Vector plasmid, escherichia coli, cell and method for overexpressing HPV18 E6 gene

Abstract

The invention discloses a vector plasmid, escherichia coli, a cell and a method for overexpressing an HPV18E6 gene. The vector plasmid comprises a screening module, a tracking module, a regulation and control module, a virus packaging module and a core functional element, the screening module comprises a prokaryotic screening sub-module and a eukaryotic screening sub-module, and the prokaryotic screening sub-module comprises an AmpR promoter and an AmpR gene; the eukaryotic screening submodule comprises a PGK promoter and a PuroR gene; the tracking module comprises a ZsGreen1 fluorescent protein gene; the regulation and control module comprises a double-starting subsystem and an enhancement element; the double-promoter system comprises a CMV (cytomegalovirus) strong promoter and the PGK promoter; the enhancement element comprises a WPRE sequence and an HIV RRE sequence; the virus packaging module comprises an HIV-1psi packaging signal, a 3 'LTR sequence and a lac promoter; the core functional element comprises an HPV18E6 gene. The vector plasmid provided by the invention has both prokaryotic amplification and eukaryotic expression capabilities, and is suitable for overexpression research of the HPV18E6 gene, virus packaging and establishment of a stable cell line.

Description

Technical Field

[0001] The present invention relates to the field of biological technology, and in particular to a vector plasmid, Escherichia coli, cells and a method for overexpressing the HPV18 E6 gene. Background Art

[0002] U14 cells are a mouse cervical cancer cell line that is widely used in cancer research, especially in tumor biology, immunotherapy, and drug screening. U14 cells themselves cannot express any HPV genes. In order to enable U14 cells to better simulate the process of HPV gene-induced carcinogenesis in mouse models for the development of HPV vaccines, it is necessary to artificially design plasmid vectors carrying HPV genes to transfect U14 cells. However, the existing technology lacks vector plasmids that can be transfected into U14 cells and can effectively express HPV genes.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0004] The present invention provides a vector plasmid, Escherichia coli, cells and method for overexpressing the HPV18 E6 gene, aiming to solve the technical problems existing in the prior art mentioned in the above background technology.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The first aspect of the present invention provides a vector plasmid for overexpressing the HPV18 E6 gene, comprising a screening module, a tracking module, a regulatory module, a viral packaging module and core functional elements; the screening module comprises a prokaryotic screening submodule and a eukaryotic screening submodule, the prokaryotic screening submodule comprising an AmpR promoter and an AmpR gene; the eukaryotic screening submodule comprising a PGK promoter and a PuroR gene; the tracking module comprising a ZsGreen1 fluorescent protein gene; the regulatory module comprising a dual promoter system and an enhancing element; the dual promoter system comprising a strong CMV promoter and the PGK promoter; the enhancing element comprising a WPRE sequence and an HIV RRE sequence; the viral packaging module comprising an HIV-1ψ packaging signal, a 3'LTR sequence and a lac promoter; and the core functional elements comprising the HPV18 E6 gene.

[0007] The second aspect of the present invention provides an Escherichia coli comprising the vector plasmid for overexpressing the HPV18E6 gene according to the first aspect of the present invention.

[0008] A third aspect of the present invention provides a method for producing U14 cells that overexpress the HPV18 E6 gene, comprising:

[0009] Providing Escherichia coli carrying the vector plasmid for overexpressing the HPV18 E6 gene according to the first aspect of the present invention;

[0010] Extracting the vector plasmid from the Escherichia coli;

[0011] The vector plasmid was transfected into 293T cells to produce virus;

[0012] The virus was used to infect U14 cells to obtain U14 cells overexpressing the HPV18 E6 gene.

[0013] In an optional embodiment of the third aspect of the present invention, the extracting the vector plasmid from the Escherichia coli comprises:

[0014] The Escherichia coli was inoculated into LB medium containing ampicillin and cultured;

[0015] The cultured E. coli is collected, and the vector plasmid is extracted from the cultured E. coli using an endotoxin-free plasmid miniprep kit to obtain the vector plasmid.

[0016] In an optional embodiment of the third aspect of the present invention, transfecting the vector plasmid into 293T cells to produce virus comprises:

[0017] Passaging the 293T cells into a culture flask, and waiting for the 293T cells to grow to a predetermined size before transfection;

[0018] When the amount of vector remains unchanged, the ratio of the vector plasmid to the auxiliary vector is adjusted according to the size of the vector plasmid;

[0019] Mixing the mixed vector and the transfection reagent according to the requirements of the transfection reagent, and performing transfection of the 293T cells;

[0020] After culturing for a first preset time, replacing the culture medium to culture the transfected 293T cells;

[0021] After culturing for a second preset time, collecting the viral supernatant from the culture medium of the transfected 293T cells;

[0022] After the collected virus supernatant is concentrated and purified, the virus precipitate is collected.

[0023] In an optional embodiment of the third aspect of the present invention, the method of infecting U14 cells with the virus to obtain U14 cells overexpressing the HPV18 E6 gene comprises:

[0024] U14 cells were plated on a 24-well plate and infected with the virus;

[0025] Performing puromycin killing test and HPV18 E6 gene expression test on the U14 cells after the virus infection;

[0026] The U14 cells that passed the test were subjected to monoclonal culture to obtain U14 cells that overexpressed the HPV18 E6 gene.

[0027] The fourth aspect of the present invention provides a U14 cell overexpressing the HPV18 E6 gene, which is prepared by the preparation method according to any one of the third aspects of the present invention.

[0028] Beneficial Effects: The present invention discloses a vector plasmid, Escherichia coli, cell, and method for overexpressing the HPV18 E6 gene. The vector plasmid includes a screening module, a tracking module, a regulatory module, a viral packaging module, and core functional elements. The screening module includes a prokaryotic screening submodule and a eukaryotic screening submodule, wherein the prokaryotic screening submodule includes an AmpR promoter and an AmpR gene; the eukaryotic screening submodule includes a PGK promoter and a PuroR gene; the tracking module includes a ZsGreen1 fluorescent protein gene; the regulatory module includes a dual promoter system and an enhancing element, wherein the dual promoter system includes a strong CMV promoter and the PGK promoter; the enhancing element includes a WPRE sequence and an HIV RRE sequence; the viral packaging module includes an HIV-1ψ packaging signal, a 3' LTR sequence, and a lac promoter; and the core functional element includes the HPV18 E6 gene. The vector plasmid of the present invention has both prokaryotic amplification and eukaryotic expression capabilities and is suitable for HPV18 E6 gene overexpression research, viral packaging, and stable cell line establishment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a genetic composition map of a vector plasmid for overexpressing the HPV18 E6 gene of the present invention.

[0030] Figure 2 This is a complete schematic diagram of the process for preparing a multivalently expressed Listeria monocytogenes cervical cancer vaccine of the present invention.

[0031] Figure 3 This is a schematic diagram of the qPCR detection results of U14 cells overexpressing the HPV18 E6 gene in the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] See also Figure 1In a first aspect, the present invention provides a vector plasmid for overexpressing the HPV18 E6 gene, comprising a screening module, a tracking module, a regulatory module, a viral packaging module, and core functional elements; the screening module comprises a prokaryotic screening submodule and a eukaryotic screening submodule, the prokaryotic screening submodule comprising an AmpR promoter and an AmpR gene; the eukaryotic screening submodule comprising a PGK promoter and a PuroR gene; the tracking module comprising a ZsGreen1 fluorescent protein gene; the regulatory module comprising a dual promoter system and an enhancing element; the dual promoter system comprising a strong CMV promoter and the PGK promoter; the enhancing element comprising a WPRE sequence and an HIV RRE sequence; the viral packaging module comprising an HIV-1ψ packaging signal, a 3'LTR sequence, and a lac promoter; and the core functional elements comprising the HPV18 E6 gene.

[0034] Specifically, in the present invention, the AmpR promoter + AmpR gene is used to support ampicillin resistance screening in Escherichia coli; the PGK promoter drives the PuroR gene (co-expressed with ZsGreen through T2A self-cleaving peptide) to achieve puromycin selection in mammalian cells; the ZsGreen1 fluorescent protein gene is used to cooperate with PuroR to achieve dual labeling through T2A: green fluorescence is used to observe transfection efficiency, and PuroR is used for stable cell line screening; the CMV strong promoter is used to drive the efficient expression of the HPV18 E6 oncogene; the PGK constitutive promoter is used to maintain continuous expression of the selection marker; WPRE is used to enhance mRNA stability; the HIVRRE element is used to promote RNA nuclear export; the HIV-1ψ packaging signal is used to ensure the correct assembly of viral particles; the 3'LTR sequence is used to mediate genome integration into the host chromosome; the prokaryotic replication elements (lac promoter, etc.) are used to support bacterial amplification; HPV18 E6 gene (NCBI No. NC_001357.1): used for viral carcinogenic mechanism research or gene function analysis, and the nucleotide sequence of the vector plasmid of the present invention is shown in SEQ ID NO: 1.

[0035] The vector plasmid of this invention utilizes a lentiviral backbone, enabling both prokaryotic amplification (in E. coli) and eukaryotic expression (in mammalian cells), making it suitable for gene overexpression research, viral packaging, and stable cell line establishment. The component layout is meticulously designed, and each functional module achieves spatiotemporal coordinated expression through self-cleaving peptides and specific regulatory elements.

[0036] The second aspect of the present invention provides an Escherichia coli comprising the vector plasmid for overexpressing the HPV18E6 gene according to the first aspect of the present invention.

[0037] See also Figure 2 The third aspect of the present invention provides a method for producing U14 cells that overexpress the HPV18 E6 gene, comprising:

[0038] S100. Provide Escherichia coli carrying the vector plasmid for overexpressing the HPV18 E6 gene according to the first aspect of the present invention; specifically, the vector plasmid includes a screening module, a tracking module, a regulatory module, a viral packaging module, and core functional elements; the screening module includes a prokaryotic screening submodule and a eukaryotic screening submodule, the prokaryotic screening submodule including an AmpR promoter and an AmpR gene; the eukaryotic screening submodule including a PGK promoter and a PuroR gene; the tracking module including a ZsGreen1 fluorescent protein gene; the regulatory module including a dual promoter system and an enhancing element; the dual promoter system including a strong CMV promoter and the PGK promoter; the enhancing element including a WPRE sequence and an HIV RRE sequence; the viral packaging module including an HIV-1ψ packaging signal, a 3'LTR sequence, and a lac promoter; and the core functional elements including the HPV18 E6 gene.

[0039] S200, extracting the vector plasmid from the E. coli; in an optional embodiment of the third aspect of the present invention, extracting the vector plasmid from the E. coli comprises: inoculating the E. coli into LB culture medium containing ampicillin and culturing; collecting the cultured E. coli, and extracting the vector plasmid from the cultured E. coli using an endotoxin-free plasmid miniprep kit. Exemplarily, this step can be: inoculating the bacterial liquid into approximately 50 mL of LB culture medium containing 50 mg / L of Amp, and culturing at 37°C, 200 rpm, with shaking for approximately 18 hours. Collecting the bacterial cells, and extracting the plasmid using an endotoxin-free plasmid miniprep kit (Tian Gen: DP118).

[0040] S300, transfecting the vector plasmid into 293T cells to produce virus; in an optional embodiment of the third aspect of the present invention, transfecting the vector plasmid into 293T cells to produce virus includes: passaging the 293T cells into a culture flask, and waiting for the 293T cells to grow to a preset level for transfection; adjusting the ratio of the vector plasmid to the auxiliary vector according to the size of the vector plasmid while keeping the vector dosage unchanged; mixing the mixed vector and the transfection reagent according to the requirements of the transfection reagent, and transfecting the 293T cells; after culturing for a first preset time, replacing the culture medium and culturing the transfected 293T cells; after culturing for a second preset time, collecting the viral supernatant from the culture medium of the transfected 293T cells; concentrating and purifying the collected viral supernatant to collect the viral precipitate.

[0041] For example, the steps can be as follows: Cell culture: one day before transfection, the grown 293T cells are subcultured into T75 culture flasks at an appropriate ratio, and transfection is prepared when the cells grow to about 80%. Plasmid transfection: when the total amount of vector remains unchanged, the ratio of shuttle vector to auxiliary vector is adjusted according to the size of shuttle vector (i.e., vector plasmid). TM (Biyuntian: C0521) According to the requirements of the transfection reagent, mix the plasmid and transfection reagent, and perform cell transfection. Replace the culture medium: 18 to 24 hours after transfection, carefully aspirate the cell culture medium and discard it in a conical flask, then seal it and sterilize it under high temperature and high pressure. The culture medium replacement here provides a culture medium with sufficient nutrients for virus production, and at the same time, the transfection reagent, plasmid, etc. can be removed. Culture medium collection: 24 hours after the medium change, aspirate the cell culture supernatant into a 50mL centrifuge tube and add fresh culture medium. At 48 hours, aspirate the cell culture supernatant into a 50mL centrifuge tube, discard the cells, and filter the supernatant with a 0.45μm sterile filter and transfer it to a new centrifuge tube. Virus concentration: Concentrate and purify the lentivirus according to the PEG lentivirus purification reagent (Yinmao Shengye: P1201). Finally, dissolve the lentivirus precipitate with PBS, divide it into 200uL / tube, and store it at -80℃.

[0042] S400. Infect U14 cells with the virus to obtain U14 cells that overexpress the HPV18 E6 gene. In an optional embodiment of the third aspect of the present invention, infecting U14 cells with the virus to obtain U14 cells that overexpress the HPV18 E6 gene includes: plating U14 cells on a 24-well plate and then infecting the U14 cells with the virus; performing a puromycin killing test and an HPV18 E6 gene expression test on the virus-infected U14 cells; and performing monoclonal culture on the U14 cells that pass the test to obtain U14 cells that overexpress the HPV18 E6 gene.

[0043] Exemplarily, this step can be specifically as follows: Cell infection: 5x104 cells are plated on a 24-well plate the day before infection, and the corresponding amount of virus is taken to infect the cells according to the MOI. Drug killing: 24 hours after infection, use a culture medium containing a final concentration of Puro of 1 μg / mL to kill the cells for 2 days, and then change to a culture medium without Puro to continue culturing. Cell genome detection: After the cell suspension is centrifuged at 1500 rpm for 5 minutes, the supernatant is removed, and cell lysis solution is added to obtain the genome. The genome sample is PCR amplified using sequencing primers. When a band is obtained by PCR amplification, it is sent for sequencing and compared with the vector overexpression sequence. Single clone: After the cell genome sequencing result matches the vector overexpression sequence, after counting, the cell concentration is controlled at 1-1.5 cells per 100 μL by limiting dilution. The cell suspension is inoculated into a 96-well plate. Picking single clones: After culturing for about two weeks, observe the monoclonal clones growing in the 96-well plate under a microscope, and mark the wells where the fluorescent monoclonal clones are located. After digestion, discard the digestion solution and take 200 μL of culture medium to resuspend the cells for passage. 6 When the cells were grown, half of them were sent for precipitation and qPCR detection, and the other half were expanded and kept as seeds.

[0044] The results of the qPCR test of the present invention can be shown as Figure 3 As shown, from Figure 3 It can be seen that the HPV18 E6 gene is not expressed in the background of U14 cells. The CT value of the target gene in the monoclonal cell line is only about 1.2 lower than the CT value of the internal reference, indicating that E6 is highly expressed in the monoclonal cell line.

[0045] In addition, the fourth aspect of the present invention provides a U14 cell overexpressing the HPV18 E6 gene, which is prepared by the preparation method described in any one of the third aspects of the present invention.

[0046] In summary, the present invention discloses a vector plasmid, Escherichia coli, cells, and method for overexpressing the HPV18 E6 gene. The vector plasmid includes a screening module, a tracking module, a regulatory module, a viral packaging module, and core functional elements. The screening module includes a prokaryotic screening submodule and a eukaryotic screening submodule, wherein the prokaryotic screening submodule includes the AmpR promoter and the AmpR gene; the eukaryotic screening submodule includes the PGK promoter and the PuroR gene; the tracking module includes the ZsGreen1 fluorescent protein gene; the regulatory module includes a dual promoter system and an enhancing element; the dual promoter system includes a strong CMV promoter and the PGK promoter; the enhancing element includes a WPRE sequence and an HIV RRE sequence; the viral packaging module includes the HIV-1ψ packaging signal, 3' LTR sequence, and lac promoter; and the core functional elements include the HPV18 E6 gene. The vector plasmid of the present invention has both prokaryotic amplification and eukaryotic expression capabilities and is suitable for HPV18 E6 gene overexpression research, viral packaging, and stable cell line establishment.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A vector plasmid for overexpressing the HPV18 E6 gene, characterized in that: It includes a screening module, a tracking module, a regulatory module, a virus packaging module and core functional elements; the screening module includes a prokaryotic screening submodule and a eukaryotic screening submodule, the prokaryotic screening submodule includes an AmpR promoter and an AmpR gene; the eukaryotic screening submodule includes a PGK promoter and a PuroR gene; the tracking module includes a ZsGreen1 fluorescent protein gene; the regulatory module includes a dual promoter system and an enhancing element; the dual promoter system includes a CMV strong promoter and the PGK promoter; the enhancing element includes a WPRE sequence and an HIV RRE sequence; the virus packaging module includes an HIV-1ψ packaging signal, a 3'LTR sequence and a lac promoter; and the core functional elements include an HPV18 E6 gene.

2. An Escherichia coli, characterized in that Comprising the vector plasmid for overexpressing the HPV18 E6 gene according to claim 1.

3. A method for producing U14 cells overexpressing the HPV18 E6 gene, characterized in that: include: Providing Escherichia coli carrying the vector plasmid for overexpressing the HPV18 E6 gene according to claim 1; Extracting the vector plasmid from the Escherichia coli; The vector plasmid was transfected into 293T cells to produce virus; The virus was used to infect U14 cells to obtain U14 cells overexpressing the HPV18 E6 gene.

4. The method for producing U14 cells overexpressing the HPV18 E6 gene according to claim 3, characterized in that: The extracting of the vector plasmid from the Escherichia coli comprises: The Escherichia coli was inoculated into LB medium containing ampicillin and cultured; The cultured E. coli is collected, and the vector plasmid is extracted from the cultured E. coli using an endotoxin-free plasmid miniprep kit to obtain the vector plasmid.

5. The method for producing U14 cells that overexpress the HPV18 E6 gene with multivalent expression according to claim 3, characterized in that: The transfection of the vector plasmid into 293T cells to produce virus comprises: Passaging the 293T cells into a culture flask, and waiting for the 293T cells to grow to a predetermined size before transfection; When the amount of vector remains unchanged, the ratio of the vector plasmid to the auxiliary vector is adjusted according to the size of the vector plasmid; Mixing the mixed vector and the transfection reagent according to the requirements of the transfection reagent, and performing transfection of the 293T cells; After culturing for a first preset time, replacing the culture medium to culture the transfected 293T cells; After culturing for a second preset time, collecting the viral supernatant from the culture medium of the transfected 293T cells; After the collected virus supernatant is concentrated and purified, the virus precipitate is collected.

6. The method for producing U14 cells that overexpress the HPV18 E6 gene with multivalent expression according to claim 3, characterized in that: The method of using the virus to infect U14 cells to obtain U14 cells that overexpress the HPV18 E6 gene comprises: U14 cells were plated on a 24-well plate and infected with the virus; Performing puromycin killing test and HPV18 E6 gene expression test on the U14 cells after the virus infection; The U14 cells that passed the test were subjected to monoclonal culture to obtain U14 cells that overexpressed the HPV18 E6 gene.

7. A U14 cell overexpressing the HPV18 E6 gene, characterized in that: Prepared by the preparation method according to any one of claims 3 to 6.

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