PCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector as well as construction method and application thereof

Abstract

The invention discloses a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector as well as a construction method and application of the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector. The construction method comprises the following steps: mixing an mCherry gene segment and an IRES2 gene segment which are respectively obtained by PCR (Polymerase Chain Reaction) amplification in equal mass, then carrying out fusion amplification to obtain an IRES2-mCherry fusion gene segment, and carrying out homologous recombination on the IRES2-mCherry fusion segment and a pCDNA-3xHA-hTERT linearization vector. On the basis of a pCDNA-3xHA-hTERT stent vector, a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector with red fluorescence is successfully constructed, and the fusion expression vector can be used as a fusion expression vector of the pCDNA-3xHA-hTERT-IRES2. The construction method is simple and efficient, enzyme digestion is not needed, and the constructed pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector can be subsequently used for transfecting bovine endometrial epithelial cells, so that the immortalized bovine endometrial epithelial cells are obtained.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically involving the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector, its construction method, and its application. Background Technology

[0002] Uterine epithelial cells are a large class of functional cells in the uterus, playing a series of roles including regulating the endometrial microenvironment, substance exchange, immune protection, and secreting essential hormones and related products. Primary endometrial epithelial cells have limited passage counts, are prone to dedifferentiation, and exhibit unstable biological functions such as responses to estradiol and progesterone, leading to poor experimental reproducibility and hindering long-term systematic research and technology transfer. Achieving cell immortalization through genetic engineering techniques (such as introducing the hTERT gene) can overcome the Hayflick limit, maintaining epithelial cell-specific phenotypes and normal hormone responses during long-term passages, while preserving stable proliferative activity and biological functions. Immortalized cell lines can serve as standardized experimental models, widely applied in research on the pathogenesis of endometrial-related diseases, exploration of embryo-endometrial interactions in reproductive medicine, and high-throughput drug screening and toxicology assessment. This provides stable and reproducible cellular tools for subsequent innovative technology transfer, addressing the long-term research and large-scale application needs that primary cells cannot meet.

[0003] Telomerase reverse transcriptase (hTERT), the core catalytic subunit of telomerase, enables cells to proliferate indefinitely by maintaining telomere length. To ensure that the hTERT gene effectively performs its immortalization function, a fluorescent reporter gene can be added after the hTERT gene to form a fusion protein. After the vector is transfected into cells, fluorescence can be directly observed under a microscope, indicating that the vector can express the hTERT gene in the cells.

[0004] In existing technologies, GFP (green fluorescent protein) has been co-expressed in vectors carrying the hTERT gene to achieve real-time tracking of the hTERT gene. However, compared to the commonly used green fluorescent protein (GFP), red fluorescent protein (Red Fluorescent Protein) has significant advantages in long-term live-cell imaging: First, the endogenous fluorescent substances in mammalian cells (such as riboflavin) mostly emit light in the blue-green band, leading to strong background interference in GFP imaging. Red fluorescence, on the other hand, operates in a band where cell autofluorescence is weaker, resulting in clearer images with a higher signal-to-noise ratio. Second, the photon energy of red light is lower than that of green light, causing less phototoxicity and less interference with cellular physiological states, making it more suitable for long-term dynamic observations lasting several days or even weeks. Furthermore, red fluorescence provides an ideal complementary channel for multicolor labeling experiments; when used in conjunction with GFP, it offers better spectral separation and effectively avoids cross-color interference. Therefore, red fluorescence is a better choice for long-term studies such as cell immortalization, but no relevant research has been found to date.

[0005] Based on the above research background, the present invention aims to construct a vector expressing red fluorescence and carrying the hTERT gene for the immortalization of endometrial epithelial cells.

[0006] Current traditional vector construction methods involve restriction endonuclease digestion and T4 DNA ligase ligation. However, these methods lack flexibility and the ligation direction is uncontrollable. For single or partial double digestion, the direction of the inserted fragment cannot be controlled, leading to a large number of erroneous clones with reverse insertion, further reducing the proportion of valid positive clones. Furthermore, the commonly used scaffold vector pCDNA-3xHA-hTERT carrying the hTERT gene lacks suitable restriction enzyme sites, making it difficult to construct the target vector using traditional digestion methods. Therefore, this invention proposes a digestion-free method to construct a red fluorescent vector carrying the hTERT gene. Summary of the Invention

[0007] The first objective of this invention is to provide a method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector.

[0008] A second objective of this invention is to provide a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector.

[0009] The third objective of this invention is to provide the application of the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector.

[0010] The first objective of this invention is achieved by the following technical solution: The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector is characterized by comprising the following steps: S1. Using a plasmid containing the mCherry gene as a template, the mCherry gene fragment was amplified by PCR. As a monomeric protein modified from DsRed, mCherry has a stronger tissue penetration in its excitation spectrum, making it suitable for deep tissue or in vivo imaging. Its resistance to photobleaching is significantly better than that of green fluorescent proteins such as EGFP, and it can withstand long-term excitation light exposure, enabling cell dynamic tracking for more than 72 hours. Its low phototoxicity avoids light-induced cell stress death. Especially when fused with hTERT, the red light of mCherry can avoid interference from cell autofluorescence, providing a high signal-to-noise ratio and long-term non-invasive tracing solution for anti-aging research of rare cells or stem cells in the tumor microenvironment. S2. Using a vector containing the IRES2 sequence as a template, the IRES2 gene fragment was amplified by PCR. S3. Perform fusion PCR: Mix the mCherry gene fragment obtained in S1 and the IRES2 gene fragment obtained in S2 in equal quantities to obtain a mixture of amplified gene products; use the mixture of amplified gene products as a template to amplify the IRES2-mCherry fusion gene fragment. S4. The IRES2-mCherry fusion gene fragment obtained in S3 is inserted into a linearized vector containing the hTERT gene through homologous recombination to obtain the pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid. S5. The pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid was transformed into Escherichia coli DH5α competent cells, cultured, and then sequenced to identify the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector.

[0011] hTERT can extend cell lifespan and overcome the bottleneck of senescence and apoptosis in primary cell culture; mCherry provides non-invasive, real-time cell localization and proliferation monitoring, simplifies experimental procedures, and can achieve the dual functions of cell immortalization and visual tracking.

[0012] Furthermore, in S1, the primers used for PCR amplification of the mCherry gene fragment include forward primer F1 and reverse primer R1; the sequence of F1 is shown in SEQ ID No. 1, and the sequence of R1 is shown in SEQ ID No. 2.

[0013] Furthermore, in S2, the primers used for PCR amplification of the IRES2 gene fragment include forward primer F2 and reverse primer R2; the sequence of F2 is shown in SEQ ID No. 3, and the sequence of R2 is shown in SEQ ID No. 4.

[0014] Furthermore, in S3, the primers used to amplify the IRES2-mCherry fusion gene fragment include forward primer F3 and reverse primer R3; the sequence of F3 is shown in SEQ ID No. 7, and the sequence of R3 is shown in SEQ ID No. 8.

[0015] Further, in S4, the method for preparing the linearized vector containing the hTERT gene includes: using pCDNA-3xHA-hTERT plasmid as a template, performing reverse PCR amplification, verifying the amplification product by 1% agarose gel electrophoresis and recovering it from the gel, to obtain the pCDNA-3xHA-hTERT linearized vector.

[0016] Furthermore, the primers used for the reverse PCR amplification include forward primer F4 and reverse primer R4; the sequence of F4 is shown in SEQ ID No. 9, and the sequence of R4 is shown in SEQ ID No. 10.

[0017] The second objective of this invention is achieved by the following technical solution: The pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector was constructed using the method described above.

[0018] The third objective of this invention is achieved by the following technical solution: The application of the above-mentioned pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector in cell immortalization specifically involves transfecting bovine endometrial epithelial cells with the pCDNA-3xHA-hTERT-IRES2-mCherry vector and culturing them to obtain immortalized bovine endometrial epithelial cells.

[0019] Advantages of the present invention: The present invention constructs the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector by linking the IRES2-mCherry fusion fragment to fusion PCR and then performing homologous recombination of the IRES2-mCherry fusion fragment with the pCDNA-3xHA-hTERT linearized vector.

[0020] The construction method is simple, requiring no enzyme digestion. The mCherry red fluorescent protein gene fragment is directly inserted into a linearized vector containing the hTERT gene via PCR, successfully constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector. Furthermore, experiments have verified that the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector can be used to construct immortalized endometrial epithelial cells. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is an agarose gel electrophoresis image of the mCherry gene fragment in Example 1; Figure 2This is an agarose gel electrophoresis image of the IRES2 gene fragment in Example 1; Figure 3 This is an agarose gel electrophoresis image of the IRES2-mCherry fusion gene fragment in Example 1; Figure 4 This is an agarose gel electrophoresis image of the pCDNA-3xHA-hTERT linearized vector in Example 1; Figure 5 This is the gene map of the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector in Example 1; Figure 6 Electron micrographs of P0, P15, and P30 generation immortalized bovine endometrial epithelial cells from Example 2. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The overall concept of this invention is as follows: Traditional vector construction methods involve restriction endonuclease digestion and T4 DNA ligase ligation. However, limitations in restriction sites and low ligation efficiency lead to time-consuming and low-success-rate vector construction: 1. Traditional restriction enzyme digestion and ligation methods result in fixed cloning sites. Once a site is selected, any subsequent modifications or gene sequence adjustments are extremely difficult, lacking flexibility. T4 DNA ligase itself has low ligation efficiency. When ligating more than two fragments simultaneously (e.g., one vector and two insert genes), the probability of correct end-to-end collision and ligation decreases significantly, directly resulting in a low positive clone rate. 2. Vector self-ligation: Linearized vectors, if incompletely digested or dephosphorylated, are prone to self-circularization, creating a large amount of empty vector background. This severely interferes with subsequent positive clone screening, wasting time and reagents. 3. Uncontrollable ligation direction: For single or partial double digestion, the direction of the insert fragment cannot be controlled, leading to a large number of erroneous clones with reverse insertion, further reducing the proportion of effective positive clones.

[0025] The scaffold vector pCDNA-3xHA-hTERT lacks suitable restriction enzyme sites. Therefore, this invention provides a digestion-free vector construction method using PCR and homologous recombination, offering a new approach for constructing vectors without suitable restriction enzyme sites. This method completely eliminates restriction enzyme dependence; the linearization process no longer requires any restriction endonucleases, thus being unrestricted by the number, location, or reproducibility of restriction enzyme sites on the vector. Theoretically, this method allows for linearization at any position on the vector, providing insertion flexibility for multiple genes, especially complex regulatory elements. The final assembled vector does not introduce any extra bases at the ligation site (depending on primer design), achieving "seamless" cloning. The homologous recombination enzyme-driven reaction is far more efficient than the random ligation of T4 DNA ligase. Because homologous arms are specific, it ensures that each gene fragment is inserted into the predetermined position in the correct orientation, avoiding common problems of empty vectors and mismatches in enzyme digestion and ligation.

[0026] Example 1: Construction of pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector 1. PCR amplification of a single gene Using DNA from a plasmid containing the mCherry gene as a template, the mCherry gene fragment was amplified using forward primer F1 (sequence shown in SEQ ID No. 1) and reverse primer R1 (sequence shown in SEQ ID No. 2). Using DNA from a vector containing the IRES2 sequence as a template, the IRES2 gene fragment was amplified using forward primer F2 (sequence shown in SEQ ID No. 3) and reverse primer R2 (sequence shown in SEQ ID No. 4). The amplification products were identified by 1% agarose gel electrophoresis, and the gel was then recovered to obtain the target fragment. The mCherry gene fragment (sequence shown in SEQ ID No. 5) is shown below. Figure 1 As shown (711bp), the IRES2 gene fragment (its sequence is shown in SEQ ID No. 6) is as follows: Figure 2 As shown (587bp).

[0027] The plasmid containing the mCherry gene was purchased from Addgene (Plasmid#176016); the vector containing the IRES2 sequence was purchased from Shenzhen BGI Genomics Co., Ltd. as the synthetic plasmid IRES2-FAD3.

[0028] 2. PCR amplification of fusion genes The amplified mCherry gene fragment and IRES2 gene fragment were mixed in a 1:1 ratio to obtain an amplified gene product mixture. Using this mixture as a template, the IRES2-mCherry fusion gene fragment was amplified using forward primer F3 (sequence shown in SEQ ID No. 7) and reverse primer R3 (sequence shown in SEQ ID No. 8). The amplified product was verified as IRES2-mCherry (approximately 1.4 kb) by 1% agarose gel electrophoresis and purified. Figure 3 As shown in the electrophoresis results, the IRES2-mCherry fusion fragment was successfully amplified.

[0029] The PCR amplification reaction systems for single gene PCR amplification and fusion gene PCR amplification in the above steps are shown in Table 1, the reaction procedures are shown in Table 2, and the primer sequences used are shown in Table 3.

[0030] Table 1 PCR amplification reaction system

[0031] Table 2 PCR amplification reaction procedure

[0032] Table 3 Primer sequences involved in PCR amplification of single genes and PCR amplification of fusion genes

[0033] 3. Construction of expression carrier (1) The vector pCDNA-3xHA-hTERT (Addgene#51637) was linearized using reverse PCR. Using pCDNA-3xHA-hTERT plasmid as a template, amplification was performed using forward primer F4 (sequence shown in SEQ ID No. 9) and reverse primer R4 (sequence shown in SEQ ID No. 10). The primer sequences used are shown in Table 4. The reverse PCR reaction system is shown in Table 1, and the reaction conditions are shown in Table 2. The amplified products were verified by 1% agarose gel electrophoresis, and the linearized vector was recovered from the gel. Figure 4 As shown.

[0034] Table 4 Reverse PCR Primer Sequences

[0035] (2) The IRES2-mCherry fusion fragment was inserted into the linearized vector containing the hTERT gene obtained in (1) (pCDNA-3xHA-hTERT linearized vector) via homologous recombination to form the pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid, as shown in the figure. Figure 5As shown in Table 5, the homologous recombination reaction system was subjected to a reaction at 50°C for 15 min.

[0036] Table 5 Homologous recombination reaction system

[0037] In Table 5, X = 0.02 × number of base pairs of pCDNA-3xHA-hTERT linearized vector (ng), and Y = 0.04 × number of base pairs of IRES2-mCherry fusion gene fragment (ng).

[0038] The reagents 2x Phanta Flash Master Mix in the PCR amplification reaction system in Table 1 and 2x ClonExpree Mix in the homologous recombination reaction system in Table 5 were both purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0039] (3) Transform 5 μL of the recombinant pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid into 50 μL of Escherichia coli DH5α competent cells, gently mix, incubate on ice for 30 min, heat shock at 42℃ for 60 s, and immediately place on ice for 3 min.

[0040] (4) Then add 500 μL of antibiotic-free liquid LB medium and incubate at 37°C for 1 h.

[0041] (5) Centrifuge the culture medium at 5000 rpm for 5 min, discard a portion of the supernatant, retain 100 μL, resuspend the retained portion by gentle tumbling and spread it on a solid LB medium plate with AMP resistance, and incubate at 37℃ for 12~16 h.

[0042] (6) Select single clones of the bacterial strain and culture them in LB medium with AMP resistance at 37°C for 12-16 h to obtain competent bacterial culture.

[0043] (7) Take 500 μL of competent bacterial culture and send it to the company for sequencing. Add glycerol to the remaining culture and store it at -80℃. Finally, the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector with correct sequencing was successfully obtained.

[0044] (8) The bacterial strain carrying the correctly sequenced pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector was expanded and cultured. After the plasmid was extracted, it was transfected into the cells by liposomes. The expression of red light could be observed under a microscope, indicating that the cells also expressed the hTERT gene.

[0045] The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector of the present invention completely eliminates enzyme digestion dependence and successfully constructs the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector containing the mCherry gene.

[0046] Example 2: Immortality of Bovine Endometrial Epithelial Cells 1. Immortality treatment of bovine endometrial epithelial cells Since bovine endometrial epithelial cells (BEECs) have a limited number of passages under in vitro conditions, they were immortalized by transfecting the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector with liposomes to ensure stable passage.

[0047] The specific steps are as follows: (1) Before transfection, primary bovine endometrial epithelial cells were seeded into 6-well plates. When the confluence reached 70-80%, the F12 culture medium containing FBS was discarded, the cells were washed twice with PBS, and then replaced with FBS-free DMEM / F12 cell culture medium. The cells were then treated at 37°C and 5% CO2 for 2 hours. (2) According to the manufacturer’s instructions, Lipofectamine 2000 (Invitrogen, 11668019) was used for transfection; the pCDNA-3xHA-hTERT-IRES2-mCherry vector successfully constructed in Example 1 was transfected into bovine endometrial epithelial cells and cultured at 37°C and 5% CO2 saturated humidity. (3) After culturing for 5 h, the medium was replaced with DMEM / F12 cell culture medium containing 400 μg / mL G418 and cultured at 37℃ and 5% CO2. The medium was changed every 2 days for the first 5 days. From the sixth day, the number of dead cells increased significantly. The concentration of G418 was halved. On the 10th day, there were obvious colonies of positive clones. The positive clone colonies were continued until they increased significantly. Single clones were selected and cultured. The resulting cells were immortalized bovine endometrial epithelial cells (denoted as BEECshTERT). They were stored in liquid nitrogen for later use.

[0048] 2. Effect Verification The BEECshTERT prepared by the above steps was continuously cultured at 37℃ and 5% CO2, and its growth status was observed by electron microscopy.

[0049] BEECshTERT is growing well and has been stably propagated to more than 30 generations. Figure 6As shown, the 15th and 30th generation cells showed no significant morphological differences compared to the primary cells, all exhibiting a "pebble" growth pattern with tight connections between cells, indicating that the prepared BEECshTERT cells have unlimited passage capacity, thus obtaining an immortalized bovine endometrial epithelial cell line.

[0050] The pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector constructed by the method provided in this invention can be used to immortalize bovine endometrial epithelial cells.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector, characterized in that, Includes the following steps: S1. Using a plasmid containing the mCherry gene as a template, amplify the mCherry gene fragment by PCR; S2. Using a vector containing the IRES2 sequence as a template, the IRES2 gene fragment was amplified by PCR. S3. Perform fusion PCR: Mix the mCherry gene fragment obtained in S1 and the IRES2 gene fragment obtained in S2 in equal quantities to obtain an amplified gene product mixture; use the amplified gene product mixture as a template to amplify the IRES2-mCherry fusion gene fragment. S4. The IRES2-mCherry fusion gene fragment obtained in S3 is inserted into a linearized vector containing the hTERT gene by homologous recombination to obtain the pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid. S5. The pCDNA-3xHA-hTERT-IRES2-mCherry recombinant plasmid was transformed into Escherichia coli DH5α competent cells, cultured, and then sequenced to identify the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector.

2. The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector according to claim 1, characterized in that, In S1, the primers used for PCR amplification of the mCherry gene fragment include forward primer F1 and reverse primer R1; the sequence of F1 is shown in SEQ ID No. 1, and the sequence of R1 is shown in SEQ ID No.

2.

3. The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector according to claim 1, characterized in that, In S2, the primers used for PCR amplification of the IRES2 gene fragment include forward primer F2 and reverse primer R2; the sequence of F2 is shown in SEQ ID No. 3, and the sequence of R2 is shown in SEQ ID No.

4.

4. The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector according to claim 1, characterized in that, In S3, the primers used to amplify the IRES2-mCherry fusion gene fragment include forward primer F3 and reverse primer R3; the sequence of F3 is shown in SEQ ID No. 7, and the sequence of R3 is shown in SEQ ID No.

8.

5. The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector according to claim 1, characterized in that, In S4, the method for preparing the linearized vector containing the hTERT gene includes: using pCDNA-3xHA-hTERT plasmid as a template, performing reverse PCR amplification, verifying the amplification product by 1% agarose gel electrophoresis and recovering it from the gel, and obtaining the pCDNA-3xHA-hTERT linearized vector.

6. The method for constructing the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector according to claim 5, characterized in that, The primers used for the reverse PCR amplification include forward primer F4 and reverse primer R4; the sequence of F4 is shown in SEQ ID No. 9, and the sequence of R4 is shown in SEQ ID No.

10.

7. The pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector constructed by the method of any one of claims 1-6.

8. The application of the pCDNA-3xHA-hTERT-IRES2-mCherry fusion expression vector of claim 7 in cell immortalization, characterized in that, Immortalized bovine endometrial epithelial cells were obtained by transfecting bovine endometrial epithelial cells with the pCDNA-3xHA-hTERT-IRES2-mCherry vector.

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