A nucleic acid molecule encoding a red fluorescent protein

Abstract

The present application relates to the field of bioengineering, and particularly relates to a kind of artificial codon optimization nucleic acid molecule of the red fluorescent protein coding.The present application provides nucleic acid molecule of red fluorescent protein coding, and the red fluorescent protein is mCherry, compared with the existing red fluorescent protein nucleotide sequence, the number of CpG site is increased from 56 to 92, the content of the third position of codon G / C (GC3) is not less than 99%.The nucleotide sequence provided by the present application is the mCherry synonymous codon substitution sequence obtained by combining the increase of the number of CpG sites and the use of optimal codon strategy, which is 1.3-2.4 times higher than the fluorescence intensity of the sequence before modification in mammalian (human / mouse) cells;The present application uses the method of codon optimization to obtain the nucleic acid molecule of the red fluorescent protein that does not exist in nature, which can encode the red fluorescent protein with stronger fluorescence intensity, and promotes the development of live cell imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, and particularly relates to a nucleic acid molecule encoding a red fluorescent protein. BACKGROUND

[0002] Fluorescent proteins have promoted the progress of biological research due to their rich fluorescence spectrum and diverse photochemical properties. Natural fluorescent proteins and their homologues are widely used as fluorescent tags of specific proteins. In vitro, a vector containing a fusion gene of a target protein and a fluorescent protein is constructed (which usually does not affect the normal localization and function of the target protein), and then the vector is transferred into cells and organisms for expression, so as to realize the visualization of the target protein to explore protein expression, tracking, localization, translocation, interaction, etc. Even co-imaging can be performed using multiple fluorescent proteins. Targeting specific proteins in various subcellular compartments using a fluorescent protein fusion vector can realize subcellular localization. Under the drive of a specific genetic background and promoter, the visualization of specific cell types in the whole animal, organ, tissue and cell culture can also be realized. In recent years, fluorescent proteins have also been applied to the development of new fields, such as deep tissue imaging, patient blood component analysis, biosensors, etc.

[0003] The wavelength of red fluorescent protein fluorescence is in the far infrared region of the visible spectrum. Due to the advantages such as less light tissue scattering, stronger penetration, less damage, low background and lower phototoxicity of biological tissues in this range, red fluorescent protein is widely used in fluorescence imaging of living cells and tissues and organs. mCherry is a new type of red fluorescent protein obtained by directed evolution of the chromophore residues of mRFP1 by Shaner et al. Among the obtained red fluorescent protein monomers, the excitation wavelength of mCherry is 587 nm, the emission wavelength is 610 nm, the extinction coefficient is 72,000 M -1 cm -1 -1 , pKa<4.5, with the characteristics of longer wavelength, stronger light stability, faster maturation and higher pH resistance, but the brightness is low, so it is of great significance to optimize the red fluorescent protein for fluorescence imaging of living cells.

[0004] Codon optimization can be used to modulate gene expression without changing the protein sequence. Codon optimization is a genetic engineering method that replaces the original codons in a nucleotide sequence with synonymous codons to increase protein production. The use of different synonymous codons to encode the same polypeptide can have a significant impact on protein expression levels. Some studies have shown that codon optimization can affect protein conformation and function, and the optimized sequence may encode a protein with stronger function than the native sequence. The main strategies for codon optimization are: 1) to speed up translation, such as using the most frequently used synonymous codons in the host to make the codon adaptation index (CAI) of the sequence as high as possible; 2) to increase mRNA stability, such as increasing the proportion of GC in all bases (GC content), increasing the proportion of codons with G or C at the 3rd position (GC3 content), avoiding certain restriction enzyme cleavage sites, etc. Different codon optimization methods are often not compatible, such as the optimal codon for arginine (R) is AGA, while the codon with the highest GC content is CGG or CGC. Selecting the optimal codon will affect the GC content and GC3 content of the nucleotide sequence, and selecting the codon with high GC content and GC3 content will affect the CAI of the nucleotide sequence. Most existing codon optimization tools use a relatively single standard to design the sequence, such as only using the optimal codon or only using the codon with the highest GC and GC3 content, which may not be the best codon optimization scheme.

[0005] A CG dinucleotide sequence refers to a cytosine (C) and guanine (G) and the phosphate group connecting them in a nucleotide sequence, also known as a CpG site. Most DNA methylation occurs on the cytosine before the guanine nucleotide or CpG site, and the C residue is easily deaminated to T (thymine) spontaneously, and methylation changes the activity of the DNA fragment without changing the sequence. In addition to the GC and CpG-rich sequence segments in the mammalian genome forming CpG islands that are not easily methylated, the remaining CpG sites are distributed throughout the genome and are generally methylated. Related studies have shown that CpG site methylation in gene therapy vectors inhibits transgene expression in vivo by mediating transcriptional repression and promoting innate immune responses. Based on the above reported negative effects, CpG sites are usually avoided when codon optimizing expression constructs for recombinant protein production and gene therapy. However, different proteins have different structures and functions, and a universal strategy may not be suitable for all protein optimization. The ultimate goal of protein optimization is to obtain a function and application that is suitable for the target protein.

[0006] The present application carries out codon optimization on the existing red fluorescent protein, obtains a nucleotide sequence which does not exist in nature, and expresses a red fluorescent protein with stronger fluorescence intensity in mammalian (human / mouse) cells, thereby promoting the development of live cell imaging technology. SUMMARY

[0007] Therefore, the present application provides a nucleic acid molecule encoding a red fluorescent protein. The mCherry_Plus sequence provided by the present application is a nucleotide sequence obtained by increasing CpG sites and using optimal codons in the existing red fluorescent protein mCherry without changing the protein sequence thereof; the sequence is subjected to whole gene synthesis and an expression vector is constructed to transfect mammalian (human / mouse) cells, which are observed under a fluorescence microscope and analyzed by flow cytometry, and the red fluorescent protein expressed after mCherry_Plus transfection has a higher average fluorescence intensity.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0009] The present application provides a nucleic acid molecule encoding a red fluorescent protein, wherein the red fluorescent protein is mCherry, the number of CpG sites of the nucleic acid molecule is not less than 90, and the content of GC3 is not less than 99%.

[0010] In some embodiments of the present application, the number of CpG sites of the nucleic acid molecule is 92, and the content of GC3 is 99.6%.

[0011] In some embodiments of the present application, the nucleic acid molecule has:

[0012] (I) a nucleotide sequence as shown in SEQ ID NO: 1; or

[0013] (II) a nucleotide sequence complementary to the nucleotide sequence as described in (I); or

[0014] (III) a nucleotide sequence encoding the same protein as the nucleotide sequence as described in (I) or (II), but different from the nucleotide sequence as described in (I) or (II) due to the degeneracy of genetic code; or

[0015] (IV) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as described in (I), (II) or (III), and a nucleotide sequence having the same or similar function as the nucleic acid molecule as described in (I), (II) or (III); or

[0016] (V) a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence as described in (I), (II), (III) or (IV).

[0017] The present invention also provides an expression cassette comprising: the aforementioned nucleic acid molecule and an acceptable gene element.

[0018] In some embodiments of the present invention, the gene element in the expression cassette includes an attL site and a resistance gene.

[0019] In some embodiments of the present invention, the resistance gene in the above expression cassette includes the Kana resistance gene.

[0020] The present invention also provides an expression vector comprising: the above-described nucleic acid molecule and / or the above-described expression cassette.

[0021] In some embodiments of the present invention, the expression vector further includes a promoter.

[0022] In some embodiments of the present invention, the promoter in the above expression vector includes: a CMV promoter.

[0023] In some embodiments of the present invention, the promoter in the above expression vector further includes a step of fusion with a pUp plasmid; the pUp plasmid includes: an attL site, an attR site, and a Kana resistance gene.

[0024] In some embodiments of the present invention, the expression vector further includes a target vector; the target vector includes an attB site, an attR site, a CmR gene, a ccdB gene, and an Amp resistance gene.

[0025] In some embodiments of the present invention, the target vector in the above expression vector includes: an Escherichia coli cloning vector.

[0026] The present invention also provides a host, transformation and / or transfection of the above expression vector.

[0027] In some embodiments of the present invention, the transformed and / or transfected cells in the host include mammalian cells.

[0028] In some embodiments of the present invention, the mammalian cells in the host include human cells and / or mouse cells.

[0029] The present invention also provides products comprising: the above-described nucleic acid molecule, the above-described expression cassette, the above-described expression vector and / or the above-described host, as well as acceptable adjuvants, excipients and / or devices.

[0030] The present invention also provides the application of the above-mentioned nucleic acid molecules, expression cassettes, expression vectors, hosts and / or products in enhancing fluorescence intensity.

[0031] The present invention also provides the application of the above-mentioned nucleic acid molecules, expression cassettes, expression vectors, hosts and / or products in mammalian fluorescent protein imaging.

[0032] The present invention provides a nucleic acid molecule encoding a red fluorescent protein, wherein the red fluorescent protein is mCherry, and the nucleic acid molecule has not less than 90 CpG sites and not less than 99% GC3 content.

[0033] The beneficial effects of this invention include:

[0034] (1) Without altering the protein sequence of mCherry, this invention significantly improves the brightness of mCherry expression in mammalian (human / mouse) cells, further optimizing the application of mCherry red fluorescent protein in mammalian fluorescent protein imaging;

[0035] (2) The codon optimization strategy of increasing CpG sites in the nucleotide sequence used in this invention is different from the commonly used strategies such as using only the codon with the best preference, relatively low mRNA secondary structure free energy, or removing CpG, and provides a new idea for codon optimization strategy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 A comparison diagram of the wild-type and optimized nucleotide sequences of mCherry;

[0038] Figure 2 A schematic diagram showing the construction of expression vectors for wild-type and optimized nucleotide sequences of fluorescent proteins;

[0039] Figure 3 The image shows fluorescence images of 293T cells transfected with expression vectors containing wild-type and optimized nucleotide sequences of mCherry; from left to right: mCherry_Wt, mCherry_Remove CG, mCherry_Plus, mCherry_LinearDesign; top: 20×, Red 80ms; bottom: 20×, Blue 80ms.

[0040] Figure 4 Statistical graph showing flow cytometry data of wild-type and optimized mCherry nucleotide sequence expression vectors transfected into 293T cells;

[0041] Figure 5Fluorescence images of BHK21 cells transfected with expression vectors containing wild-type and optimized nucleotide sequences of mCherry; from left to right: mCherry_Wt, mCherry_Remove CG, mCherry_Plus, mCherry_LinearDesign; top: 20×, Red 80ms; bottom: 20×, Blue 80ms.

[0042] Figure 6 Statistical graph showing flow cytometry data of wild-type and optimized mCherry nucleotide sequence expression vectors transfected into BHK21 cells;

[0043] Figure 7 Fluorescence images of adeno-associated virus-transduced 293T cells loaded with wild-type and optimized nucleotide sequences of mCherry are shown. From left to right: mCherry_Wt, mCherry_Remove CG, mCherry_Plus, mCherry_LinearDesign; Row 1, MOI = 100; Row 2, MOI = 200; Row 3, MOI = 500; Row 4, MOI = 1000; 20×, Red 80ms.

[0044] Figure 8 Flow cytometry data of 293T cells transduced with adeno-associated virus loaded with wild-type and optimized nucleotide sequences of mCherry are presented. Detailed Implementation

[0045] This invention discloses a nucleic acid molecule encoding a red fluorescent protein.

[0046] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0047] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0048] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0049] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0050] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0051] This invention provides a nucleotide sequence optimized using the wild-type nucleotide sequence of mCherry (mCherry_Wt) as a template, which can express a red fluorescent protein with higher fluorescence intensity. In this wild-type sequence, CpG sites within and between codons are increased through synonymous codon substitution. Subsequently, based on the DNA codon preferences of humans and mice (http: / / www.kazusa.or.jp / codon / ), while maintaining a high number of CpG sites, codons that were not involved in CpG site modification and optimization are replaced with optimal human and mouse codons to obtain the mCherry_Plus sequence with high CpG site content.

[0052] This invention provides a codon-optimized mCherry gene, the optimized nucleotide sequence of which is as follows:

[0053]

[0054] (As shown in SEQ ID NO:1)

[0055] The bold italicized portion represents the codons in the mCherryPlus gene that have been designed and replaced according to the strategy of increasing CpG sites and adopting codon preferences in humans and mice. According to the applicant's experiments, the codon-optimized mCherry nucleotide sequence can be used in mammalian cell imaging.

[0056] In addition to the optimized mCherry sequences with multiple CpG sites mentioned above, we also provide the following sequences for control experiments:

[0057] The wild-type nucleotide sequence (mCherry_Wt) of the red fluorescent protein mCherry was used as a template;

[0058] 1. mCherry_Remove CG sequence: In codon optimization, optimized sequences that do not contain CpG dinucleotides are often selected. In the wild-type sequence, synonymous codons with the composition NCG or CGN and codons with C as the third codon before GNN are replaced. Then, the optimal codons of humans and mice are selected to replace the non-optimal codons at other positions. For example, if the optimal codon composition NNC needs to be followed by the GNN codon, then the second-best codon is selected for replacement. Finally, a nucleotide sequence that does not contain CpG sites is obtained and named the mCherry_Remove CG sequence (as shown in SEQ ID NO:4).

[0059] 2. mCherry_LinearDesign sequence: The mCherry_Wt sequence was codon optimized using the online tool LinearDesign (https: / / rna.baidu.com / app / vaccine / linear-design / forecast). LinearDesign uses computational linguistics methods, combining mRNA stability and optimal codons for calculation, and finally obtains a nucleotide sequence with low secondary structure free energy (MFE) and high codon fitness index (CAI), named the mCherry_LinearDesign sequence (as shown in SEQ ID NO:5).

[0060] Sequence alignment of mCherry_Wt, mCherry_Plus, mCherry_Remove CG, and mCherry_Linear Design as follows: Figure 1 As shown.

[0061] In Examples 1 to 4 of this invention, all raw materials and reagents used can be purchased from the market.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: A codon-optimized nucleotide sequence of a red fluorescent protein

[0064] Studies have shown that most synonymous codons in naturally occurring genomes are not used at equal frequencies; this effect is known as codon usage bias. Codon usage frequencies differ between species; frequently used codons are called high-frequency codons / optimal codons, while infrequently used codons are called low-frequency codons / rare codons. Based on the known standard human codon table and human codon preferences, CpG sites were first analyzed, revealing that the wild-type mCherry sequence (mCherry_Wt) contains 56 CpG sites (in bold), as shown below:

[0065]

[0066] (As shown in SEQ ID NO:2)

[0067] (1) The applicant first replaced the codons without CpG sites in the sequence of codons containing CpG sites within synonymous codons, thereby increasing the CpG content in the codons by increasing the CpG sites within the codons themselves (bold and italicized parts); secondly, the first codon of the GNN codon was replaced with a synonymous codon with a C base at the 3rd position to increase the CpG sites between codons (bold and underlined parts), as shown below:

[0068]

[0069]

[0070] (As shown in SEQ ID NO:3)

[0071] (2) The obtained sequence contains 92 CpG sites; after the above manual optimization, and combined with CAI, the codons of other sites not involved in the optimization are replaced with the optimal codons by humans and mice, as shown below (bold and italic parts), finally obtaining the mCherry_Plus sequence:

[0072]

[0073]

[0074] (As shown in SEQ ID NO:1)

[0075] (3) The inventors used the mCherry optimized sequence with all CpG sites removed (mCherry_RemoveCG) and the sequence designed using the online tool LinearDesign (mCherry_LinearDesign) as controls in their experiments. The characteristics of the mCherry optimized sequence are shown in Table 1:

[0076] Table 1

[0077]

[0078] The alignment of the four nucleotide sequences of mCherry is as follows: Figure 1 After artificial optimization, compared with the wild-type sequence, mCherry_Plus has an increased number of CpG sites, increased GC3 content and GC content, and a decreased secondary structure free energy (MFE).

[0079] Example 2: Construction of a vector containing a codon-optimized red fluorescent protein gene

[0080] (1) Replace the Gene in the pDown plasmid containing the AttL1-Gene-attL2 sequence with the optimized mCherry sequence obtained in Example 1, and obtain pDown-mCherry vectors containing different optimized mCherry sequences through whole-genome synthesis. The core structure is as follows: Figure 2 As shown, this includes the attL site, the optimized mCherry sequence, and the Kana resistance gene.

[0081] (2) Select the CMV promoter and fuse the CMV sequence with the pUp plasmid as in step (1). The core structure of the resulting pUp-CMV plasmid is as follows: Figure 2 As shown, it mainly includes the attL site, attR site, CMV gene, and Kana resistance gene.

[0082] (3) An E. coli plasmid cloning vector, pRp.Des2d, was selected as the backbone vector, and its core structure is as follows: Figure 2 The expression vector mainly includes the attB site, attR site, CmR gene, ccdB gene, and Amp resistance gene. Among them, the CmR gene is the chloramphenicol resistance gene; the ccdB gene encodes the toxic protein ccdB in the ccd operon of E. coli. This toxic protein can inhibit DNA gyrase to repair broken DNA, ultimately leading to cell death.

[0083] (4) The pUp-CMV plasmid, pRp.Des2d backbone plasmid, and pDown-mCherry_Wt, pDown-mCherry_RemoveCG, pDown-mCherry_Plus, and pDown-mCherry_LinearDesign vectors were respectively loaded into... The reaction was carried out at 25°C for 5 hours under the catalysis of IIPlus enzyme. The reaction system is shown in Table 2. After freezing and thawing on ice for 30 seconds, competent cells can be transformed. After thawing and culturing, a small amount was spread on a plate containing Amp and incubated upside down at 37°C for 14-16 hours.

[0084] During the LR reaction, the pUp-CMV plasmid and pRp.Des2d backbone plasmid recombine with the pDown-mCherry_Wt, pDown-mCherry_RemoveCG, pDown-mCherry_Plus, and pDown-mCherry_LinearDesign plasmids through homologous arm recombination, respectively, to obtain the following results: Figure 2 The target vectors shown are named pRP-CMV>mCherry_Wt, pRP-CMV>mCherry_Remove CG, pRP-CMV>mCherry_LinearDesign, and pRP-CMV>mCherry_Plus.

[0085] Table 2

[0086]

[0087] (5) Pick single colonies from the Amp plates cultured in step (4) and expand them on liquid culture medium. Those colonies that can grow in Amp-resistant medium but not in Kana-resistant medium are positive clones. Sequencing is performed on the positive clones to detect the att sites and the sequences between them. If the sequencing is correct, the vectors pRP-CMV>mCherry_Wt, pRP-CMV>mCherry_Remove CG, pRP-CMV>mCherry_LinearDesign, and pRP-CMV>mCherry_Plus can be successfully obtained.

[0088] Example 3: Effect of CpG site codon optimization on the expression of red fluorescent protein in human and mouse cells.

[0089] The four expression vectors obtained in Example 2 were used to transfect 293T cells (human embryonic kidney epithelial cells) and BHK21 cells (hamster kidney fibroblasts). After transfection, the expression of red fluorescent protein was observed under a fluorescence microscope and analyzed by flow cytometry.

[0090] (1) 18-24 hours before the experiment, seed enough 293T cells and BHK21 cells into 6-well cell culture dishes. Transfection can be performed when the confluence reaches 70-80%.

[0091] (2) According to the transfection reagent Trans-Hi TM The In Vitro DNA Transfection Reagent instruction manual recommends changing the culture medium 30–60 minutes before transfection and culturing the cells in an incubator.

[0092] (3) Take 5 ng of pRP-CMV>mCherry_Wt, pRP-CMV>mCherry_Remove CG, pRP-CMV>mCherry_LinearDesign, and pRP-CMV>mCherry_Plus vector plasmids, and add 2 ng of pRP-CMV>TagBFP2_Wt plasmid to each as an internal control plasmid. If the amount of plasmid is insufficient, it can be adjusted according to Trans-Hi. TM In the Vitro DNATransfection Reagent instructions, provide the culture container and recommended DNA amount, add sufficient pUC19 plasmid, mix the plasmid thoroughly, and dissolve it in serum-free culture medium.

[0093] (4) Trans-Hi TM In Vitro DNA Transfection Reagent, dissolve in an equal volume of serum-free culture medium and mix thoroughly with the plasmid solution from step (3). Incubate at room temperature for 10–15 minutes.

[0094] (5) After incubation, drop the solution from step (4) into the cell culture medium, gently shake to mix, and continue culturing at 37°C and 5% CO2.

[0095] (6) After incubating for 18-24 hours in step (5), the culture medium needs to be replaced once, and the culture should continue for 48 hours.

[0096] (7) Take a picture and record it under a fluorescence microscope 48 hours after transfection.

[0097] (8) After photographing, trypsin was used for digestion. Digestion was terminated with complete culture medium. The serum-containing culture medium was removed by centrifugation, and the cells were resuspended in PBS solution. Flow cytometry was used for analysis. The positive rates and average fluorescence intensities of red and blue fluorescence were statistically analyzed, and the results are shown in Tables 3 and 4.

[0098] Table 3

[0099]

[0100] *MFI=All cells Mean×Postive%.

[0101] Table 4

[0102]

[0103] *MFI=All cells Mean×Postive%.

[0104] The results are shown in the graph, under the same transfection conditions:

[0105] 1) Under a fluorescence microscope, after transfection with plasmids containing different mCherry nucleotide sequences, the red fluorescence of mCherry_Remove CG sequence transfection in 293T and BHK21 cells was weaker than that of mCherry_Wt sequence. mCherry_LinearDesign sequence expressed only a small amount of red fluorescence, while mCherry_Plus sequence transfection showed significantly enhanced red fluorescence compared to mCherry_Wt. Figure 3 , 4 5, 6).

[0106] 2) In 293T cells, the mean fluorescence intensity of cells transfected with the mCherry_Remove CG sequence was 0.07 times that of mCherry_Wt, the mean fluorescence intensity of cells transfected with the mCherry_LinearDesign sequence was only 0.01 times that of mCherry_Wt, while the mean fluorescence intensity of cells transfected with mCherry_Plus was 1.321 times that of mCherry_Wt. Figure 3 , 4 In BHK21 cells, the average fluorescence intensity of mCherry_Remove CG sequence transfection was 0.075 times that of mCherry_Wt, the average fluorescence intensity of mCherry_LinearDesig n sequence transfection was only 0.013 times that of mCherry_Wt, and the average fluorescence intensity of mCherry_Plus transfection was 1.491 times that of mCherry_Wt. Figure 5 , 6 );

[0107] Example 4: Effect of AAV virus loaded with mCherry_Plus transduced human cells on red fluorescent protein expression

[0108] Following the expression vector construction method in Example 2, an adeno-associated virus expression vector pAAV.Des2d was selected, and the CMV promoter and the wild-type or optimized mCherry sequence were loaded into a eukaryotic expression plasmid using the well-known LR reaction to obtain four plasmids that can be expressed in adeno-associated virus, which were named pAAV-CMV>mCherry_Wt, pAAV-CMV>mCherry_RemoveCG, pAAV-CMV>mCherry_Plus, and pAAV-CMV>mCherry_LinearDesign, respectively.

[0109] Six μg of helper plasmid pHelper, six μg of AAV2 viral capsid plasmid, and six μg of adeno-associated virus expression plasmid carrying the mCherry gene and its optimized sequence were mixed to form a plasmid mixture. The plasmid mixture and PEIMax solution (1 mg / ml) were diluted with an equal volume of serum-free basal medium. The total plasmid to PEIMax mass ratio was 1:3. After mixing, the mixture was incubated at room temperature for 10–15 minutes, and then added to 293T culture medium in a 10 cm culture dish. The cells were cultured in a 37°C, 5% CO2 cell culture incubator for 48 h. After 48 h, the cells were photographed and recorded under a fluorescence microscope. The cells were digested for flow cytometry analysis and virus collection.

[0110] After digestion, the cells were centrifuged at 4°C and 4000 rpm for 20 minutes. The cells were then lysed with lysis buffer and digested with totipotent nuclease for 2 hours. The cells were then extracted with an equal volume of chloroform and centrifuged at 4°C and 12000 rpm for 20 minutes. After centrifugation, the virus was found in the clear upper layer of solution. The cells were then aliquoted and stored at -80°C.

[0111] Before transduction, cells were seeded in 6-well plates to ensure cell confluence of approximately 50%–60% at transduction after 24 hours. AAV2 virus was diluted to an appropriate titer using calculations, and absolute quantification was performed by qPCR. Before transduction, fresh culture medium was added, and four adeno-associated viruses carrying different mCherry genes were added to 293T culture medium at MOIs of 100, 200, 500, and 1000, respectively, and cultured for an additional 48 hours. Cells were photographed under a microscope, digested, and analyzed by flow cytometry.

[0112] The results are as follows Figure 7 , 8As shown, when using four adeno-associated virus expression plasmids containing the mCherry gene for AAV2 virus packaging, the red fluorescence of mCherry_Wt and mCherry_Plus was close to saturation, while the red fluorescence of mCherry_Remove CG and mCherry_LinearDesign was weaker. The yields of the four AAV2 viruses after 48 hours of virus harvesting are shown in Table 5.

[0113] Table 5

[0114]

[0115] The results of transducing 293T with four AAV2 viruses at different MOI gradients are as follows:

[0116] Table 6

[0117]

[0118]

[0119] *MFI=All cells Mean×Postive%.

[0120] The positive rates of red fluorescent protein expressed by mCherry_Remove CG and mCherry_LinearDesign under different MOIs were lower than those of mCherry_Wt, and their average fluorescence intensities were weaker than those of mCherry_Wt. At MOI=100, there was no significant difference in the positive rates and red fluorescence intensities between mCherry_Wt and mCherry_Plus. At MOI=200 and MOI=500, the red fluorescence intensity of mCherry_Plus was significantly enhanced compared to mCherry_Wt, with the average fluorescence intensity of mCherry_Plus reaching 1.932 times and 2.395 times that of mCherry_Wt, respectively. At MOI=1000, the red fluorescence positive rates of both mCherry_Plus and mCherry_Wt reached saturation, with the average fluorescence intensity of mCherry_Plus being 1.833 times that of mCherry_Wt. AAV2 virus loaded with mCherry_Plus transduced human cells expressed a red fluorescent protein with a stronger average fluorescence intensity. Figure 7 , Figure 8 ).

[0121] The above experimental results demonstrate that this invention successfully optimized the existing red fluorescent protein mCherry. Without altering the mCherry protein sequence, by increasing the CpG site content in the wild-type nucleotide sequence and optimizing the host codon using the optimal codon, the optimized mCherry nucleotide sequence mCherry_Plus was obtained. The optimized sequence was cloned into a eukaryotic expression vector and transfected into human 293T cells and mouse BHK21 cells, successfully achieving mCherry protein expression with a significantly enhanced average fluorescence intensity compared to the wild-type. Furthermore, when mCherry_Plus was integrated into an AAV2 expression vector and packaged into an AAV2 virus for transduction of 293T cells, the red fluorescent protein expressed by mCherry_Plus exhibited a higher average fluorescence intensity than that expressed by mCherry_Wt in the same MOI transduction experiment.

[0122] In summary, this invention, through codon optimization by increasing the number of CpG sites in the nucleotide sequence and combining this with an optimal codon usage strategy, significantly improves the fluorescence intensity of mCherry protein expression in mammalian (human / mouse) cells, successfully optimizing the expression of red fluorescent protein. The mCherry_Plus sequence is unique, artificially optimized, and cannot be calculated using existing codon tools.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

1.

2. An expression box, characterized in that, include: The nucleic acid molecule as described in claim 1 and the acceptable gene element.

3. An expression carrier, characterized in that, include: The nucleic acid molecule as described in claim 1 and / or the expression cassette as described in claim 2.

4. The expression vector as described in claim 3, characterized in that, The expression vector also includes a promoter.

5. The product, characterized in that, include: The nucleic acid molecule as described in claim 1, the expression cassette as described in claim 2, and / or the expression vector as described in claim 3 or 4, as well as acceptable adjuvants, excipients, and / or devices.

6. The use of the nucleic acid molecule as described in claim 1, the expression cassette as described in claim 2, the expression vector as described in claim 3 or 4, and / or the product as described in claim 5 in enhancing fluorescence intensity.

7. The application of the nucleic acid molecule as described in claim 1, the expression cassette as described in claim 2, the expression vector as described in claim 3 or 4, and / or the product as described in claim 5 in mammalian fluorescent protein imaging.

Images