Non-coding region sequence capable of improving mRNA expression efficiency and application thereof

Abstract

The invention discloses a non-coding region sequence capable of improving mRNA (messenger ribonucleic acid) expression efficiency and application of the non-coding region sequence. Eight different non-coding regions are designed by taking a reporter gene EGFP as a target gene, an mRNA non-coding region capable of realizing high-level expression is finally screened out by comparing expression levels and transfection efficiencies of the EGFP carrying the different non-coding regions at different time points and cell lines, and the EGFP mRNA carrying the non-coding region can be quickly translated and expressed after transfecting cells, so that the expression efficiency of the cells is greatly improved. Obvious green fluorescence can be seen within 3 hours, while weak green fluorescence can be seen within 12 hours after EGFP-Control transfection, and the fluorescence intensity is relatively weak all the time. In addition, by further analyzing the secondary structure, GC content, sequence length and the like of the RNA sequence of the non-coding region and explaining the reason of the expression difference, a theoretical basis is provided for the sequence design of the efficient mRNA vaccine.

Description

Technical Field

[0001] The present invention relates to a non-coding region sequence of mRNA expression efficiency, and in particular to a non-coding region sequence capable of improving mRNA expression efficiency and its application. The present invention belongs to the field of biotechnology. Background Art

[0002] mRNA is an intermediate substance in the central dogma, carrying the coding information for synthesizing proteins, and can be further translated and processed into functional proteins. In 1990, scientists injected in vitro transcribed mRNA directly into mouse skeletal muscle cells, which could be translated and expressed, thus proving the feasibility of mRNA vaccine development. The milestone turning point of mRNA vaccines began when Hungarian biochemist Katalin Karikó and American immunologist Drew Weissman used pseudouridine to replace uridine in artificially synthesized mRNA, thereby helping the synthetic mRNA to successfully evade the cell's inherent immune defense and avoid excessive immune responses caused by activation of Toll-like receptors and RIG-I receptors, significantly enhancing the translation expression of mRNA. This major improvement won the Nobel Prize in Physiology or Medicine in October 2023.

[0003] Subsequently, mRNA-based therapies have been widely studied and applied in different types of diseases. As we all know, during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the mRNA vaccines BNT162b2 and mRNA-1273 of BioNtech in the United States and Moderna in Germany were developed at an unprecedented speed and proved to have a protection efficiency of up to 95%. They received emergency approval from the FDA and were vaccinated to millions of people around the world to fight the global outbreak of coronavirus disease COVID-19, which further stimulated the research enthusiasm of scientists around the world for mRNA vaccines.

[0004] As a representative of new vaccines, mRNA vaccines have unlimited potential and application value. However, to obtain an efficient mRNA vaccine, the sequence design of its mRNA is the key. The in vitro transcribed mRNA simulates the structure of endogenous mRNA, which is mainly composed of a 5′ cap structure (5′ Cap), non-coding regions (5′ UTR and 3′ UTR), an open reading frame (ORF) encoding the target gene, and a poly (A) tail. These structural elements are crucial to the stability and transcription efficiency of mRNA. The 5′cap of mRNA can regulate the post-transcriptional splicing, nuclear export, protein translation initiation and resistance to exonuclease degradation of mRNA. The commonly used capping technology currently uses commercial cap analogs to cap during in vitro transcription or after transcription, and the capping efficiency is as high as 95% or more; the Poly (A) tail is usually composed of 10-250 adenine ribonucleotides, which synergistically regulates the stability and translation efficiency of mRNA. The most commonly used Poly (A) in current research is composed of 100 to 120 A, connected by a 10bp linker in the middle. There are also studies reporting that a 20% ratio of C in the Poly (A) tail can significantly enhance the translation expression of the target gene. The open reading frame is the most critical component of the mRNA vaccine, encoding the target gene. Through codon optimization, the introduction of functional peptides and modifications during transcription, it can avoid excessive immune response and increase translation levels without changing the protein sequence. Non-coding regions (UTRs) are the non-coding parts of the mRNA sequence located upstream (5′UTR) and downstream (3′UTR) of the mRNA coding region. They do not directly encode proteins, but have the strongest plasticity and universality. They play a particularly important role in regulating mRNA translation and protein expression. Non-coding regions with high translation efficiency and high stability will make mRNA vaccines better used in clinical practice. Summary of the invention

[0005] One of the purposes of the present invention is to use the easily detectable reporter gene EGFP as the target gene, and design a total of 8 mRNAs carrying different non-coding regions. The optimal non-coding region is screened out by transfection efficiency and expression level, and the reasons for the differences in expression levels are further analyzed in sequence to provide a theoretical basis for the sequence design of efficient mRNA vaccines.

[0006] The second object of the present invention is to provide an mRNA sequence encoding EGFP, which can significantly increase the expression level of EGFP.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention provides a non-coding region capable of improving mRNA expression efficiency, wherein the non-coding region comprises a 5' non-coding region shown in SEQ ID NO.5 and a 3' non-coding region shown in SEQ ID NO.6, or the non-coding region comprises a 5' non-coding region shown in SEQ ID NO.12 and a 3' non-coding region shown in SEQ ID NO.13.

[0009] Furthermore, the present invention also proposes the use of the non-coding region in preparing mRNA vaccines and the use of the non-coding region in improving the expression efficiency of target gene mRNA.

[0010] In a specific embodiment of the present invention, the target gene is an enhanced green fluorescent protein gene.

[0011] Furthermore, the present invention also provides an mRNA molecule, wherein the mRNA molecule contains the non-coding region described in the present invention.

[0012] Preferably, the mRNA molecule further contains a poly(A) tail.

[0013] Preferably, the poly(A) tail consists of 120 adenines (120A).

[0014] Preferably, the mRNA molecule also contains an expression frame of enhanced green fluorescent protein.

[0015] Among them, preferably, the mRNA molecule is obtained by transcription of the nucleotide sequence shown in SEQ ID NO.16 or 21.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention designs 8 different non-coding regions with the reporter gene EGFP as the target gene. By comparing the expression levels and transfection efficiency of EGFP carrying different non-coding regions at different time points and cell lines, a mRNA non-coding region that can be expressed at a high level is finally screened out. After the EGFP mRNA carrying the non-coding region is transfected into cells, it can be rapidly translated and expressed, and obvious green fluorescence can be seen at 3h, while EGFP-Control can be seen with weak green fluorescence at 12h after transfection, and the fluorescence intensity is always relatively weak. In addition, by further analyzing the secondary structure, GC content and sequence length of the non-coding region RNA sequence and explaining the reasons for its expression differences, a theoretical basis is provided for the sequence design of efficient mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The following is the framework diagram of plasmids carrying EGFP in different non-coding regions;

[0019] The plasmid framework diagram depicts EGFP with different UTRs and multiple restriction sites; restriction endonucleases BamHI and NotⅠ are used for gene replacement, while EcoRⅤ is used for plasmid linearization; 5'UTR3 and 5'UTR4 have the same sequence and are depicted in the same color; similarly, the sequences of 3'UTR2, 3'UTR5, and 3'UTR6 are also the same and are also represented by the same color;

[0020] Figure 2 For the linearization and in vitro transcription of plasmids carrying EGFP mRNAs with different noncoding regions;

[0021] Among them, A is the result of plasmid linearization; 1: EGFP-UTR1 and its linearization; 2: EGFP-UTR2 and its linearization; 3: EGFP-UTR3 and its linearization; 4: EGFP-UTR4 and its linearization; 5: EGFP-UTR5 and its linearization; 6: EGFP-UTR6 and its linearization; 7: EGFP-UTR7 and its linearization; 8: EGFP-UTR8 and its linearization; 9: EGFP-Control and its linearization; B is the mRNA synthesized by in vitro transcription; 1: mRNA EGFP-UTR1; 2: mRNA EGFP-UTR2; 3: mRNA EGFP-UTR3; 4: mRNAEGFP-UTR4; 5: mRNAEGFP-UTR5; 6: mRNAEGFP-UTR6; 7: mRNAEGFP-UTR7; 8: mRNAEGFP-UTR8; 9: mRNAEGFP-Control;

[0022] Figure 3 Results of fluorescence microscopy observation of EGFP mRNA carrying different noncoding regions at 3, 6, 12, 24, 36, 48, and 72 h after transfection (exposure time was set to 40 ms and gain was adjusted to 1×);

[0023] Figure 4 Comparison of expression levels of mRNA-EGFP in different non-coding regions after transfection;

[0024] Among them, A is the transfection efficiency of EGFP mRNA carrying different non-coding regions; B is the average fluorescence intensity after transfection of EGFP mRNA carrying different non-coding regions; C is Western blot of EGFP mRNA carrying different non-coding regions. The theoretical size of EGFP is 27kDa, and the theoretical size of the internal reference protein β-actin is 43kDa; D is Western blot gray analysis (ns means no statistical significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0025] Figure 5 To verify the universality of mRNAEGFP-UTR3 / 8 in different cell lines;

[0026] Among them, A is the transfection efficiency of EGFP-UTR3 and EGFP-UTR8 mRNA in BHK, PK-15, Vero, Marc-145, IBRS-2, MDBK, HEK293T and F81 cells; B is the average fluorescence intensity of EGFP-UTR3 and EGFP-UTR8 mRNA after transfection of BHK, PK-15, Vero, Marc-145, IBRS-2, MDBK, HEK293T and F81 cells;

[0027] Figure 6 Synthesis and expression of FMDV mRNA P12A3C carrying UTR3 and UTR8;

[0028] Among them, A is the plasmid linearization of P12A3C-UTR3 and P12A3C-UTR8; 1: P12A3C-UTR3 and its linearization; 2: P12A3C-UTR8 and its linearization; B is the in vitro transcription of P12A3C-UTR3 and P12A3C-UTR8; 1: mRNA P12A3C-UTR3; 2: mRNA P12A3C-UTR8; C is the expression of mRNA P12A3C-UTR3 and P12A3C-UTR8 in different cells; P12A3C protein is cleaved in cells to produce three proteins of different sizes, with theoretical molecular weights between 25 and 35 kDa; the internal reference protein β-actin is 43 kDa;

[0029] Figure 7 For sequence analysis of non-coding regions;

[0030] Among them, A is the GC content of 5′UTRs; B is the GC content of 3′UTRs; C is the minimum free energy (MFE) for predicting 5′UTRs; D is the minimum free energy (MFE) for predicting 3′UTRs;

[0031] Figure 8 It is the planar structure diagram of MFE in the non-coding region;

[0032] The nucleotides are colored according to their structural type, with stems (canonical helices) in green, multiloops (junctions) in red, interior loops in yellow, hairpin loops in blue, and 5' and 3' unpaired regions in orange. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0034] Example 1 Design and screening of EGFP mRNA non-coding region

[0035] 1 Materials and methods

[0036] 1.1 Plasmids and cells

[0037] pcDNA3.1(+) was deposited by the Foot-and-Mouth Disease Prevention and Control and Technology Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0038] HEK293T (human embryonic kidney cells), Marc-145 (African green monkey embryonic kidney cells), F81 (cat kidney cells), BHK-21 (hamster kidney cells), Vero (African green monkey kidney cells), PK-15 and IBRS-2 (pig kidney cells), MDBK (bovine kidney cells) are maintained by the Foot-and-Mouth Disease Prevention and Control and Technology Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0039] 1.2 Main Reagents

[0040] Agarose was purchased from BIOWEST, Spain, E. coli culture reagents (tryptone, yeast powder, sodium chloride) and ampicillin were purchased from OMEGA. Stbl3 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. Snap Assembly reagent, BamH I, Not I, EcoR V, Xba I restriction endonucleases and DNA markers of different sizes, PrimeSTAR Max DNA Polymerase were purchased from Baori Biotechnology Co., Ltd. 6000 bp RNA marker and 10×MOPS buffer were purchased from Thermo Fisher Scientific. Plasmid sub-title kit and gel recovery kit were purchased from OMEGA, T7 in vitro reverse transcription and capping kit were purchased from Jiangsu Shenji Biotechnology Co., Ltd., lithium chloride, diethyl pyrocarbonate (DEPC), electrophoresis buffer, phenol chloroform nucleic acid extraction reagent, GelRed nucleic acid dye were purchased from Beijing Solaibao Technology Co., Ltd., 35% formaldehyde was purchased from Beijing Chemical Plant. Anti-EGFP monoclonal antibody and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Shanghai Biotech. Nitrocellulose membrane (NC) was a product of Millipore. Protein standard molecular weight was a product of Fermentas.

[0041] 1.3 Design and synthesis of EGFP mRNA containing different non-coding regions

[0042] EGFP (Enhanced Green Fluorescent Protein) was used as the target gene to construct EGFP mRNA with different non-coding regions. A total of 8 different non-coding region pairs were designed (Table 1), and poly (A) was composed of 120 adenines (120A). The EGFP coding sequence carrying different non-coding regions and poly (A) (120A) is shown in SEQ ID NO.14-21, and inserted into the pcDNA3.1 (+) vector through the restriction sites HindIII and EcoR V, and BamHI and Not I restriction sites were added on both sides of the target gene to facilitate the subsequent replacement of the target gene. The pure EGFP full gene was inserted into the empty vector of pcDNA3.1 (+) through BamHI and NotI restriction enzymes, and a control without non-coding regions and poly (A) tails was constructed. The eight plasmids containing different non-coding regions were named EGFP-UTR1, EGFP-UTR2, EGFP-UTR3, EGFP-UTR4, EGFP-UTR5, EGFP-UTR6, EGFP-UTR7, and EGFP-UTR8, and the control was named EGFP-Control. The schematic diagram of the recombinant plasmids is shown in Figure 1 .

[0043] Table 18 RNA sequences for different non-coding regions

[0044]

[0045]

[0046]

[0047]

[0048] 1.4 Plasmid linearization and purification

[0049] The ends of the linearized plasmids used for in vitro transcription are usually blunt ends or 5' ends protruding. We linearized 8 plasmids containing different non-coding regions, EGFP-UTR1, EGFP-UTR2, EGFP-UTR3, EGFP-UTR4, EGFP-UTR5, EGFP-UTR6, EGFP-UTR7, and EGFP-UTR8, using EcoR V, and the EGFP-Control plasmid was linearized using Xba I. The linearized template was confirmed to be completely linearized by gel electrophoresis, purified with phenol chloroform, and the concentration was measured, and stored at -40℃ for use.

[0050] 1.5 Preparation of mRNA

[0051] Use T7 Co-transcription RNA Synthesis Kit, NTPs as substrates, to transcribe the linearized EGFP-UTR1, EGFP-UTR2, EGFP-UTR3, EGFP-UTR4, EGFP-UTR5, EGFP-UTR6, EGFP-UTR7, EGFP-UTR8 plasmid DNA sequences obtained in Section 1.4 above, and efficiently synthesize single-stranded RNA. The kit contains CAP GAG, which is designed for co-transcription capping of mRNA to produce mRNA with natural Cap 1. After in vitro transcription, the mRNA prepared by lithium chloride purification was first identified by denaturing gel electrophoresis for its integrity, and Nanodrop was used to detect its concentration and purity, and stored at -80 degrees.

[0052] 1.6 Gel electrophoresis

[0053] Plasmids and linearized plasmids were subjected to ordinary agarose gel electrophoresis: 0.5 g agarose, 50 mL 1×TAE electrophoresis buffer, 5 μL nucleic acid dye was configured into gel, 300 ng plasmid was added to each well, 1×TAE electrophoresis buffer, 120 V, run the gel for 30 min, and then observe the image. RNA was subjected to denaturing gel electrophoresis: 0.5 g agarose, 36 mL DEPC water, 9 mL formaldehyde, 5 mL 10×MOPS buffer, configured into gel, mRNA needed to be premixed with 2×RNALoading and heated in a 70°C metal bath for 10 min, then 300 ng mRNA was added to each well, 80 V in 1×MOPS electrophoresis buffer, run for 45 min, and then observe the image.

[0054] 1.7 Expression verification of mRNA-EGFP carrying different non-coding regions

[0055] Lipofectamine TM 2000 Reagent was used to transfect the cells with the mRNA prepared above. The specific operation steps were referred to the instruction manual. The luminescence of the cells was observed under a fluorescence microscope at different time points after transfection, and samples were collected for WB detection. At different time points after transfection, the cells were digested to prepare single cell suspensions, and the transfection efficiency was detected by flow cytometry using the FITC channel.

[0056] 1.8 Construction and expression of FMDV mRNA P12A3C carrying UTR3 and UTR8

[0057] To further verify the superiority of UTR3 and UTR8, we constructed FMDVmRNAP12A3C carrying UTR3 and UTR8 respectively. The construction method was the same as 1.3, except that EGFPmRNA was replaced by FMDVmRNAP12A3C. The FMDVmRNAP12A3C prepared by in vitro transcription was transfected into multiple cell lines such as BHK, HEK293T, PK-15, IBRS-2, and MDBK to verify its expression.

[0058] 1.9 Sequence analysis of different non-coding regions

[0059] In order to further analyze the reasons for the differences in the expression of different mRNA EGFP, Geneious Prime was used to predict the GC content, U content, and sequence length of different non-coding regions; RNAfold was used to predict the minimum free energy of the secondary structure of different non-coding regions.

[0060] 2 Results

[0061] 2.1 Obtaining mRNA EGFP carrying different non-coding regions

[0062] In order to prepare mRNA EGFP carrying different non-coding regions, we first linearized the plasmids carrying different non-coding regions with EcoR V, and the control plasmid without non-coding regions with Xba I ( Figure 2 A), the linearized plasmid was purified by phenol chloroform and used as a template for in vitro transcription to prepare mRNAs carrying different non-coding regions ( Figure 2 B).

[0063] 2.2 Expression verification of mRNA-EGFP carrying different non-coding regions

[0064] To verify the expression of EGFP carrying mRNAs with different non-coding regions, we first transfected 1×10 5 BHK cells. After transfection, the luminescence was observed using a fluorescence microscope at 3, 6, 12, 24, 36, 48, and 72 hours, with a uniform exposure of 40 ms and a gain of 1×. It was visible to the naked eye that the green fluorescence of mRNA EGFP-UTR5 and mRNA EGFP-Control was almost invisible. The green fluorescence of the other mRNA EGFP transfections was visible 3 hours after transfection, and the fluorescence intensity reached a peak at 24 hours and showed a weakening trend at 72 hours, which was probably caused by cell apoptosis ( Figure 3 ).

[0065] 2.3 Comparison of expression levels of mRNA-EGFP carrying different non-coding regions

[0066] In order to further quantitatively analyze the expression efficiency of mRNA EGFP carrying different non-coding regions, we transfected 1×10 5 BHK cells were transfected and the cells were digested 24 hours after transfection. The EGFP positive rate and fluorescence intensity were analyzed by flow cytometry. It was found that the transfection efficiency of mRNA EGFP-UTR2, EGFP-UTR3, EGFP-UTR4, EGFP-UTR6, EGFP-UTR7, and EGFP-UTR8 was close to 100% ( Figure 4 A), but the average fluorescence intensity of mRNA EGFP-UTR3 and EGFP-UTR8 was significantly higher than that of other mRNAs with transfection efficiency close to 100%, EGFP-UTR2, EGFP-UTR4, EGFP-UTR6, and EGFP-UTR7 ( Figure 4 B). WB grayscale analysis of samples 24 hours after transfection also demonstrated that the expression levels of mRNA EGFP-UTR3 and EGFP-UTR8 were the highest, and there was no significant difference between them ( Figure 4 C and D)

[0067] 2.4 Expression of mRNA EGFP carrying UTR3 and UTR8 in different cell lines

[0068] We then transfected mRNA EGFP-UTR3 and EGFP-UTR8 in different cell lines, and further analyzed their expression differences in different cell lines by flow cytometry 24 hours after transfection. We found that the transfection efficiency of mRNA EGFP-UTR3 / 8 in different cell lines was as high as more than 90% ( Figure 5 A). It is noteworthy that the transfection efficiency of EGFP-UTR3 mRNA was significantly higher than that of EGFP-UTR8 mRNA in IBRS-2, HEK293T, and F81 cells. However, analysis of mean fluorescence intensity showed that there was no difference between EGFP-UTR3 and EGFP-UTR8 mRNA in IBRS-2 cells. In HEK293T cells, the mean fluorescence intensity of EGFP-UTR3 mRNA was significantly greater than that of EGFP-UTR8 mRNA. In contrast, in F81 cells, the mean fluorescence intensity of EGFP-UTR3 mRNA was significantly lower than that of EGFP-UTR8 mRNA ( Figure 5 B). Overall, these two mRNA structures showed significant expression in multiple cell lines and have the potential to design effective mRNA vaccines.

[0069] 2.5 Construction and expression of FMDV mRNAP12A3C carrying UTR3 and UTR8

[0070] The results of electrophoresis of plasmid linearization of P12A3C-UTR3 and P12A3C-UTR8 are shown in Figure 6 As shown in A, the in vitro transcription results of P12A3C-UTR3 and P12A3C-UTR8 are shown in Figure 6 As shown in B, the expression results of P12A3C-UTR3 and P12A3C-UTR8 in different cells are as shown in Figure 6 C. P12A3C protein is cleaved in cells to produce three proteins of different sizes, with theoretical molecular weights ranging from 25 to 35 kDa; the internal reference protein β-actin is 43 kDa.

[0071] 2.6 Sequence analysis of non-coding regions

[0072] Sequences in different non-coding regions have different base characteristics and structures. Among them, GC content and minimum free energy of secondary structure are closely related to the mRNA expression of different non-coding regions. We used genous Prime to analyze and count the GC content of UTRs and found that the GC content of the 5'UTR of the highly expressed mRNA EGFP-UTR3 and mRNA EGFP-UTR8 was between 45-50% ( Figure 7 A), while the GC content of the 3'UTR is between 35-40% ( Figure 7 B). Among mRNA EGFP-UTR2, mRNA EGFP-UTR5, and mRNA EGFP-UTR6 with the same 3'UTR, the 5'UTR of the low-expressed mRNA EGFP-UTR5 has a very high level of secondary structure free energy (-421.71 kcal / mol), while the secondary structure free energy of the 5'UTR of mRNA EGFP-UTR2 and mRNA EGFP-UTR6 is between -20 and -40 kcal / mol ( Figure 7 C). Among mRNA EGFP-UTR3 and mRNA EGFP-UTR4 with the same 5'UTR, the secondary structure free energy of the 3'UTR of mRNA EGFP-UTR3 with excellent expression level (-65.28 kcal / mol) is smaller than the secondary structure free energy of the 3'UTR of mRNA EGFP-UTR4 (-81.50 kcal / mol) ( Figure 7 D) RNAfold predicts the minimum free energy of the secondary structure of different non-coding region sequences. The minimum free energy structure diagram is shown in Figure 8 .

Claims

1. A non-coding region capable of improving mRNA expression efficiency, characterized in that: The non-coding region includes the 5' non-coding region shown in SEQ ID NO.5 and the 3' non-coding region shown in SEQ ID NO.6, or the non-coding region includes the 5' non-coding region shown in SEQ ID NO.12 and the 3' non-coding region shown in SEQ ID NO.

13.

2. Use of the non-coding region of claim 1 in the preparation of mRNA vaccines.

3. Use of the non-coding region according to claim 1 in improving the expression efficiency of target gene mRNA.

4. The use according to claim 3, characterized in that The target gene is an enhanced green fluorescent protein gene.

5. An mRNA molecule, characterized in that The mRNA molecule contains the non-coding region according to claim 1.

6. The mRNA molecule according to claim 5, characterized in that The mRNA molecule also contains a poly(A) tail.

7. The mRNA molecule according to claim 6, characterized in that The poly(A) tail is composed of 120 adenines.

8. The mRNA molecule according to any one of claims 5 to 7, characterized in that The mRNA molecule also contains an expression frame of enhanced green fluorescent protein.

9. The mRNA molecule of claim 8, wherein The mRNA molecule is obtained by transcription of the nucleotide sequence shown in SEQ ID NO.16 or 21.

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