HPV18 recombinant lentiviral vector, cell and construction method and application of mouse tumor model

Abstract

The invention discloses an HPV18 recombinant lentiviral vector, a cell and a construction method and application of a mouse tumor model. The recombinant lentiviral vector has an HPV18E6 expression cassette and an E7 expression cassette; the structures of the HPV18E6 and E7 expression cassettes are a first promoter, an E6 first 2A peptide, a first selection marker, a second promoter, an E7 second 2A peptide and a second selection marker; the first promoter and the second promoter are different and are selected from a CMV promoter, an EF1a promoter, an SV40 promoter, a beta-actin promoter, a CAG promoter and a PGK promoter; the first 2A peptide and the second 2A peptide are each independently selected from a P2A peptide and a T2A peptide; one of the first selection marker and the second selection marker is a fluorescent gene, and the other one is an antibiotic gene. According to the invention, high expression of HPV18E6 and E7 proteins is realized through HPV18E6 and E7 expression cassettes.

Description

Technical Field

[0001] The present invention relates to recombinant vectors, cells, and the construction of tumor models, etc., and specifically relates to an HPV18 recombinant lentiviral vector, cells, and a construction method and application of a mouse tumor model. Background Art

[0002] As one of the main subtypes of high-risk human papillomavirus (HPV), HPV18 is closely related to the occurrence of cervical cancer. Research has found that HPV18 infects cervical cancer epithelial cells and integrates into the host genome, continuously expressing the oncoproteins E6 and E7, interfering with the cell cycle regulation of host cells (such as degrading the tumor suppressor genes p53 and Rb), resulting in abnormal cell proliferation and carcinogenesis. Epidemiological data shows that HPV16 and HPV18 together cause approximately 70% of cervical cancer cases and are closely related to poor tumor invasion and prognosis. Since the HPV18 E6 and E7 proteins are only expressed in tumors and not in normal tissues, they may become potential targets for the research of HPV18-related cervical cancer therapeutic vaccines.

[0003] An important link in the research of HPV18 tumor therapeutic vaccines and their immune agonists is to establish a suitable homologous tumor model. Currently, TC-1 cells can be used for the non-clinical efficacy evaluation of HPV16 therapeutic vaccines because they themselves express the E6 and E7 proteins of HPV16. TC-18 is obtained by transfecting the fusion gene of HPV18 E6 and E7 on the basis of TC-1. Although it can theoretically be used for the preclinical efficacy evaluation of HPV16 / HPV18 bivalent vaccines, there may be cross-interference between the E6 and E7 proteins of HPV16 and HPV18, affecting the determination of the efficacy results and being not conducive to the clarification of the mechanism of action. In view of the current lack of relevant cell lines, it is urgent to construct a tumor mouse-derived cell line stably transfected with the HPV18 E6 and E7 genes as a tool cell for the research and development of new drugs and the study of mechanisms. Summary of the Invention

[0004] In order to solve the problem of the lack of relevant cell lines, the present invention first discloses a recombinant lentiviral vector, which has an HPV18 E6 and E7 expression cassette; the structure of the HPV18 E6 and E7 expression cassette is a first promoter - E6 - a first 2A peptide - a first selection marker - a second promoter - E7 - a second 2A peptide - a second selection marker.

[0005] In some embodiments, the first promoter and the second promoter are different and are each independently selected from the CMV promoter, EF1a promoter, SV40 promoter, β-actin promoter, CAG promoter, and PGK promoter; the first 2A peptide and the second 2A peptide are each independently selected from the P2A peptide and the T2A peptide; one of the first selection marker and the second selection marker is a fluorescent gene, and the other is an antibiotic gene.

[0006] In some embodiments, the fluorescent gene is selected from GFP, RFP, YFP, BFP, CFP, and mCherry; the antibiotic gene is selected from puromycin, neomycin (G418), bleomycin, and hygromycin B.

[0007] In some embodiments, the recombinant lentiviral vector is prepared by inserting the HPV18 E6 and E7 expression cassette DNA fragment into the original plasmid pCDH.

[0008] In some embodiments, the structure of the HPV18 E6 and E7 expression cassette is selected from the following table:

[0009]

[0010]

[0011] In some embodiments, the structure of the HPV18 E6 and E7 expression cassette is CMV-E6-P2A-GFP-EF1a-E7-T2A-puro, and the nucleic acid sequence is as shown in SEQ ID NO:1. Further, the recombinant lentiviral vector is pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro.

[0012] In some embodiments, the nucleic acid sequences of E6 and E7 are as shown in SEQ ID NO:2 and 3, respectively.

[0013] In some embodiments, the amino acid sequences of the P2A peptide and the T2A peptide are as shown in SEQ ID NO:4 and 5, respectively.

[0014] Secondly, the present invention also discloses corresponding cells, which are LLC1 cells and / or EO771 cells infected with lentivirus packaged by the recombinant lentiviral vector as described above; the cells can express the E6 and E7 proteins of HPV18. LLC1 cells are a kind of mouse lung cancer cells; EO771 cells are mouse myeloid breast cancer cells.

[0015] In some embodiments, the recombinant lentiviral vector is a vector obtained by inserting the DNA fragments of the HPV18 E6 and E7 expression cassettes into pCDH; the recombinant lentiviral vector is co-transfected with the packaging plasmids pMD2.G and psPAX2 into HEK-293T cells to package lentivirus; the lentivirus is used to infect LLC1 cells and / or EO771 cells, and the cells are obtained after antibiotic drug screening in the HPV18 E6 and E7 expression cassettes.

[0016] Third, the present invention also discloses a method for constructing an HPV18 mouse tumor model, that is, the above-mentioned cells are subcutaneously inoculated into mice, and the HPV18 mouse tumor model is obtained after several days of feeding; the number of inoculated cells is 0.5×10^6 to 1×10^6 and is inoculated into mice after mixing with Matrigel. "After several days of feeding" should be understood as feeding for more than 2 to 9 days.

[0017] In some embodiments, the inoculated cells are mixed with Matrigel at a volume ratio of 1:1, and each volume is 200±10 uL, preferably 200 uL. Further, the Matrigel is low growth factor Matrigel.

[0018] In some embodiments, the HPV18 mouse tumor model is an LLC1-HPV18 tumor model and / or an EO771-HPV18 tumor model.

[0019] Fourth, the present invention also discloses the application of the above-mentioned cells in the efficacy evaluation of an HPV18 therapeutic tumor vaccine, that is, after immunizing mice with the HPV18 therapeutic tumor vaccine to be evaluated, the above-mentioned cells are subcutaneously inoculated into the mice, and after a period of time, the tumor volume of the mice is detected. The smaller the tumor volume, the better the efficacy of the HPV18 therapeutic tumor vaccine to be evaluated. Here, "a period of time" is determined according to experimental needs. For example, it can be 7 to 30 days later, or 12 to 25 days later, or 13 to 21 days later, or 14 to 18 days later, or 15 days later.

[0020] Fifth, the present invention also discloses an HPV18 therapeutic tumor vaccine, which is an RNA vaccine and has a nucleic acid sequence as shown in SEQ ID NO: 6.

[0021] Aiming at the cell lines constructed by conventional methods, in which genes are not expressed, tumors do not form in vivo, or tumors are prone to shrinkage after formation, the present invention optimizes the codons of HPV18 E6 and E7 proteins and connects a tag protein through a 2A peptide. The present invention realizes the high expression of HPV18 E6 and E7 proteins through the HPV18 E6 and E7 expression cassettes. At the same time, it also realizes the smooth growth of tumors in the body of a mouse tumor model constructed by cells carrying this expression cassette.

[0022] In the present invention, the HPV18 E6 and E7 genes are respectively expressed in the parental cells LLC1 and EO771 that do not express the HPV16 E6 and E7 proteins, which not only avoids the cross-interference of the HPV16 E6 and E7 carried by the TC-1 cells themselves on the preclinical efficacy evaluation of the HPV18 therapeutic vaccine and affects the result determination, but also can respectively identify the expression of the HPV18 E6 and E7 proteins in the tumor, which helps to clarify the mechanism of action of the drug to be tested.

[0023] The beneficial effects of the present invention are as follows:

[0024] (1) The expression efficiency of the HPV18 E6 and E7 proteins is low. Using the mouse-derived codon-optimized sequences (such as SEQ ID NO: 2 and 3) and dual promoters can improve their expression efficiency.

[0025] (2) The design of the HVP18 E6 and E7 expression cassettes can efficiently screen positive monoclonal cells. E6 is linked to green fluorescent protein (GFP) through the P2A peptide. On the one hand, it can solve the problem of no good antibodies for detecting the E6 protein, and at the same time, the expressed protein will be cleaved at the P2A site, without affecting the function of E6. E7 is linked to the puromycin (abbreviated as puro) gene through the T2A peptide, and the expressed protein will be cleaved at the T2A site, without affecting the function of E7. The combination of GFP and puromycin can improve the screening efficiency of positive monoclonal cells.

[0026] (3) The cells constructed in the present invention (especially LLC1-HPV18 cells and EO771-HPV18 cells) can respectively detect the expression of E6 and E7 proteins, which helps to clarify the mechanism of action of the subsequent drugs.

[0027] (4) Selecting LLC1 and EO771 cells that do not express the HPV16 E6 and E7 proteins can avoid the interference of the HPV16 E6 and E7 proteins on the efficacy evaluation and affect the result determination, and also helps to clarify the mechanism of action of the drug. Selecting appropriate parental tumor cells is also an important reason for the success of this tumor model. LLC1 and EO771 cells are parental cells that have been screened and tested multiple times and can stably form tumors in mice and express the HPV18 E6 and E7 proteins. Cells such as B16F10, MC38, RM1, 4T1, and EMT-6 either do not grow tumors, or the tumors shrink after growth, or the target genes cannot be detected in the tumors after growth. These cells that cannot stably form tumors in mice and continuously express the HPV18 E6 and E7 proteins cannot achieve the purpose of the present invention.

[0028] (5) The HPV18 mouse tumor model has a good tumor formation effect in vivo, without the phenomena of no tumor growth and tumor shrinkage (such as shown by curve A in Example 2, Figure 2 ).

[0029] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. Description of the Drawings

[0030] Figure 1 Western blot (A) and FACS (B) were used to identify the expression of HPV18 E6 and E7 proteins in the constructed cell lines. β-actin was used as β-actin. N represents the negative control.

[0031] Figure 2 This is the detection result of the construction of the HPV18 mouse tumor model in Example 2. Among them, A shows the tumor growth and body weight changes of LLC1-HPV18 and EO771-HPV18 cells in vivo; B and C respectively show the expression of HPV18 E6 and E7 proteins in ex vivo tumors at the end of the experiment detected by FACS and Western blot. Each peak and lane represents an ex vivo tumor sample of a mouse, and the numbers are the labels of the mice.

[0032] Figure 3A This is a schematic diagram of the animal experiment design.

[0033] Figure 3B These are the tumor growth curves and body weight change curves of the LLC1-HPV18 and EO771-HPV18 tumor models.

[0034] Figure 3C Flow cytometry was used to detect memory effector CD8+ T cells in the blood, and IFN-γ ELISpot was used to detect the specific immunity to HPV18 E6 and E7 proteins.

[0035] Figure 4 This is the detection result of tumorigenesis in vivo and gene expression in tumors after tumorigenesis of B16F10-HPV18 cells. Each peak and lane represents an ex vivo tumor sample of a mouse, and the numbers are the labels of the mice.

[0036] Figure 5 This is the detection result of tumorigenesis in vivo and gene expression in tumors after tumorigenesis of 4T1-HPV18 cells. Each peak and lane represents an ex vivo tumor sample of a mouse, and the numbers are the labels of the mice.

[0037] Figure 6 This is the detection result of tumorigenesis in vivo and gene expression in tumors after tumorigenesis of EMT6-HPV18 cells. Each lane represents an ex vivo tumor sample of a mouse, and the numbers are the labels of the mice. Detailed Embodiments

[0038] For the convenience of those skilled in the art to understand, some terms appearing in this text are explained and described.

[0039] In this text, the singular forms of "a", "an" and "the" also include their plural forms, unless the context otherwise indicates.

[0040] In this text, unless otherwise specified, the terms "comprising", "including" or "having" mean containing the listed numerical values, steps or components, but do not exclude also containing other numerical values, steps or components.

[0041] In this text, "A and / or B" means that it can be any of the three cases: A and B, A alone, and B alone.

[0042] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0043] In some embodiments, the main object of the present invention is to optimize the codons of the HPV18 E6 and E7 genes and design the expression cassette structure, so as to improve the gene expression efficiency while constructing a tumor model that only expresses the HPV18 E6 and E7 proteins, thereby filling the blank that there is no homologous tumor model available for the efficacy evaluation of HPV18-related tumor immunotherapy drugs.

[0044] Example 1. Design of recombinant lentiviral vector and construction of cell line

[0045] Due to the low expression efficiency of HPV18 E6 and E7 proteins, the amino acid sequences of E6 and E7 proteins were first optimized for mouse codons to improve the expression efficiency of E6 and E7 proteins, and were respectively linked to GFP and Puro through P2A and T2A peptides. At the same time, in order to maintain good expression efficiency of both proteins, CMV and EF1a promoters were respectively selected to express E6 and E7 proteins in the experiment, and then the recombinant lentiviral vector pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro was obtained. The nucleic acid sequences obtained after the mouse codon optimization of E6 and E7 proteins are shown in SEQ ID NO:2 and 3 respectively. The structure of the HPV18 E6 and E7 expression cassettes in the recombinant lentiviral vector pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro is CMV-E6-P2A-GFP-EF1a-E7-T2A-puro, and the nucleic acid sequence is shown in SEQ ID NO:1. The original plasmid pCDH of the recombinant lentiviral vector was purchased from Miaoling Biology.

[0046] The pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro was co-transfected with the packaging plasmids pMD2.G (purchased from Miaoling Biology) and psPAX2 (purchased from Miaoling Biology) into HEK-293T cells (from ATCC) to package lentiviruses, which were then concentrated and purified. The lentiviruses were used to infect LLC1 cells (from ATCC) and EO771 cells (from ATCC) respectively. After puromycin drug screening, the expression of E6 and E7 proteins was identified by immunoblotting (Western blot) and fluorescence-activated cell sorting (FACS), and monoclonal cells were selected by limiting dilution method. After secondary identification by Western blot and FACS, LLC1-HPV18 and EO771-HPV18 cells were obtained respectively. As Figure 1 shown, both LLC1-HPV18 and EO771-HPV18 cells can express E6 and E7 proteins of HPV18.

[0047] Example 2. Construction of HPV18 mouse tumor model

[0048] Due to the high immunogenicity of HPV18 E6 and E7 proteins, in order to increase the tumorigenicity of cells, the inventor mixed 0.5×10^6 200uL LLC1-HPV18 cells [with Matrigel (low-growth factor Matrigel from Corning) in a 1:1 equal volume mixture], and 1×10^6 200uL EO771-HPV18 cells (mixed with Matrigel (the same as above) in a 1:1 equal volume mixture), and subcutaneously inoculated them at the upper right shoulder position of female C57BJ / 6J mice (from Vital River). The tumor size and body weight were measured 3 times a week, and the mice were euthanized at the end of the experiment. The tumors were collected, and the expression of HPV18 E6 and E7 proteins in the tumors was identified by Western blot and FACS. As Figure 2 shown, both cell lines could form tumors in mice, and the in vitro tumor detection results showed that the E6 and E7 proteins of HPV18 were still expressed in the tumors at the end of the experiment. The above results indicate that the HPV18 mouse tumor model was successfully established.

[0049] Example 3. Design and efficacy verification of self-replicating RNA encoding HPV18 E6 and E7 proteins

[0050] The amino acids of HPV18 E6 and E7 proteins were optimized for mouse codons. At the same time, cysteine at positions 65 and 108 of the E6 protein was mutated to glycine; cysteine at position 27 of the E7 protein was mutated to glycine, glutamate at position 29 was mutated to glycine, and cysteine at position 98 was mutated to glycine, so as to improve the protein expression efficiency and inhibit its tumorigenicity. This sequence was sent to a third-party company to synthesize HPV18 self-replicating RNA (HPV18saRNA, the nucleic acid sequence is shown in SEQ ID NO:6).

[0051] To verify the practicability of the HPV18 mouse tumor model established in Example 2, the inventor injected 100ul 80ug / mL HPV18 saRNA (12 mice) and an equal volume of phosphate-buffered saline (PBS) (12 mice) into 24 female C57BL / 6J mice (18 - 22g, 6 - 8 weeks old) by intramuscular injection on the 0th day and the 7th day respectively. On the 7th day, 6 mice were taken from each of the 2 treatment groups and subcutaneously inoculated with LLC1-HPV18 cells (0.5×10^6, 200uL; plus Matrigel with a volume ratio of 1:1) and 6 mice from each of the other groups were subcutaneously inoculated with EO771-HPV18 cells (1×10^6, 200uL; plus Matrigel with a volume ratio of 1:1). The tumor growth was observed, and the spleen and blood were collected at the end of the experiment to detect the specific immune level. Among them, the Matrigel was low-growth factor Matrigel from Corning. The technical route is as follows Figure 3AAs shown in the figure. From the results of tumor growth, it can be seen that HPV18 saRNA immunization can significantly inhibit tumor growth ( Figure 3B ). From the analysis results of immune cells in the blood and spleen, it can be seen that the proportions of memory effector CD8+ T cells in the HPV18 saRNA immunization group were 16.6%, 23.6% and 18.9%, 23.4% respectively, which were significantly higher than 4.9%, 5.6% and 4.0%, 4.7% in the control group, indicating that HPV18 saRNA induced the body to produce a high level of memory effector CD8+ T cells ( Figure 3C ). The spleen cells were analyzed by IFN-γ solid-phase enzyme-linked immunosorbent spot assay (ELIspot) (using the INF-γ ELISpot kit from Mabtech), and stimulated with HPV18 E6 and E7 proteins (both synthesized by Nanjing Genscript, and their sequences are the amino acid sequences translated from the nucleic acid sequences shown in SEQ ID NO:2 and 3 respectively) for 18 h. The experimental results showed that more immune spots could be produced by the stimulation of HPV18 E6 and E7 protein polypeptides in the HPV18 saRNA immunization group, indicating that HPV18 saRNA immunization induced the body to produce immunity specific to HPV18 E6 and E7 proteins. The above experimental results prove that the constructed LLC1-HPV18 and EO771-HPV18 tumor models can be used for the efficacy evaluation of HPV18 therapeutic tumor vaccines. The LLC1-HPV18 and EO771-HPV18 tumor models are the HPV18 mouse tumor models established in Example 2.

[0052] Comparative examples: B16F10 cells, 4T1 cells and EMT6 cells were used as the cells receiving lentiviral infection respectively

[0053] The recombinant lentiviral vector pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro obtained in Example 1 was used for lentivirus packaging, and the method was the same as that in Example 1. That is, this pCDH-CMV-E6-P2A-GFP-EF1a-E7-T2A-puro was co-transfected with the packaging plasmids pMD2.G (purchased from Miaoling Biology) and psPAX2 (purchased from Miaoling Biology) into HEK-293T cells (from ATCC) to package into lentivirus, and then concentrated and purified. Then, these lentiviruses were used to infect B16F10 cells, 4T1 cells and EMT6 cells (all these 3 kinds of cells are from ATCC), respectively. After puromycin drug screening, Western blot and Fluorescence-Activated Cell Sorting (FACS) were used to identify the expression of E6 and E7 proteins, and monoclonal cells were selected by the limiting dilution method, and were re-identified by Western blot and FACS to obtain B16F10-HPV18, 4T1-HPV18 and EMT6-HPV18 cells respectively. As Figures 4-6 shown, the effects of B16F10-HPV18, 4T1-HPV18 and EMT6-HPV18 cells were significantly inferior to those of LLC1-HPV18 cells and EO771-HPV18 cells.

[0054] In this article, specific examples were used to elaborate on the principle and implementation mode of the present invention. The description of the above examples is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation mode and application scope. It is possible to make changes and improvements to the present invention without exceeding the concept and scope defined by the claims. To sum up, the content of the embodiments in this specification should not be construed as a limitation to the present invention.

Claims

1. A recombinant lentiviral vector, characterized in that, Having HPV18 E6 and E7 expression cassettes; the structures of the HPV18 E6 and E7 expression cassettes are the first promoter - E6 - the first 2A peptide - the first selection marker - the second promoter - E7 - the second 2A peptide - the second selection marker; the first promoter and the second promoter are different and are each selected from the CMV promoter, EF1a promoter, SV40 promoter, β-actin promoter, CAG promoter, and PGK promoter; the first 2A peptide and the second 2A peptide are each independently selected from the P2A peptide and T2A peptide; one of the first selection marker and the second selection marker is a fluorescent gene, and the other is an antibiotic gene.

2. The recombinant lentiviral vector according to claim 1, wherein The fluorescent gene is selected from GFP, RFP, YFP, BFP, CFP, and mCherry; the antibiotic gene is selected from puromycin, neomycin (G418), bleomycin, and hygromycin B.

3. The recombinant lentiviral vector according to claim 1, characterized in that, The recombinant lentiviral vector is prepared by inserting the DNA fragment of the HPV18 E6 and E7 expression cassettes into the original plasmid pCDH.

4. The recombinant lentiviral vector according to claim 1 or 3, characterized in that, The structures of the HPV18 E6 and E7 expression cassettes are selected from the following table:

5. The recombinant lentiviral vector according to claim 4, characterized in that, The structure of the HPV18 E6 and E7 expression cassette is CMV - E6 - P2A - GFP - EF1a - E7 - T2A - puro, and the nucleic acid sequence is as shown in SEQ ID NO:

1.

6. The recombinant lentiviral vector according to claim 1, characterized in that, The nucleic acid sequences of E6 and E7 are as shown in SEQ ID NO:2 and 3 respectively.

7. A cell, characterized in that, The cells are LLC1 cells and / or EO771 cells infected with lentivirus packaged by the recombinant lentiviral vector according to any one of claims 1 - 6; the cells can express the E6 and E7 proteins of HPV18.

8. A method for constructing an HPV18 mouse tumor model, characterized in that, Subcutaneously inoculating the cells according to claim 7 into mice, and obtaining the HPV18 mouse tumor model after breeding for several days; the number of inoculated cells is 0.5×10^6 - 1×10^6 and is inoculated into mice after being mixed with Matrigel.

9. The method for constructing an HPV18 mouse tumor model according to claim 8, characterized in that, The inoculated cells are mixed with Matrigel at a volume ratio of 1:1, and each volume is 200 ± 10 uL; the Matrigel is low - growth - factor Matrigel.

10. Use of the cell according to claim 7 in the efficacy evaluation of an HPV18 therapeutic tumor vaccine, characterized in that, By immunizing mice with the to - be - evaluated HPV18 therapeutic tumor vaccine and then subcutaneously inoculating the cells into the mice, detecting the tumor volume of the mice after a period of time, the smaller the tumor volume, the better the efficacy of the to - be - evaluated HPV18 therapeutic tumor vaccine.

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