A vector backbone, a test plasmid, and a construction method and application thereof

Abstract

The present invention discloses a vector backbone and a method for constructing the same. The present invention also discloses a test plasmid and a method for constructing the same. The autonomous replication and stable existence characteristics of the episome of the present invention complement the efficient integration ability of the Piggybac transposon system. The episome provides more copies of foreign genes, while the transposon system ensures that these copies can be accurately and efficiently integrated into the host genome. The two work together to greatly improve the success rate and efficiency of foreign gene integration, overcoming some limitations existing in traditional gene integration methods, such as low integration efficiency and unstable gene expression. The mutation efficiency of the present invention is high, up to 65% at most, and the mutation frequency of the present invention is stable.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a vector backbone, a test plasmid, and a construction method and application thereof. Background Art

[0002] Transposon systems, also known as jumping genes, are a class of DNA sequences that can insert and excise within the genome and change their own positions. According to different transposition methods, transposons can be divided into two major categories: DNA transposons and retrotransposons. DNA transposons follow the "cut-and-paste" mechanism, while retrotransposons use the "copy-and-paste" mechanism for transposition. The diversity and functions of transposons make them important tools for genome research, molecular genetics, and bioengineering.

[0003] The PiggyBac (PB) transposon system is a mobile genetic element belonging to DNA transposons. It was initially discovered in the cabbage looper ( Hyloicus pinastri ), and the "Piggy" in its name comes from this moth. As an efficient transposon system, the PB transposon transposes between vectors and chromosomes through the "cut-and-paste" mechanism. In this process, the PB transposase can recognize the specific inverted terminal repeat sequences (ITRs) located at both ends of the transposon vector, and effectively move the content between these sequences from the original site, and then precisely integrate it into the TTAA site of the host genome. This powerful transposition activity enables the target gene between the two ITRs in the PB vector to be easily transferred into the target genome, providing a powerful tool for gene therapy and functional research. Most studies on the PB transposon separate the PB transposon module from the transposase, insert the target gene to be transposed between the repeat sequences at both ends of the transposon, and express the transposase on another vector, which can efficiently induce the transposase to regulate the transposition of the transposon.

[0004] Currently, the randomness of the integration sites of the Piggybac transposon system may lead to the uncertainty of gene expression and differences in integration efficiency in different cell types. In some cell types with a higher degree of differentiation, due to the influence of chromatin structure and epigenetic modifications, its integration efficiency is low, etc., which limits its wide application in genetic engineering. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a vector backbone and a method for constructing the same. The vector backbone of the present invention contains an episome and a PB transposon. Among them, the episome is a genetic element that can replicate autonomously in cells independently of chromosomes. Combining the episome with the PB transposon system is expected to overcome the deficiencies of the PB transposon system when used alone, improve the integration efficiency and expression stability of foreign genes, and provide a more effective tool for genomic research, molecular genetics, and bioengineering.

[0006] Another technical problem to be solved by the present invention is to provide a test plasmid and a method for constructing the same.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned vector backbone and the above-mentioned test plasmid in the preparation of diagnostic standards.

[0008] Technical Solution: To solve the above problems, the present invention provides a vector backbone, which includes a 5'-end inverted terminal repeat sequence, a selection marker 1 for screening and identification, a promoter sequence, a sequence for transcriptional termination, a selection marker 2 for subsequent selection or visualization of transformed cells, a 3'-end inverted terminal repeat sequence, an episome sequence, a resistance gene sequence, and the gene sequence of the piggyBac transposon with a pUC replication origin. The episome is the OriP and EBNA1 genes.

[0009] Among them, the selection marker 1 includes a visual detection gene, the promoter includes a CAG promoter, the sequence for transcriptional termination includes a rabbit β-globin polyadenylation signal sequence, and the selection marker 2 includes one or several of the drug selection markers puromycin, geneticin, or hygromycin.

[0010] Among them, the resistance gene includes an ampicillin resistance gene.

[0011] The present invention also includes a test plasmid, which includes the above-mentioned vector backbone.

[0012] Among them, the test plasmid also includes a mutation sequence.

[0013] The present invention also includes a method for combining an episome with a piggyBac transposon, which includes the following steps:

[0014] 1) PCR amplification to obtain the target sequence;

[0015] 2) Construct the above-mentioned vector backbone.

[0016] The present invention also includes a method for constructing a test plasmid, which includes introducing a mutation sequence into the above-mentioned vector backbone to obtain it.

[0017] Among them, the mutant sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The autonomous replication and stable existence characteristics of the episome of the present invention complement the efficient integration ability of the Piggybac transposon system. The episome provides more copies of foreign genes, while the transposon system ensures that these copies can be accurately and efficiently integrated into the host genome. The two work together to greatly improve the success rate and efficiency of foreign gene integration, overcoming some limitations existing in traditional gene integration methods, such as low integration efficiency and unstable gene expression. This combination method of the present invention can also expand the applicable scope of gene integration technology. For example, in different types of cells or organisms, the performance of the episome and the Piggybac transposon system may be different, but after combination, efficient foreign gene integration can be achieved in a wider range of cell types and species, providing a more general and effective technical means for gene therapy, the establishment of transgenic animal models, and other genetic engineering applications, further reflecting its value in improving integration efficiency. The mutation efficiency of the present invention is high, up to 65% at most, and the mutation frequency of the present invention is stable. Description of the Drawings

[0019] Figure 1 Electrophoresis diagram of the PCR product for obtaining the cloned target sequence in Example 1;

[0020] Figure 2 Mutation frequency result diagram of the episome plus PB transposon in Example 1;

[0021] Figure 3 Mutation frequency result diagram of only the PB transposon in Example 1;

[0022] Figure 4 Mutation frequency result diagram of only the episome in Example 1;

[0023] Figure 5 Vector backbone map for constructing a vector containing a pure PB transposon plus mutant sequence 1;

[0024] Figure 6 Vector backbone map for constructing a vector containing a pure episome plus mutant sequence 1;

[0025] Figure 7 Vector backbone map for constructing a vector containing an episome plus PB transposon plus mutant sequence 1. Detailed Embodiments

[0026] Introduction of each element of the vector map in the embodiments of the present invention:

[0027] 5' ITR: 5' inverted terminal repeat sequence. When a DNA sequence is flanked by two ITRs, the PiggyBac transposon can recognize them and insert the flanked region (including the two ITRs) into the host genome.

[0028] Selection marker 1: A visually detectable gene (such as EGFP), the fluorescence of which can be used to screen and identify successfully transfected or transformed cells.

[0029] CAG promoter: The CAG promoter is an artificially constructed composite promoter composed of the cytomegalovirus early enhancer and the chicken β-actin promoter, which is used to drive the broad expression of downstream marker genes.

[0030] rBG pA: Rabbit β-globin polyadenylation signal, which helps the transcriptional termination of the upstream ORF.

[0031] Selection marker 2: Drug selection markers (such as puromycin (Puro), geneticin (Neo), hygromycin (Hygro)), which allow cells transduced with the vector to be selected or visualized.

[0032] 3' ITR: 3' inverted terminal repeat sequence.

[0033] OriP: OriP is a specific region in the Epstein-Barr virus (EBV) genome. It serves as the origin of replication and plays a central role in replicating and maintaining the genome during the latent phase of EBV. It consists of the family repeat (FR) element and the dyad symmetry (DS) element, and these two elements coordinately regulate the replication process of viral DNA.

[0034] EBNA1: EBNA1 is a key nuclear protein encoded by EBV. It plays a crucial role in the latent and lytic replication phases of the virus. It maintains and replicates the EBV genome by directly binding to the OriP region. During the lytic replication period, EBNA1 redistributes to specific nuclear replication regions and participates in the replication process of newly synthesized viral DNA together with viral replication proteins.

[0035] AmpiciIIin: Ampicillin resistance gene.

[0036] pUC ori: pUC origin of replication. Plasmids carrying this origin exist in Escherichia coli at high copy numbers. Example 1

[0037] 1. Construction of vector backbone and preparation of test plasmid

[0038] Construct a transposon vector: Construct a vector backbone of the PiggyBac transposon containing EBNA1 and orip (episome).

[0039] 1) Obtain the cloned target sequence by PCR

[0040] Configure the reaction system with VB163 plasmid (Suzhou Haixing Biotechnology Co., Ltd., shown in SEQ ID NO.3), including the upstream primer G54663-1F and downstream primer G54663-1R shown in Table 3, Q5® Ultra-Fidelity DNA Polymerase (NEB, catalog number M0491V, 2,000 U / mL), 5*Q5 buffer, 10 mM dNTP, and ddH2O. The reaction system is shown in Table 1, and then perform amplification according to the set reaction conditions, which include steps such as pre-denaturation at 95°C, denaturation at 95°C, annealing at 60°C, and extension at 72°C. The specific conditions are shown in Table 2.

[0041] Table 1 Reaction System

[0042]

[0043] Table 2 PCR Reaction Conditions

[0044]

[0045] Table 3 Specific Primer Sequences

[0046]

[0047] Perform electrophoresis analysis on the PCR product. After verifying the product band, perform gel cutting and recovery to verify the product band. Meeting the conditions of "unique band, clear band" meets the requirements. Perform gel cutting and recovery on the qualified PCR product (refer to the Axygen product manual), and use the recovered PCR product for the next ligation experiment. The results are shown in Figure 1 .

[0048] Mix the circular empty plasmid VB158 (Suzhou Haixing Biotechnology Co., Ltd., shown in SEQ ID NO.4) with the restriction endonuclease and perform enzymatic digestion reaction at 37°C for 60 min to obtain the linearized plasmid; the enzymatic digestion system is shown in Table 4.

[0049] Table 4 Enzymatic Digestion System

[0050]

[0051] Mix the gel-cut and recovered PCR product with the linearized plasmid, add the recombinase ClonExpress II (Nanjing Novoprotein Scientific Co., Ltd., catalog number C112-01) for ligation reaction, incubate in a water bath at 52°C for 40 min, and let it stand for 5 min to reduce the temperature. The ligation system is shown in Table 5.

[0052] Table 5 Ligation System

[0053]

[0054] Transfer the ligation product into competent cells. After ice-bathing for 10 min, heat-shock at 42 °C for 90 s, immediately ice-bathe for 3 min, add 800 µL of antibiotic-free LB medium and culture for 1 h. Centrifuge the cultured sample to remove part of the supernatant, mix the remaining and spread it on a culture dish, then incubate it upside down overnight to screen for positive clones. Finally, pick single colonies, add 2×Phanta Max MasterMix (Nanjing Novoprotein Scientific Inc., catalog number P515-01) for colony PCR. The reaction system for colony PCR is shown in Table 6, and the reaction conditions are shown in Table 7.

[0055] Table 6 Reaction system for colony PCR

[0056]

[0057] Table 7 Reaction conditions

[0058]

[0059] After electrophoretically identifying the positive clones, extract the plasmid and perform sequencing verification, and send it to Saisuofei Biotechnology Co., Ltd. for Sanger sequencing. The finally obtained plasmid has correct enzyme digestion bands, and the sequencing results show that the sequence (shown in SEQ ID NO.5) is 100% correct, and a PB vector backbone containing an episome is obtained.

[0060] Determine the mutation sites on the reference sequence, synthesize mutant sequence 1 and mutant sequence 2, and clone mutant sequence 1 and mutant sequence 2 into the PB vector backbone containing an episome, the PB vector backbone (VB163 plasmid, shown in SEQ ID NO.3), and the episomal vector backbone (VB158 plasmid, shown in SEQ ID NO.4) respectively to form test plasmids and control plasmids (this process is sent to Saisuofei Biotechnology Co., Ltd. to complete). Specifically, clone mutant sequence 1 and mutant sequence 2 between the BamHⅠ and SmaⅠ restriction enzyme sites of the PB vector backbone to obtain control plasmid 1 and control plasmid 2; clone mutant sequence 1 and mutant sequence 2 between the BamHⅠ and EcoRⅠ restriction enzyme sites of the episomal vector backbone to obtain control plasmid 3 and control plasmid 4; clone mutant sequence 1 and mutant sequence 2 at the BamHⅠ restriction enzyme site of the PB vector backbone containing an episome to obtain test plasmid 1 and test plasmid 2. And perform transfection-grade large-scale extraction of nucleic acids with low endotoxin on the obtained plasmids.

[0061] Mutant sequence 1 (shown in SEQ ID NO.1):

[0062] AAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGtcGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGA

[0063] Mutated sequence 2 (shown as SEQ ID NO.2):

[0064] AAGGTGAGTTTGTATTAAAAGGTACTGGTGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAGGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGaaGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGA

[0065] 2. Cell treatment and electroporation

[0066] Take HEK293T cells in the logarithmic growth phase, digest, centrifuge to remove the supernatant, resuspend with PBS, count, and take 1×10 6Transfer the cells to an EP tube and centrifuge to obtain cell pellets. Resuspend the cells with electroporation buffer (Opti-MEM™ I, Thermo Fisher Scientific, catalog number 31985-070), and add the plasmid (test plasmid 1 or test plasmid 2: PBase plasmid (PUF07 from Suzhou Haixing Biotechnology Co., Ltd.) at a ratio of 2:1) and mix well. Input the optimal electroporation parameters of 480 V for electroporation, and electroporate the EGFP plasmid (PUC01 from Suzhou Haixing Biotechnology Co., Ltd., catalog number) as a control group under the same parameters. Transfer the electroporated cells to high-glucose DMEM (DMEM-H) medium containing 10% fetal bovine serum (FBS) for culture. Observe the cell status after 24 h, and judge the electroporation efficiency according to the EGFP control group. After 48 h, use puromycin (Puro) at a concentration of 4.0 μg / mL for drug screening, and the drug screening period is 3 d. After the drug screening is completed, remove the drug-containing medium and replace it with fresh medium, and culture for 24 - 48 h to restore the cell status.

[0067] 3. Mutation Detection and Integration Efficiency Evaluation

[0068] First, passage the cells at generations 1, 3, 5, 9, 12, 15, and 18, and extract DNA for subsequent experiments. Use an animal tissue DNA kit (Hangzhou Xinjing Biological Reagent Development Co., Ltd., catalog number 3101050) to process the animal tissue samples. The sample processing is carried out according to the operation instructions of the SIMGEN animal tissue DNA kit. After DNA extraction, use Nano-400A to detect the sample concentration and record it, and dilute the sample to 50 ng / μl.

[0069] Next, design and synthesize primers for the upstream and downstream of the mutation site on the NCBI website. After design and synthesis, place an order for synthesis at Ascent Biotechnology Co., Ltd. The primer sequences are shown in Table 8:

[0070] Table 8 Primer Sequences

[0071]

[0072] The enzyme used for PCR identification is the PCR special enzyme 2 × TaqMaster Mix (catalog number: P112-01) from Novoprotein Scientific Inc. According to the instructions of the PCR enzyme, configure the PCR reaction system as shown in the following table. Calculate the total system according to the total amount of the sample for configuration and aliquoting. This step must be operated on a 96-well metal ice box.

[0073] Place the required upstream and downstream primers, ddH2O, and P112 enzyme in an ice box. Take out a 96-well PCR plate, mark the sample information, operation date, etc. on the bottom of the plate, and mark the added template in the upper right corner of the wells with the pre-prepared system. Load the samples according to the markings. After completion, centrifuge and mix well using a 96-well plate low-speed centrifuge. After centrifugation, place the PCR plate in the preheated PCR instrument, cover the thermal lid, set the reaction program, and then click start. The 25 μL reaction system and reaction program are shown in Tables 9 and 10.

[0074] Table 9 Reaction System

[0075]

[0076] Table 10 Reaction Program

[0077]

[0078] Take out the agarose gel from the mold, soak it in TBE solution and wait for the bubbles in the pores to overflow, then take out the gel and place it on the operating table. Pipette 5 μL of the PCR product and load it into the pores in the specified order. Use StarMarker D5000 from Kangrun Biotech for the Marker. After loading, place the gel in the electrophoresis tank, set the electrophoresis voltage to 300 V, and the electrophoresis time to 15 min. After 15 min, take out the agarose gel and take a UV photo. According to the electrophoresis pattern, compare whether the electrophoresis bands match the theoretical size and whether the bands are clear and bright. If the electrophoresis bands are qualified, it proves that the PCR product is qualified. Pipette the PCR product and the upstream and downstream primers used for amplification, and send them to Saisuofei Biotech Co., Ltd. for Sanger sequencing. Perform Sanger sequencing on the PCR amplification products of the identified samples and wild-type samples (control group), and compare the obtained sequencing results on the ICE website (https: / / ice.synthego.com / # / ), and reliable target site mutation frequency data can be obtained. Integration efficiency evaluation: Evaluate and compare the integration efficiency of the system by comparing the mutation frequencies of different test plasmids. A high mutation frequency indicates a high integration efficiency.

[0079]

[0080] The mutation frequencies in this example are shown in Table 11 and Figure 2 as follows.

[0081] Table 11 Mutation Frequencies of Episome Plus PB Transposon

[0082]

[0083] Comparative Example 1

[0084] The control plasmids 1 and 2 constructed in Example 1 were tested for mutation frequency, and the results are shown in Table 12 and Figure 3 as follows.

[0085] Table 12 Mutation frequencies of individual PB transposons containing mutant sequences

[0086]

[0087] Comparative Example 2

[0088] The control plasmids 3 and 4 constructed in Example 1 were tested for mutation frequency, and the results are shown in Table 13 and Figure 4 as follows.

[0089] Table 13 Mutation frequencies of individual episomal vector backbones containing mutant sequences

[0090]

[0091] The present invention separately demonstrates individual PB transposons containing mutant sequences (control plasmids 1 and 2), individual episomes (control plasmids 3 and 4), and the combination of episomes and PB transposons (test plasmids 1 and 2). Whether it is an individual PB transposon or an individual episome, their mutation frequencies show a downward trend and eventually tend to be stable, significantly lower than the case of the combination of episomes and PB transposons. In the recombinant plasmids containing mutant sequence 1, PB transposon, and episome in the present invention, the mutation frequency starts to increase gradually from the Pn+3 generation, the growth rate of the mutation frequency significantly accelerates at the Pn+9 generation, and by the Pn+12 generation, the mutation frequency has exceeded 50% and continues to increase, exceeding 60% at the Pn+18 generation. Similarly, in the recombinant plasmids containing mutant sequence 2, PB transposon, and episome, the mutation frequency starts to increase from the Pn+3 generation, the upward trend becomes significant at the Pn+5 generation, the mutation frequency has exceeded 50% at the Pn+9 generation, and remains above 50% at the Pn+18 generation, reaching 65%.

Claims

1. A carrier skeleton, characterized in that, The vector backbone sequentially includes the 5'-terminal inverted terminal repeat sequence, selection marker 1 for screening and identification, promoter sequence, sequence for transcriptional termination, selection marker 2 for the selected or visualized cells after subsequent transformation, 3'-terminal inverted terminal repeat sequence, episomal sequence, resistance gene sequence, and the gene sequence of the piggyBac transposon with the pUC replication origin, and the episome is the OriP and EBNA1 genes.

2. The carrier skeleton according to claim 1, wherein The selection marker 1 includes a visual detection gene, the promoter includes the CAG promoter, the sequence for transcriptional termination includes the rabbit β-globin polyadenylation signal sequence, and the selection marker 2 includes one or more of the drug selection markers puromycin, geneticin, or hygromycin.

3. The carrier skeleton according to claim 1, characterized in that, The resistance gene includes the ampicillin resistance gene.

4. The method for constructing the carrier skeleton according to any one of claims 1 to 3, characterized in that, It includes the following steps: 1) PCR amplification to obtain the target sequence; 2) Construct the vector backbone according to any one of claims 1 to 3.

5. A test plasmid, characterized in that, The test plasmid includes the vector backbone according to any one of claims 1 to 3.

6. The test plasmid according to claim 5, wherein The test plasmid further includes a mutant sequence.

7. A method for constructing a test plasmid, characterized in that, The construction method includes introducing the mutant sequence into the vector backbone according to any one of claims 1 to 3 to obtain it, and the mutant sequence is shown as SEQ ID NO.1 or SEQ ID NO.

2.

8. Use of the vector backbone according to any one of claims 1 to 3 and the test plasmid according to claim 5 or 6 in the preparation of a diagnostic reference standard.

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