of definitions are provided below. Moreover, unless specifically stated 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 this application belongs.

[0042] The use of the word "a" or "an" when used in the context of the patent claims and / or specification shall not be construed to mean "one and only one" unless specifically stated so. Similarly, the use of the term "another" when used in the context of the patent claims and / or specification shall not be construed to mean "one and only one" unless specifically stated so.

[0043] The use of the words "include", "includes" and "including", "comprising", "comprises" and "comprising", "have" (and any form of have including "has" and "having") "include" (and any form of include, including "includes" and "including") used herein, are including and open ended and do not exclude additional, unrecited elements or method steps.

[0044] As used herein, "recombinant phagemid packaging plasmid" or "Phagemid" are used interchangeably to refer to a special type of vector artificially constructed to contain single-stranded phage packaging sequences, replicon as well as plasmid replicon, cloning sites, marker genes.

[0045] As used herein, "minicircle DNA," "mcDNA," "miniCircle," and "miniCircle DNA" are used interchangeably. Minicircle DNA is a novel small circular supercoiled expression cassette that lacks bacterial backbone sequences such as resistance marker genes, enhancing safety for clinical applications. Compared to viral vectors, plasmid vectors, minicircle DNA reduces the possibility of inflammation and gene silencing, expresses for a longer period, and enhances gene expression intensity by 10-1000 times, whether in vivo or in vitro.

[0046] EXAMPLE: The present application will be more readily understood by reference to the following examples, which are offered by way of illustration and are not intended to limit the scope of the present application.

[0047] 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 this application 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 application.

[0048] While the present application has been described in detail with respect to the embodiments thereof, it should be understood that the embodiments are illustrative only and are not limiting of the present application as defined by the appended claims. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the present application disclosed herein.

[0049] Experimental methods not specifically described in the present application are performed according to the specific methods in the book "Molecular Cloning Laboratory Manual" (Fourth Edition) by J. Sambrook, or according to the relevant product instructions. As used herein, unless otherwise specified, all terms in the present application should be understood according to their ordinary meanings known in the art. Biological reagents used in the present application, unless otherwise specified, can be obtained commercially, and the main experimental materials are shown in Table 1.

[0050] Table 1 Main materials

[0051]

[0052] Example 1: Generation of circular single-stranded DNA from pMPdID2C

[0053] 1) Recombinant phage packaging plasmid pMPdID2C was obtained by PCR subcloning, consisting of a start sequence, a target sequence and a stop sequence in this order. The start sequence is the full-length start sequence as shown in SEQ ID NO: 1, with a sequence length of 381 nt, the stop sequence is the full-length stop sequence as shown in SEQ ID NO: 2, with a sequence length of 330 nt, and the target sequence is as shown in SEQ ID NO: 4.

[0054] 2) Transformation of the recombinant phage packaging plasmid and the helper plasmid

[0055] 2.1. Take competent cells DH5a (Novagen) from -80 °C and quickly place on ice to thaw.

[0056] 2.2. Add 25 ng of phagemid pMPdID2C and 50 ng of helper plasmid to be transformed to 100 μL of competent cells, mix by flicking the tube wall and incubate on ice for 30 min.

[0057] 2.3. After 45 sec of heat shock in a 42 °C water bath, quickly place on ice for 2 min.

[0058] 2.4. Add + 900 μL of LB or SOC liquid medium (without antibiotics) to the centrifuge tube, mix and place in a 30 °C, 220 rpm shaker for 1 h.

[0059] 2.5. Take 100 μL of bacterial solution and evenly spread on LB solid medium plates containing chloramphenicol and kanamycin antibiotics.

[0060] 2.6. Invert the plates and incubate overnight.

[0061] 2.7. Label the bacterial batch number and clone number.

[0062] 2.8. Pick single colonies from the plates into 10 mL of 2 x YT liquid medium (with antibiotics) and incubate at 30 °C, 270 rpm for 16-24 h.

[0063] 3) Extraction of phage single-stranded DNA

[0064] 3.1. Centrifuge the bacterial culture at 4000 ref, 4 °C for 15 min.

[0065] 3.2. In a new centrifuge tube, weigh PEG8000 4 g / 100 mL; NaCl 3 g / 100 mL and add to the supernatant after centrifugation, avoiding disturbing the sediment below. Mix at room temperature until the solids are completely dissolved.

[0066] 3.3. Incubate on ice for 30 min.

[0067] 3.4. 5000 rcf, 4 °C, centrifugal 30 min, discard supernatant.

[0068] 3.5. Resuspend the precipitate with 133 μL / TE.

[0069] 3.6. Add 2 times volume of P2 of plasmid extraction kit, mix gently.

[0070] 3.7. Add 1.5 times volume of P3 of plasmid extraction kit, mix gently.

[0071] 3.8. Add RNase A, incubate at room temperature for 10 min, then ice bath for 10 min, 16000 rcf, 4 °C, centrifugal 30 min.

[0072] 3.9. Take the supernatant and add equal volume of anhydrous ethanol pre-cooled at -20 °C, precipitate overnight at -20 °C.

[0073] 3.10. 16000 rcf, 4 °C, centrifugal 30 min, discard supernatant.

[0074] 3.11. Wash: add 70% ethanol, 16000 rcf, 4 °C, centrifugal 30 min, discard supernatant.

[0075] 3.12. Repeat the above step.

[0076] 3.13. Perform agarose gel electrophoresis and sequencing on the extracted M13 phage genome.

[0077] The total length of pMPdID2C-css DNA is 4968 nt, of which the target sequence is 4587 nt, and the generated M13 ori sequence is the wild type M13 ori full length sequence as shown in SEQ ID NO: 1, with a sequence length of 381 nt. Agarose gel electrophoresis is shown in Figure 3 .

[0078] Example 2: pMT2d5C9 generates circular single-stranded DNA and linear single-stranded DNA

[0079] 1) Circular single-stranded DNA

[0080] The recombinant phage packaging plasmid pMT2d5C9 was obtained by PCR subcloning, and was composed of a start sequence, a ribozyme cleavage sequence, a target sequence and a termination sequence in order. The start sequence was a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt. The start sequence shown in SEQ ID NO: 5 was deleted from the sequence at positions 1-290 of the full-length start sequence shown in SEQ ID NO: 1, and was consistent with the sequence at positions 291-381. The termination sequence was a deletion termination as shown in SEQ ID NO: 6, with a sequence length of 312 nt. The termination sequence shown in SEQ ID NO: 6 was deleted from the sequence at positions 313-330 of the full-length termination sequence shown in SEQ ID NO: 2, and was consistent with the sequence at positions 1-312. The ribozyme cleavage sequence was as shown in SEQ ID NO: 3, and the target sequence was as shown in SEQ ID NO: 4. The other steps refer to Example 1.

[0081] The total length of pMT2d5C9-cssDNA was 5058 nt, of which the ribozyme cleavage sequence was 82 nt, the target sequence was 4595 nt, and the M13 ori was still a full-length sequence. The start sequence was as shown in SEQ ID NO: 5, with a sequence length of 381 nt. Agarose gel electrophoresis is shown in Figure 3 .

[0082] 2) Linear single-stranded DNA

[0083] The pMT2d5C9-cssDNA was further subjected to ribozyme cleavage, and the reaction system is shown in Table 2. After preparation, the system was thoroughly mixed and centrifuged briefly to eliminate bubbles. The reaction was carried out at 37°C for 30 min.

[0084] Table 2 Enzymatic cleavage reaction system

[0085]

[0086] The enzymatic cleavage reaction product was subjected to single-strand verification. The reaction solution of the reaction system was directly hydrolyzed by exonuclease Exo V, and then gel electrophoresis was used for detection. The detection results are shown in Figure 5 . It can be seen from Figure 5 that the product after enzymatic cleavage can be hydrolyzed by exonuclease, i.e., pMT2d5C9-ssDNA was successfully prepared.

[0087] Example 3: Generation of circular single-stranded DNA by pMT2d5C9M10

[0088] The recombinant phage packaging plasmid pMT2d5C9M10 was obtained by PCR subcloning, and was composed of a start sequence, a target sequence and a stop sequence in order. The start sequence was a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted of the sequence from 1 to 290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with the sequence from 291 to 381. The stop sequence was a deletion stop sequence as shown in SEQ ID NO: 7, with a sequence length of 302 nt, in which the stop sequence as shown in SEQ ID NO: 7 was deleted of the sequence from 151 to 160 and the sequence from 313 to 330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with the sequence from 1 to 150 and the sequence from 161 to 312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0089] The total length of the pMT2d5C9M10-css DNA was 4966 nt, in which the target sequence was 4595 nt, and the deleted M13 ori was as shown in SEQ ID NO: 8, which was deleted of 10 nucleotides at the position from 151 to 160 compared with the wild-type M13 ori, with a length of 371 nt, and the agarose gel electrophoresis is shown in Figure 3 .

[0090] Example 4: pMT2d5C9R10 generates circular single-stranded DNA

[0091] The recombinant phage packaging plasmid pMT2d5C9R10 was obtained by PCR subcloning, and was composed of a start sequence, a target sequence and a stop sequence in order. The start sequence was a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted of the sequence from 1 to 290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with the sequence from 291 to 381. The stop sequence was a deletion stop sequence as shown in SEQ ID NO: 9, with a sequence length of 302 nt, in which the stop sequence as shown in SEQ ID NO: 9 was deleted of the sequence from 253 to 262 and the sequence from 313 to 330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with the sequence from 1 to 252 and the sequence from 263 to 312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0092] The total length of the pMT2d5C9R10-css DNA was 4966 nt, in which the target sequence was 4595 nt, and the deleted M13 ori was as shown in SEQ ID NO: 10, which was deleted of 10 nucleotides at the position from 253 to 262 compared with the wild-type M13 ori, with a length of 371 nt, and the agarose gel electrophoresis is shown inFigure 3 .

[0093] Example 5: pMT2d5C9MRd generates circular single-stranded DNA

[0094] The recombinant phage packaging plasmid pMT2d5C9MRd 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 a deleted start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted from 1-290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-381. The stop sequence was a deleted stop sequence as shown in SEQ ID NO: 11, with a sequence length of 200 nt, in which the stop sequence as shown in SEQ ID NO: 11 was deleted from 151-262 and 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-150 and 263-312. The target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0095] The total length of pMT2d5C9MRd-cssDNA was 4864 nt, in which the target sequence was 4595 nt. The deleted M13 ori was as shown in SEQ ID NO: 12, which was deleted from 151-262 of the wild-type M13 ori, with a length of 269 nt. The agarose gel electrophoresis is shown in Figure 3 .

[0096] Example 6: pMT2d5C9MRdM75 generates circular single-stranded DNA

[0097] The recombinant phage packaging plasmid pMT2d5C9MRdM75 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 a deleted start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted from 1-290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-381. The stop sequence was a deleted stop sequence as shown in SEQ ID NO: 13, with a sequence length of 125 nt, in which the stop sequence as shown in SEQ ID NO: 13 was deleted from 76-232 and 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-75 and 233-312. The target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0098] pMT2d5C9MRdM75R8-css DNA total length 4781 nt, of which the target sequence is 4595 nt, the deleted M13 ori is shown as SEQ ID NO: 16, which is 195 nucleotides deleted compared with the wild-type M13 ori from 76 to 270, and the length is 186 nt, agarose gel electrophoresis is shown in Figure 3 .

[0099] Example 7: pMT2d5C9MRdM75R8 generates circular single-stranded DNA

[0100] The recombinant phage packaging plasmid pMT2d5C9MRdM75R8 is obtained by PCR subcloning, which is composed of a start sequence, a target sequence and a stop sequence in order. The start sequence is a deleted start sequence shown as SEQ ID NO: 5, the sequence length is 91 nt, and the start sequence shown as SEQ ID NO: 5 is deleted from 1 to 290 of the full-length start sequence shown as SEQ ID NO: 1, which is consistent with the sequence from 291 to 381; the stop sequence is a deleted stop sequence shown as SEQ ID NO: 15, the sequence length is 117 nt, and the stop sequence shown as SEQ ID NO: 15 is deleted from 76 to 270 and 313 to 330 of the full-length stop sequence shown as SEQ ID NO: 2, which is consistent with the sequence from 1 to 75 and 271 to 312, and the target sequence is shown as SEQ ID NO: 4. Other steps refer to Example 1.

[0101] pMT2d5C9MRdM75R8-css DNA total length 4781 nt, of which the target sequence is 4595 nt, the deleted M13 ori is shown as SEQ ID NO: 16, which is 195 nucleotides deleted compared with the wild-type M13 ori from 76 to 270, and the length is 186 nt, agarose gel electrophoresis is shown in Figure 3 .

[0102] Example 8: pMT2d5C9MRdM75R16 generates circular single-stranded DNA

[0103] The recombinant phage packaging plasmid pMT2d5C9MRdM75R16 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 a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted from 1-290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-381; the stop sequence was a deletion stop sequence as shown in SEQ ID NO: 17, with a sequence length of 109 nt, in which the stop sequence as shown in SEQ ID NO: 17 was deleted from 76-278 and 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-75, 279-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0104] The total length of pMT2d5C9MRdM75R16-cssDNA was 4773 nt, in which the target sequence was 4595 nt, and the deletion M13 ori was as shown in SEQ ID NO: 18, which was deleted from 76-278 of the wild-type M13 ori, with 203 nucleotides, and the length was 178 nt, which was shown by agarose gel electrophoresis Figure 3 .

[0105] Example 9: pMT2d5C9MRdM75R16dS generates circular single-stranded DNA

[0106] The recombinant phage packaging plasmid pMT2d5C9MRdM75R16dS 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 a deletion start sequence as shown in SEQ ID NO: 19, with a sequence length of 39 nt, in which the start sequence as shown in SEQ ID NO: 18 was deleted from 1-290 and 330-381 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-329; the stop sequence was a deletion stop sequence as shown in SEQ ID NO: 17, with a sequence length of 109 nt, in which the stop sequence as shown in SEQ ID NO: 17 was deleted from 76-278 and 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-75, 279-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0107] pMT2d5C9MRdM75R16dS-cssDNA total length 4704 nt, target sequence 4595 nt, deleted M13 ori as shown in SEQ ID NO: 20, compared with wild type M13 ori, 203 nucleotides from 76-278 and 69 sequences from 313-381 are deleted, length 109 nt, agarose gel electrophoresis is shown in Figure 4 .

[0108] Example 10: pMT2d5C9MRdM83R16 generates circular single-stranded DNA

[0109] Recombinant phage packaging plasmid pMT2d5C9MRdM83R16 is obtained by PCR subcloning, consisting of a start sequence, a target sequence and a stop sequence in order. The start sequence is a deleted start sequence as shown in SEQ ID NO: 5, sequence length 91 nt, SEQ ID NO: 5 deletes 1-290 sequences from the full-length start sequence shown in SEQ ID NO: 1, consistent with 291-381 sequences; the stop sequence is a deleted stop sequence as shown in SEQ ID NO: 21, sequence length 94 nt, SEQ ID NO: 21 deletes 68-278 sequences and 313-330 sequences from the full-length stop sequence shown in SEQ ID NO: 2, consistent with 1-67, 279-312 sequences, the target sequence is as shown in SEQ ID NO: 4. Other steps refer to Example 1.

[0110] pMT2d5C9MRdM83R16-cssDNA total length 4765 nt, target sequence 4595 nt, deleted M13 ori as shown in SEQ ID NO: 22, compared with wild type M13 ori, 211 nucleotides from 68-278 are deleted, length 170 nt, agarose gel electrophoresis is shown in Figure 3 , sequencing map is shown in Figure 6 .

[0111] Example 11: pMT1d3C2

[0112] The recombinant phage packaging plasmid pMT1d3C2 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 a complete start sequence as shown in SEQ ID NO: 1, with a sequence length of 381 nt; the stop sequence was a deleted stop sequence as shown in SEQ ID NO: 23, with a sequence length of 262 nt, wherein the stop sequence as shown in SEQ ID NO: 23 was deleted from 263-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-262; and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis is shown in Figure 7 It can be known from Figure 7 that the phage packaging plasmid pMT2d5C10 did not successfully package the circular single-stranded DNA.

[0113] Example 12: pMT1d5C1

[0114] The recombinant phage packaging plasmid pMT1d5C1 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 a complete start sequence as shown in SEQ ID NO: 1, with a sequence length of 381 nt; the stop sequence was a deleted stop sequence as shown in SEQ ID NO: 24, with a sequence length of 280 nt, wherein the stop sequence as shown in SEQ ID NO: 24 was deleted from 281-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-280; and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1, and the agarose gel electrophoresis of the final product is shown in Figure 7 . It can be known from Figure 7 that the phage packaging plasmid pMT1d5C1 did not successfully package the circular single-stranded DNA.

[0115] Example 13: pMT2d5C10

[0116] The recombinant phage packaging plasmid pMT2d5C10 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 a deletion start sequence as shown in SEQ ID NO: 25, with a sequence length of 81 nt, in which the start sequence as shown in SEQ ID NO: 25 was deleted from 1-300 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 301-381; the stop sequence was a deletion stop sequence as shown in SEQ ID NO: 6, with a sequence length of 312 nt, in which the stop sequence as shown in SEQ ID NO: 6 was deleted from 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis of the final product is shown in Figure 7 It can be seen from Figure 7 that the phage packaging plasmid pMT2d5C10 did not successfully package the circular single-stranded DNA.

[0117] Example 14: pMT2d3C1

[0118] The recombinant phage packaging plasmid pMT2d3C1 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 a deletion start sequence as shown in SEQ ID NO: 26, with a sequence length of 331 nt, in which the start sequence as shown in SEQ ID NO: 26 was deleted from 332-381 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 1-331; the stop sequence was a deletion stop sequence as shown in SEQ ID NO: 6, with a sequence length of 312 nt, in which the stop sequence as shown in SEQ ID NO: 6 was deleted from 313-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis of the final product is shown in Figure 7 . It can be seen from Figure 7 that the phage packaging plasmid pMT2d5C10 did not successfully package the circular single-stranded DNA.

[0119] Example 15: pMT2d5C9F10

[0120] ​The recombinant phage packaging plasmid pMT2d5C9F10 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 a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted from 1-290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-381; the stop sequence was a deletion stop as shown in SEQ ID NO: 27, with a sequence length of 302 nt, in which the stop sequence as shown in SEQ ID NO: 27 was deleted from 51-60 and 312-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-50, 61-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis of the final product is shown in Figure 7 It can be seen from Figure 7 that the phage packaging plasmid pMT2d5C9F10 did not successfully package the circular single-stranded DNA.

[0121] Example 16: pMT2d5C9MRdR25

[0122] The recombinant phage packaging plasmid pMT2d5C9MRdR25 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 a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the start sequence as shown in SEQ ID NO: 5 was deleted from 1-290 of the full-length start sequence as shown in SEQ ID NO: 1, and was consistent with 291-381; the stop sequence was a deletion stop as shown in SEQ ID NO: 28, with a sequence length of 175 nt, in which the stop sequence as shown in SEQ ID NO: 28 was deleted from 151-287 and 312-330 of the full-length stop sequence as shown in SEQ ID NO: 2, and was consistent with 1-150, 288-312, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis of the final product is shown in Figure 7 It can be seen from Figure 7 that the phage packaging plasmid pMT2d5C9MRdR25 did not successfully package the circular single-stranded DNA.

[0123] Example 17: pMT2d5C9MRdM90R16

[0124] ​The recombinant phage packaging plasmid pMT2d5C9MRdM90R16 was obtained by PCR subcloning, and was composed of a start sequence, a target sequence and a stop sequence in order. The start sequence was a deletion start sequence as shown in SEQ ID NO: 5, with a sequence length of 91 nt, in which the sequence of 1-290 of the full-length start sequence as shown in SEQ ID NO: 1 was deleted, and the sequence of 291-381 was kept unchanged; the stop sequence was a deletion stop as shown in SEQ ID NO: 29, with a sequence length of 94 nt, in which the sequences of 61-278 and 312-330 of the full-length stop sequence as shown in SEQ ID NO: 2 were deleted, and the sequences of 1-60 and 179-312 were kept unchanged, and the target sequence was as shown in SEQ ID NO: 4. Other steps refer to Example 1. The agarose gel electrophoresis of the final product is shown in Figure 7 It can be seen from Figure 8 that the phage packaging plasmid pMT2d5C9MRdM90R16 did not successfully package the circular single-stranded DNA.

[0125] Example 18: Preparation of mini-circle DNA

[0126] The single-primer PCR method was used for asymmetric amplification of the css DNA by taking the circular single-stranded DNA (pMPdID2C-cssDNA) as a template. Then, the DNA double strands and DNA single strands in the product were digested by exonuclease V (RecBCD), and the circular (with a cut) cdsDNA was reserved. Finally, the product was purified by column purification and quantified.

[0127] 1) Asymmetric amplification of css DNA

[0128] 1.1 Asymmetric PCR amplification was performed on pMPdID2C-cssDNA, and the amplification system is shown in Table 3. After preparation, the system was mixed thoroughly and centrifuged briefly by a mini centrifuge to eliminate bubbles in the system. The PCR amplification program is shown in Table 4.

[0129] Table 3 Asymmetric PCR amplification system of css DNA

[0130]

[0131] Table 4 PCR amplification program

[0132]

[0133] 2) Digestion reaction solution

[0134] The reaction solution obtained in the previous step was digested, the digestion system is shown in Table 5, and centrifuged for 5 s. Then, it was placed in a 37 ℃ constant temperature incubator and digested for 30 min.

[0135] Table 5 Digestion system

[0136]

[0137] 3) Purification of the reaction solution

[0138] The reaction solution was purified using a general DNA product purification kit from Tian Gen. Before use, anhydrous ethanol was added to the rinse solution PW, and the volume was referred to the label on the bottle.

[0139] 3.1 Column equilibration: 500 μL of equilibration solution BL was added to the adsorption column CB2 (the adsorption column was placed in the collection tube), centrifuged at 12,000 rpm for 1 min, and the waste liquid in the collection tube was discarded.

[0140] 3.2 5 times the volume of PB was added to the reaction solution and mixed well.

[0141] 3.3 The solution obtained in the previous step was added to the adsorption column CB2 (the adsorption column was placed in the collection tube), and was placed at room temperature for 2 min, centrifuged at 12,000 rpm for 1 min, and the waste liquid in the collection tube was discarded. The adsorption column CB2 was placed in the collection tube.

[0142] 3.4 600 μL of rinse solution PW was added to the adsorption column CB2, centrifuged at 12,000 rpm for 1 min, and the waste liquid in the collection tube was discarded. The adsorption column CB2 was placed in the collection tube.

[0143] 3.5 Repeat operation step 3.4.

[0144] 3.6 The adsorption column CB2 was placed back into the collection tube and centrifuged at 12,000 rpm for 2 min. Then, the adsorption column CB2 was placed at room temperature for several minutes and completely air-dried.

[0145] 3.7 A certain amount of enzyme-free water (about 30-60 μl) was added to the adsorption column to elute, and was placed for 2 min, and then centrifuged at 12,000 rpm for 2 min.

[0146] 3.8 The product after centrifugation was detected by agarose gel electrophoresis.

[0147] After the above experimental process, ​ The agarose gel electrophoresis results showed that only one bright band was obtained after digestion and purification, i.e., the circular single-stranded DNA was converted from single-stranded to double-stranded.

[0148] The present application discloses a recombinant bacteriophage packaging plasmid, which is capable of packaging circular single-stranded DNA. The recombinant bacteriophage packaging plasmid provided by the present application has a shortened initiation sequence, which can be shortened to the sequence shown in positions 291-329 of SEQ ID NO: 1. However, the termination sequence of the recombinant bacteriophage packaging plasmid must at least contain two sequences shown in positions 1-67 and 279-312 of SEQ ID NO: 2, so as to successfully package the circular single-stranded DNA. The circular single-stranded DNA prepared by the recombinant bacteriophage packaging plasmid provided by the present application has a shorter fixed phase sequence compared with the traditional full-length M13 ori sequence, which can reduce the influence of M13 ori as a backbone sequence on the target sequence. The preparation of microcircular DNA by the circular single-stranded DNA provided by the present application is relatively simple and convenient, and has a lower cost compared with the traditional method.

[0149] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as limiting the scope of the patent of the present application. It should be noted that those skilled in the art can make various changes, modifications and substitutions without departing from the spirit of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

[0150] Although the present application is described in detail with reference to the embodiments thereof, the embodiments are provided for illustration and not limitation. Other embodiments obtained according to the principles of the present application all belong to the scope defined by the claims of the present application.

Claims

1. A recombinant bacteriophage 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 is a sequence of x-v in SEQ ID NO: 1, the termination sequence is composed of sequences of 1-y and z-w in SEQ ID NO: 2, and x ≤ 291, 329 ≤ v ≤ 381, 67 ≤ y < z ≤ 279, w = 312, wherein x, y, z, v are positive integers, and the starting sequence is not the sequence of SEQ ID NO: 1 and / or the termination sequence is not the sequence of SEQ ID NO:

2.

2. The recombinant bacteriophage packaging plasmid according to claim 1, wherein, The recombinant phage packaging plasmid further comprises at least one deoxyribozyme cleavage site or a restriction endonuclease cleavage site.

3. The recombinant bacteriophage packaging plasmid of claim 1, wherein, The x = 291, the v = 329, and the starting sequence comprises at least a sequence of 291-329 in SEQ ID NO:

1.

4. The recombinant bacteriophage packaging plasmid of claim 1, wherein, The y = 67, the z = 279, the w = 312, and the termination sequence is a sequence of 1-67 and 279-312 in SEQ ID NO:

2.

5. The recombinant bacteriophage packaging plasmid of claim 1, wherein, The starting sequence is a sequence of SEQ ID NO: 5 or SEQ ID NO:

19.

6. The recombinant bacteriophage packaging plasmid of claim 1, wherein, The termination sequence is selected from any one of the sequences of 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 a circular single-stranded DNA, characterized by, The preparation method comprises designing and constructing the recombinant phage packaging plasmid of any one of claims 1-6, transforming a host cell with the recombinant phage packaging plasmid and a helper plasmid for replication, and extracting the circular single-stranded DNA generated in the host cell.

8. A circular single-stranded DNA, characterized in that, The circular single-stranded DNA comprises a target sequence and a deleted M13 ori sequence, and the deleted M13 ori sequence is composed of sequences of 1-y and z-312 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, wherein 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 by, The circular single-stranded DNA of claim 9 is cleaved with a deoxyribozyme or a restriction endonuclease to obtain a linear single-stranded DNA.

11. A method for preparing a minicircle DNA, characterized by, The preparation method comprises preparing the circular single-stranded DNA of claim 8 or 9, and performing in vitro complementation on the circular single-stranded DNA to obtain a mini-circle DNA.

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