CN118879751A - A molecular tool vector expression system and its application - Google Patents

Abstract

The present application relates to the field of biotechnology, and in particular to a molecular tool vector expression system and its application; the molecular tool vector expression system comprises an endothelial-specific gene promoter sequence and a backbone sequence for expressing snoRNA, wherein the endothelial-specific gene promoter sequence is derived from the ICAM2 gene, and the backbone sequence is derived from the No. 2 intron region of SNORD32A in the rpl13a gene; the endothelial-specific promoter derived from the ICAM2 gene is used to replace the traditional U6 or CMV promoter, so as to specifically initiate the expression of snoRNA, and in addition, the backbone sequence derived from the No. 2 intron region of SNORD32A in the rpl13a gene is designed to promote the presence of multiple splicing sites in the backbone sequence, thereby achieving accurate and efficient stable overexpression of snoRNA.

Description

A molecular tool vector expression system and its application

Technical Field

The present application relates to the field of biotechnology, and in particular to a molecular tool vector expression system and its application.

Background Art

Non-coding RNA (ncRNA) is an RNA molecule that is transcribed from DNA but not translated into protein. It mainly includes micro RNA (miRNA), transfer RNA (tRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and piwi-interacting RNA (piRNA). Among them, small nucleolar RNA (snoRNAs) is a class of medium-length non-coding small RNAs, which are generally in the range of 60nt to 300nt in length. SnoRNAs mainly guide the site-specific modification of nucleotides in target RNA through short base pairing regions; snoRNAs are widely present in the nucleolus of eukaryotic cells and play an important role in the modification of rRNA. Current studies have shown that snoRNAs are also involved in the modification of tRNA and mRNA. In addition, current studies have shown that many snoRNAs are not only dysregulated in tumors, but also their expression levels are related to clinical prognosis. Among these studies, the study of endothelial-specific snoRNA overexpression is the most prominent. The main process for overexpressing snoRNA vectors is: first directly PCR amplify the mature snoRNA sequence from the cells, construct it into a vector containing a U6 or CMV promoter, and finally use this virus to infect human umbilical vein endothelial cells to achieve endothelial-specific high expression of snoRNA.

However, the current research on endothelial-specific snoRNA overexpression has the following difficulties: first, the promoters of common high-expression vectors are U6 promoters or CMV promoters. These promoters are difficult to accurately express snoRNA in a certain type of organ and can only express snoRNA widely throughout the body, which leads to the actual expression effect not being consistent with expectations; secondly, since snoRNA is mainly encoded by the intron regions of protein-coding genes and non-protein-coding genes, the introns released during gene splicing need to be further processed to form mature snoRNA. Therefore, based on the above two points, the current stable overexpression of snoRNA has the problems of poor accuracy and low efficiency.

Summary of the invention

The present application provides a molecular tool vector expression system and its application to solve the following technical problem: how to achieve accurate and efficient stable overexpression of snoRNA.

In the first aspect, the present application provides a molecular tool vector expression system, which includes an endothelial-specific gene promoter sequence and a backbone sequence for expressing snoRNA, wherein the endothelial-specific gene promoter sequence is derived from the ICAM2 gene, and the backbone sequence is derived from the intron 2 region of SNORD32A in the rpl13a gene.

Optionally, the nucleotide sequence of the endothelial-specific gene promoter sequence is shown in SEQ ID NO.1.

Optionally, the backbone sequence includes, in sequence, an upstream sequence of intron region No. 2, intron region No. 2, and a downstream sequence of intron region No. 2, wherein the upstream sequence is a sequence of a preset length upstream of the 3’ end of intron region No. 2, and the downstream sequence is a sequence of a preset length from SNORD32A to downstream of the 5’ end of intron region No. 2.

Optionally, the preset length is 30bp.

Optionally, the nucleotide sequence of the upstream sequence is shown as SEQ ID NO.2, the nucleotide sequence of the intron region No. 2 is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream sequence is shown as SEQ ID NO.4.

Optionally, the nucleotide sequence of the backbone sequence is shown in SEQ ID NO.5.

In a second aspect, the present application provides a molecular tool vector, which has the molecular tool vector expression system described in the first aspect.

Optionally, the tool carrier includes at least one of the following:

Eukaryotic expression vectors, lentiviral vectors, adenoviral vectors and retroviral vectors.

In a third aspect, the present application provides a method for preparing the molecular tool carrier according to the second aspect, the method comprising:

Using the HUVEC cell genome as a template and using the first amplification primer set for PCR amplification, followed by purification, to obtain an ICAM2 fragment;

The ICAM2 fragment and the adeno-associated virus vector with a CMV promoter are subjected to a first double restriction digestion, followed by purification and T4 ligase ligation to obtain a first recombinant plasmid;

Using the mouse BC16 cell genome as a template and using the second amplification primer set for PCR amplification, followed by purification, to obtain a DNA sequence that overexpresses snoRNA;

Performing a second double restriction digestion on the DNA sequence overexpressing snoRNA and the first recombinant plasmid, followed by purification and T4 ligase ligation to obtain a second recombinant plasmid;

Using homologous recombination technology, the SNORD15A, SNORD118 and Dsred sequences are respectively inserted into the second recombinant plasmid, and then sequencing and screening are performed to obtain a molecular tool vector;

Wherein, the first amplification primer set has a first restriction enzyme cutting site set, and the first restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the first double restriction enzyme cutting;

The second amplification primer set has a second restriction enzyme cutting site set, and the second restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the second double restriction enzyme cutting;

The first restriction enzyme cleavage site group and the second restriction enzyme cleavage site group are different.

Optionally, the first restriction enzyme cleavage site group includes an AvrII site and a BspEI site; the second restriction enzyme cleavage site group includes an ECORⅠ site and an EagⅠ site.

The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

A molecular tool vector expression system provided in an embodiment of the present application comprises an endothelial-specific gene promoter sequence and a backbone sequence for expressing snoRNA, wherein the endothelial-specific gene promoter sequence is derived from the ICAM2 gene, and the backbone sequence is derived from the intron 2 region of SNORD32A in the rpl13a gene; compared with the traditional molecular tool vector for overexpressing snoRNA, the endothelial-specific promoter derived from the ICAM2 gene is used to replace the traditional U6 or CMV promoter, so that the expression of snoRNA can be specifically initiated, and in addition, the backbone sequence derived from the intron 2 region of SNORD32A in the rpl13a gene is designed. Since there are natural splicing site sequences on both sides of the intron 2 region, multiple splicing sites can be caused to exist in the backbone sequence, and under the premise of not introducing additional nucleic acid base sequences, the relevant enzymes in the expression stage can accurately splice the snoRNA sequence while also causing the backbone sequence to efficiently express snoRNA, thereby achieving accurate and efficient stable overexpression of snoRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

FIG1 is a plasmid map of the pscAAV-ICAM2-SNORD32A molecular tool vector provided in the examples of the present application;

FIG2 is a schematic diagram of a process for transforming the plasmid pscAAV-CMV-GFP into a molecular tool vector of pscAAV-ICAM2-GFP and pscAAV-ICAM2-SNORD32A by molecular biological means provided in an embodiment of the present application;

FIG3 is a diagram showing the effect of the specific detection promoter ICAM2 specifically promoting the expression of GFP protein provided in the examples of the present application;

FIG4 is a diagram showing the effect of the PCR detection molecular tool vector provided in the embodiment of the present application on overexpression of SNORD32A in cells via ICAM2;

FIG5 is a graph showing the results of PCR detection of molecular tool vectors overexpressing a variety of other snoRNA and GFP random gene fragments provided in an embodiment of the present application;

FIG6 is a diagram showing the actual effect of the molecular tool vector provided in the embodiment of the present application on high expression of SNORD32A in endothelial cells and cardiomyocytes;

FIG7 is a diagram showing the actual results of PCR detection of molecular tool vectors highly expressing SNORD32A provided in an embodiment of the present application;

FIG8 is a schematic flow chart of a method for preparing a molecular tool carrier provided in an embodiment of the present application.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

In this document, the terms including "including" and "comprising" mean "including but not limited to". Relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B can mean: A exists alone, A and B exist at the same time, and B exists alone; A and B can be singular or plural. "At least one" means one or more, "plurality" means two or more; "at least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c", can all represent: a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be single or multiple. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased on the market or prepared by existing methods.

It should be noted that, with respect to the prior art described in the background art, the inventors of the present application have found that the current main process for overexpressing snoRNA vectors has two major disadvantages: (1) From a cellular level, in the process of using molecular tool vectors containing U6 or CMV promoters to package viruses to infect endothelial cells, the non-specificity of U6 or CMV promoters may affect the subsequent infection efficiency and even greatly reduce the infection efficiency; (2) From an animal level, due to the organ diversity of mice, it is difficult to ensure that the virus can be specifically expressed in endothelial cells.

An embodiment of the present application provides a molecular tool vector expression system, which includes an endothelial-specific gene promoter sequence and a backbone sequence for expressing snoRNA, wherein the endothelial-specific gene promoter sequence is derived from the ICAM2 gene, and the backbone sequence is derived from the intron 2 region of SNORD32A in the rpl13a gene.

In some optional embodiments, the nucleotide sequence of the endothelial-specific gene promoter sequence is shown as SEQ ID NO.1.

In these embodiments, the specific nucleotide sequence of the endothelial-specific gene promoter sequence is clarified, and the expression of the backbone sequence can be further specifically promoted to achieve accurate and efficient stable overexpression of snoRNA.

In some optional embodiments, the backbone sequence includes, in sequence, an upstream sequence of intron region 2, intron region 2, and a downstream sequence of intron region 2, wherein the upstream sequence is a sequence of a preset length upstream of the 3’ end of intron region 2, and the downstream sequence is a sequence of the preset length from SNORD32A to downstream of the 5’ end of intron region 2.

In these embodiments, the backbone sequence may include, in sequence, the upstream sequence of intron region 2, intron region 2, and the downstream sequence of intron region 2, and the upstream sequence is a sequence of a preset length upstream of the 3' end of intron region 2, and the downstream sequence is a sequence of a preset length from SNORD32A to the 5' end downstream of intron region 2. The source of the backbone sequence and the specific splicing sites in the backbone sequence can be clearly identified, so that without introducing additional nucleic acid base sequences, the relevant enzymes in the expression stage can accurately splice the snoRNA sequence while also enabling the backbone sequence to efficiently express snoRNA.

In some optional embodiments, the preset length is 30 bp.

In these embodiments, the preset length may be 30 bp, which may further clarify the specific splicing sites in the backbone sequence.

In some optional embodiments, the nucleotide sequence of the upstream sequence is shown as SEQ ID NO.2, the nucleotide sequence of the intron region No. 2 is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream sequence is shown as SEQ ID NO.4.

In some optional embodiments, the nucleotide sequence of the backbone sequence is shown as SEQ ID NO.5.

In these embodiments, under the premise of controlling the preset length to 30bp, the specific nucleotide sequences of the upstream sequence, the intron region No. 2, and the downstream sequence in the backbone sequence are further refined, so that the snoRNA expression region and other functional regions in the backbone sequence can be clarified, and the specific splicing sites can also be clarified, so that without introducing additional nucleic acid base sequences, the relevant enzymes in the expression stage can accurately splice the snoRNA sequence while also promoting the backbone sequence to efficiently express snoRNA.

FIG1 exemplarily shows a plasmid map of the pscAAV-ICAM2-SNORD32A molecular tool vector provided in the examples of the present application;

Based on a general inventive concept, as shown in FIG1 , an embodiment of the present application provides a molecular tool vector having the molecular tool vector expression system.

The molecular tool vector is realized based on the above-mentioned molecular tool vector expression system. The specific composition and information of the molecular tool vector expression system can be referred to the above-mentioned embodiments. Since the molecular tool vector adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

In some optional embodiments, the tool carrier includes at least one of the following:

Eukaryotic expression vectors, lentiviral vectors, adenoviral vectors and retroviral vectors.

In these embodiments, the tool vector may include at least one of a eukaryotic expression vector, a lentiviral vector, an adenoviral vector and a retroviral vector, and may cover most biological molecule vectors, so that the molecular tool vector expression system can be used in various expression systems such as ordinary eukaryotic expression, lentiviral expression, adenoviral expression, retroviral expression, and prokaryotic expression.

FIG2 exemplarily shows a schematic diagram of a process for transforming the plasmid pscAAV-CMV-GFP into a molecular tool vector of pscAAV-ICAM2-GFP and pscAAV-ICAM2-SNORD32A by molecular biological means provided in an embodiment of the present application;

FIG8 exemplarily shows a schematic flow chart of a method for preparing a molecular tool carrier provided in an embodiment of the present application;

As shown in FIG. 2 and FIG. 8 , based on a general inventive concept, an embodiment of the present application provides a method for preparing the molecular tool carrier, the method comprising:

S1. Using the HUVEC cell genome as a template and using the first amplification primer set to perform PCR amplification, followed by purification to obtain an ICAM2 fragment;

S2. performing a first double restriction digestion of the ICAM2 fragment and the adeno-associated virus vector having a CMV promoter, followed by purification and T4 ligase ligation to obtain a first recombinant plasmid;

S3. using the mouse B16 cell genome as a template and using the second amplification primer set for PCR amplification, followed by purification, to obtain a DNA sequence overexpressing snoRNA;

S4. performing a second double restriction digestion on the DNA sequence overexpressing snoRNA and the first recombinant plasmid, followed by purification and T4 ligase ligation to obtain a second recombinant plasmid;

S5. Using homologous recombination technology, inserting SNORD15A, SNORD118 and Dsred sequences into the second recombinant plasmid, and then performing sequencing screening to obtain a molecular tool vector;

Wherein, the first amplification primer set has a first restriction enzyme cutting site set, and the first restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the first double restriction enzyme cutting;

The second amplification primer set has a second restriction enzyme cutting site set, and the second restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the second double restriction enzyme cutting;

The first restriction enzyme cleavage site group and the second restriction enzyme cleavage site group are different.

This method is a method for preparing the above-mentioned molecular tool carrier. The specific information of the molecular tool carrier can be referred to the above-mentioned embodiment. Since this method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be repeated here one by one.

It should be noted that the adeno-associated virus vector with a CMV promoter can be the plasmid pscAAV-GFP.

In some optional embodiments, the first restriction enzyme cleavage site group includes an AvrII site and a BspEI site; the second restriction enzyme cleavage site group includes an ECORⅠ site and an EagⅠ site.

In these embodiments, the first restriction site group may include an AvrII site and a BspEI site, and the second restriction site group may include an ECORⅠ site and an EagⅠ site, which can be used to promote the ICAM2 fragment and the DNA sequence for overexpressing snoRNA to enter the corresponding plasmid vector respectively based on high-fidelity PCR technology, so as to ultimately achieve accurate and efficient stable overexpression of snoRNA.

The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

Example 1

The endothelial-specific gene promoter sequence was designed based on the human ICAM2 gene, with a size of 0.33 Kb. The specific steps are as follows:

According to the Genebank gene database, the transcription start site of the human ICAM2 gene was analyzed, and the endothelial-specific gene promoter was screened according to the characteristic sequence of the molecular biology promoter transcription regulation gene expression. The specific nucleotide sequence of the screened endothelial-specific gene promoter sequence is:

CCAGGCATGACTCCAACAATGCATCCCATGGGATTTGGGGTTCCCCAGATCTGGGGCTTGTAGGCCTGACTCTCCCCTGTGCACACGTCTCATACACGCATGCGTGCACCCATTGCCTTGCCCCGCCCCTTGCACAGGGAGTCAGCAGGGAGGACTGGGTTATGCCCTGCTTATCAGCAGCTTCCCAGCTTCCTCTGCCTGGATTCTTAGAGGCCTGGGGTCCTAGAACGAGCTGGTGCACGTGGCTTCCCAAAGATCTCTC AGATAATGAGAGGAAATGCAGTCATCAGTTTGCAGAAGGCTAGGGATTCTGGGCCATAGCTCAGACCTGCGCCCACCATCTCCCTCCAGGCAGCCCTTGGCTGGTCCCTGCGAGCCCGTGGAGACTGCCAG (SEQ ID NO. 1).

In addition, the backbone sequence was designed based on the intron 2 region of SNORD32A in the mouse rpl13a gene, the 30 bp upstream of the 3' end of the exon in the region, and the 30 bp from the intron 2 region to the 5' end of exon 3. The specific steps are as follows:

According to the Ensembl gene database, SNORD32A was determined to be in the intron 2 region of rpl13a, and then the expression backbone sequence was designed based on the rules and principles of gene alternative splicing regulation.

The specific nucleotide sequence of the upstream sequence in the backbone sequence is:

CGGCCATTGTGGCCAAGCAGGTACTTCTGG(SEQ ID NO.2);

The specific nucleotide sequence of the intron 2 region in the backbone sequence is:

GTAAGTTTCATTCACCATTTACCTTTGCCTGGGAGTCCATGATGAGCAACACTCAC

CATCTTTCGTTTGAGTCTCACGACTGTGAGATCAACCCATGCACCGCTCTGAG

ACTCGCCAGCCCCTGCTTTCTTGGGACCCCTGGCCTAAAAATACTTTTGGAGCAAG

AGATCTGCCTTCCAAGAGGCTTTGCTGACTAATCTCCAACTCCCTGTTAACTCTAG (SEQ ID NO. 3), wherein the bold region is a replaceable region, which can be replaced by any mature snoRNA or DNA fragment;

The specific nucleotide sequence of the downstream sequence in the backbone sequence is:

GCCGGAAGGTGGTGGTCGTACGCTGTGAAG (SEQ ID NO. 4).

Therefore, the specific nucleotide sequence of the backbone sequence is:

CGGCCATTGTGGCCAAGCAGGTACTTCTGGGTAAGTTTCATTCACCATTTACCTTTGCCTGGGAGTCCATGATGAGCAACACTCACCATTCTTCGTTTGAGTCTCACGACTGTGAGATCAACCCATGCACCGCTCTGAGACTCGCCAGCCCCTGCTTTCTTGGGACCCCTGGCCTAAAAATACTTTTGGAGCAAGAGATCTGCCTTCCAAGAGGCTTTGCTGACTAATCTCCAACTCCCTGTTAACTCTAGGCCGGAAG GTGGTGGTCGTACGCTGTGAAG (SEQ ID NO. 5).

Example 2

Based on the endothelial-specific gene promoter sequence and backbone sequence prepared in Example 1, the following operations were further performed:

As shown in Figure 2, the specific molecular tool vector is constructed as follows:

1. Overexpression promoter modification:

The promoter CMV of the plasmid pscAAV-GFP was replaced with the epidermal-specific gene promoter sequence ICAM2Promoter. The specific steps are as follows:

Based on high-fidelity PCR technology, PCR amplification was performed with the human HUVEC cell genome as a template and the first amplification primer set {including the first amplification upstream primer: F: ATACCTAGGCCAGGCATGACTCCAACAATGC, (with an AvrII site), the first amplification downstream primer: R: ATATCCGGATCTCTGGCAGTCTCCACG, (with a BspEI site)} (the specific amplification program is shown in Table 1), and the PCR product was then subjected to agarose gel electrophoresis and then gel recovery and purification to obtain the ICAM2 fragment.

Table 1 PCR amplification program of the first amplification primer set:

The ICAM2 fragment and the adeno-associated virus vector with a CMV promoter were subjected to a first double restriction enzyme digestion (the restriction enzyme digestion sites were AvrII and BspEI, and the specific restriction enzyme digestion system was shown in Table 2), and then the target fragment was recovered using Gel Extraction Kit D2500. The specific steps were performed according to the instructions for use of the kit. Then, the above-mentioned ICAM2 fragment after enzyme digestion was connected to the linear PscAAV-GFP vector with T4 ligase. The specific connection system is shown in Table 3. The obtained connection product was transformed into Stbl3 competent cells. The specific operation was as follows: take out the competent cells from the -80℃ refrigerator and thaw on ice; take 10μL of the connection product and add it to 100μL of the competent cells, mix gently, let it stand on ice for 30min, heat shock at 42℃ for 70s, then ice bath for 1min, add 900μL of antibiotic-free LB liquid culture medium, shake on a constant temperature shaker at 37℃ for 1h, the speed is about 220rpm, centrifuge on a desktop centrifuge for 1min, take 100μL and apply it on the LB solid culture medium containing ampicillin resistance, stand upright for 5min until all the liquid is absorbed, invert it in a 37℃ bacterial incubator, and leave it overnight for 12h~14h. Check the transformation result the next day. Then, positive colonies were screened by colony PCR and sent to Qingke Company for sequencing and identification, and the first recombinant plasmid pscAAV-ICAM2-GFP as shown in Figure 2 was successfully constructed;

Table 2 The first double enzyme digestion system:

The reaction temperature of the first double enzyme digestion system is 37° C., and the reaction needs to be carried out in a PCR instrument for 1 hour.

Table 3 T4 ligation system for the first enzyme digestion product:

ICAM2 sequence after restriction digestion 37.5ng PscAAV-GFP linearized plasmid after restriction digestion 50ng 10xT4 DNA Ligase buffer 2μL T4 DNA ligase 1μL Nuclease-free water Make up to 20 μL

The first digestion product needs to be kept at 4°C overnight.

2. Construction of snoRNA-specific expression vector:

Based on high-fidelity PCR technology, the mouse BC16 cell genome was used as a template and the second amplification primer set {including the second amplification upstream primer: F: CAGTGAATTCCGGCCATTGTGGCCAAGC (with ECORⅠ site), the second amplification downstream primer: R: CATACGGCCGCTTCACAGCGTACGACCAC (with EagⅠ site)} was used for PCR amplification (the specific amplification procedure is shown in Table 4). After agarose gel electrophoresis, the PCR product was subjected to agarose gel electrophoresis, and the target fragment was recovered using Gel Extraction Kit D2500. The specific steps were performed according to the instructions of the kit to obtain the DNA sequence of overexpressed snoRNA.

Table 4 PCR amplification program of the second amplification primer set

The DNA sequence overexpressing snoRNA and the first recombinant plasmid were subjected to a second double restriction enzyme digestion (the restriction enzyme digestion sites were ECORⅠ and EagⅠ, and the specific restriction enzyme digestion system was shown in Table 5), and then the target fragment was recovered using Gel Extraction Kit D2500. The specific steps were performed according to the instructions for use of the kit. Then, the above-mentioned ICAM2 fragment after enzyme digestion was connected to the linear PscAAV-GFP vector with T4 ligase. The specific connection system is shown in Table 6. The obtained connection product was transformed into Stbl3 competent cells. The specific operation was as follows: take out the competent cells from the -80℃ refrigerator and thaw on ice; take 10μL of the connection product and add it to 100μL of the competent cells, mix gently, let it stand on ice for 30min, heat shock at 42℃ for 70s, then ice bath for 1min, add 900μL of antibiotic-free LB liquid culture medium, shake on a constant temperature shaker at 37℃ for 1h, the speed is about 220rpm, centrifuge on a desktop centrifuge for 1min, take 100μL and apply it on the LB solid culture medium containing ampicillin resistance, stand upright for 5min until all the liquid is absorbed, invert it in a 37℃ bacterial incubator, and leave it overnight for 12h~14h. Check the transformation result the next day. Then, positive colonies were screened by colony PCR and sent to Qingke Company for sequencing and identification, and the second recombinant plasmid pscAAV-ICAM2-SNORD32A as shown in Figure 2 was successfully constructed;

Table 5 The second double enzyme digestion system

The reaction temperature of the second double enzyme digestion system is 37° C., and the reaction needs to be carried out in a PCR instrument for 1 hour.

Table 6 Second enzyme digestion product T4 ligation system

The second digestion product needs to be kept at 4°C overnight.

After confirming that the second recombinant plasmid was successfully constructed, the SNORD15A, SNORD118 and Dsred sequences were inserted into the second recombinant plasmid through the ECORⅠ site and the EagⅠ site using homologous recombination technology, that is, the required SNORD15A, SNORD118 and Dsred sequences were amplified by PCR technology, and then digested and purified, and then connected with T4 ligase. Sequencing screening was then performed to confirm that the plasmid was constructed correctly to obtain the molecular tool vector shown in Figure 1.

Related experiments and effect data:

1. Based on the molecular tool vector obtained in Example 2, the effect of specifically promoting the expression of GFP protein was detected. The specific steps are as follows:

The constructed PscAAV-ICAM2-GFP first recombinant plasmid was transfected with a transfection reagent Transfected into 293T cells, 48 hours after transfection, observed under a fluorescence microscope and took photos to record whether the 293T cells had fluorescence. The specific transfection process was as follows: first, 293T cells were plated in a 6-well plate; Add 2 μg of PscAAV-ICAM2-GFP to the buffer for dilution and gently blow and mix with the pipette tip; add 4 μL of Mix by gently blowing with the pipette tip; incubate at room temperature for 10 minutes; replace the cell medium in the 6-well plate with 2 mL of 1640 complete medium; add 200 μL of transfection complex dropwise to each well and add evenly; gently shake the culture plate horizontally back and forth and left and right, and incubate at 37°C; analyze after 48 hours or later.

The results are shown in FIG3 . The ICAM2 promoter can overexpress GFP protein in 293T cells, causing the cells to emit green fluorescence.

2. Based on the molecular tool vector obtained in Example 2, the PCR technique was used to detect the effect of overexpressing SNORD32A in cells through ICAM2. The specific steps were as follows:

First, the SNORD32A product was amplified according to the PCR program shown in Table 7, and then the PCR product was subjected to agarose gel electrophoresis and finally UV development.

Table 7 Detection PCR program

The results are shown in FIG4 , and the molecular tool vector can highly express SNORD32A in 293T cells.

3. Based on the molecular tool vector obtained in Example 2, PCR technology was used to detect the overexpression of various other snoRNA and GFP random gene fragments. The specific steps are as follows:

First, SNORD15A, SNORD118, and Dsred products were amplified according to the PCR program shown in Table 8, and then these PCR products were subjected to agarose gel electrophoresis and finally UV development.

Table 8 Detection PCR program

The results are shown in FIG5 . In addition to being able to specifically and highly express SNORNA sequences other than SNORD32A, SNOD15A, and SNORD118, the backbone plasmid can also specifically and highly express some small DNA sequences similar to Dsred.

4. Based on the molecular tool vector obtained in Example 2, the actual effect of the molecular tool vector on high expression of SNORD32A in endothelial cells and cardiomyocytes was investigated. The specific steps are as follows:

The constructed PscAAV-ICAM2-GFP first recombinant plasmid was packaged with adeno-associated virus packaging reagent The cells were packaged into 293T cells, and the viral supernatant was collected. Finally, the viral supernatant was used to infect endothelial cells and cardiomyocytes. After 72 hours of infection, the cells were observed for fluorescence using a fluorescence microscope and photographed for record.

The results are shown in Figure 6.

5. Based on the molecular tool vector obtained in Example 2, the actual effect of the molecular tool vector on the expression of SNORD32A in endothelial cells was investigated. The specific steps are as follows:

First, the SNORD32A product was amplified according to the following PCR procedure, and then the PCR product was subjected to agarose gel electrophoresis and finally UV development.

Table 9 Detection PCR program

The obtained results are shown in FIG7 , and the molecular tool vector can specifically and highly express SNORD32A in endothelial cells, but cannot specifically and highly express SNORD32A in cardiomyocytes.

In summary, the molecular tool vector expression system provided in the embodiment of the present application uses an endothelial-specific promoter derived from the ICAM2 gene to replace the traditional U6 or CMV promoter, which can specifically initiate the expression of snoRNA. In addition, the backbone sequence derived from the intron 2 region of SNORD32A in the mouse rpl13a gene is designed to promote the presence of multiple splicing sites in the backbone sequence, thereby achieving accurate and efficient stable overexpression of snoRNA.

In addition, a molecular tool vector expression system provided in an embodiment of the present application replaces the traditional CMV promoter with the endothelial-specific promoter ICAM2 through molecular biological means. From the effect of the detected promoter ICAM2 specifically promoting the expression of GFP protein, it can be known that it essentially solves the problem of low expression efficiency of snoRNA in endothelial cells; secondly, in the results of animal cell experiments, adeno-associated virus containing the molecular tool vector expression system was used to simultaneously infect human umbilical vein endothelial cells and cardiomyocytes, and the PCR test results showed that the molecular tool vector can specifically and efficiently express SNORD32A in endothelial cells, which shows that the molecular tool vector expression system can efficiently and accurately overexpress SNORD32A, and has the infection efficiency of the molecular tool vector of the molecular tool vector expression system.

In addition, an embodiment of the present application provides a molecular tool vector expression system, which can be integrated into various types of expression vectors, for example, it can be applied to various expression systems such as ordinary eukaryotic expression, lentiviral expression, adenoviral expression, retroviral expression, and prokaryotic expression.

In addition, a molecular tool vector provided in an embodiment of the present application can achieve efficient expression of snoRNA while accurately splicing the target fragment by adding natural splicing site sequences on both sides of the snoRNA fragment, without introducing additional nucleic acid base sequences. This enables the molecular tool vector to have the advantage of strong selectivity and can ensure the accurate expression of non-coding RNA including snoRNA.

In addition, the molecular tool vector provided in the embodiment of the present application is particularly suitable for the expression of various endothelial-specific regulated snoRNAs, and can also be used for the expression of common snoRNAs and DNA sequences, and has the advantages of high efficiency and stability of expression.

In addition, the method for preparing a molecular tool vector provided in the embodiment of the present application can simply and easily cause the molecular tool vector to express snoRNA, and the overall operation is simple and easy to promote.

The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims (10)

1. A molecular tool vector expression system, characterized in that the molecular tool vector expression system comprises an endothelial-specific gene promoter sequence and a backbone sequence for expressing snoRNA, wherein the endothelial-specific gene promoter sequence is derived from the ICAM2 gene, and the backbone sequence is derived from the intron 2 region of SNORD32A in the rpl13a gene. 2 . The molecular tool vector expression system according to claim 1 , wherein the nucleotide sequence of the endothelial-specific gene promoter sequence is as shown in SEQ ID NO.1. 3. The molecular tool vector expression system according to claim 1 is characterized in that the backbone sequence includes, in sequence, an upstream sequence of intron region No. 2, intron region No. 2, and a downstream sequence of intron region No. 2, wherein the upstream sequence is a sequence of a preset length upstream of the 3’ end of the intron region No. 2, and the downstream sequence is a sequence of the preset length from SNORD32A to the downstream of the 5’ end of the intron region No. 2. 4 . The molecular tool vector expression system according to claim 3 , wherein the preset length is 30 bp. 5. The molecular tool vector expression system according to claim 4 is characterized in that the nucleotide sequence of the upstream sequence is shown as SEQ ID NO.2, the nucleotide sequence of the intron region No. 2 is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream sequence is shown as SEQ ID NO.4. 6 . The molecular tool vector expression system according to claim 1 , wherein the nucleotide sequence of the backbone sequence is as shown in SEQ ID NO.5. 7. A molecular tool vector, characterized in that the molecular tool vector has the molecular tool vector expression system according to any one of claims 1 to 6. 8. The molecular tool carrier according to claim 7, characterized in that the tool carrier comprises at least one of the following: Eukaryotic expression vectors, lentiviral vectors, adenoviral vectors and retroviral vectors. 9. A method for preparing the molecular tool carrier according to claim 7 or 8, characterized in that the method comprises: Using the HUVEC cell genome as a template and using the first amplification primer set for PCR amplification, followed by purification, to obtain an ICAM2 fragment; The ICAM2 fragment and the adeno-associated virus vector with a CMV promoter are subjected to a first double restriction digestion, followed by purification and T4 ligase ligation to obtain a first recombinant plasmid; Using the B16 cell genome as a template and using the second amplification primer set for PCR amplification, followed by purification, to obtain a DNA sequence overexpressing snoRNA; Performing a second double restriction digestion on the DNA sequence overexpressing snoRNA and the first recombinant plasmid, followed by purification and T4 ligase ligation to obtain a second recombinant plasmid; Using homologous recombination technology, the SNORD15A, SNORD118 and Dsred sequences are respectively inserted into the second recombinant plasmid, and then sequencing and screening are performed to obtain a molecular tool vector; Wherein, the first amplification primer set has a first restriction enzyme cutting site set, and the first restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the first double restriction enzyme cutting; The second amplification primer set has a second restriction enzyme cutting site set, and the second restriction enzyme cutting site set is the same as the restriction enzyme cutting site of the second double restriction enzyme cutting; The first restriction enzyme cleavage site group and the second restriction enzyme cleavage site group are different. 10. The method according to claim 9, characterized in that the first restriction site group includes an AvrII site and a BspEI site; and the second restriction site group includes an ECORⅠ site and an EagⅠ site.