Endogenous retrovirus envelope protein p_env of sebastodes hemispinosus, lentivirus vector and application thereof

Abstract

The present application relates to a kind of Sebastes schlegelii endogenous retrovirus envelope protein P_env, lentivirus vector and its application, belong to the genetic breeding field of molecular biology, the amino acid sequence of the envelope protein P_env is as shown in SEQ ID NO.1.The present application simultaneously provides the membrane fusion protein plasmid of containing the envelope protein P_env, the in vitro assembly system of lentivirus particle, transformant and kit, and the application of the envelope protein P_env, the P_env protein of the present application is replaced VSVG protein using hard fish source, will effectively improve the transduction efficiency of lentivirus particle to hard fish cell, realize efficient gene delivery.Meanwhile, as endogenous Env protein, overcome the immune rejection of host to exogenous Env protein, also have the potential of in-vivo application.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding in molecular biology, specifically relating to the endogenous retroviral envelope protein P_env of the rockfish, lentiviral vectors, and their applications. Background Technology

[0002] Gene delivery is the process of artificially introducing specific genes into cells or organisms using certain vectors or specific technologies. It has wide applications in gene therapy, gene function verification, and gene editing. Generally, gene delivery systems can be divided into two main categories: viral vector gene delivery systems and non-viral vector gene delivery systems. Compared to non-viral vector gene delivery systems, viral vector gene delivery systems have several advantages, including but not limited to: the ability to widely infect animal recipient cells, the ability to precisely integrate the carried genetic material into the recipient cell genome, the ability to efficiently express the target gene, and the ability to control vector spread and avoid expressing viral proteins after infection. Therefore, viral vector gene delivery systems have become the preferred method for gene delivery.

[0003] Currently, viral vector gene delivery systems mainly include three types: lentivirus, adenovirus, and adeno-associated virus. Among them, lentivirus is widely used due to its advantages such as rapid expression rate, high expression level, and ability to integrate into the cellular genome. Lentiviral vectors are mainly derived from HIV, and their basic structure includes functional proteins such as lentiviral gag, pol, and rev, lentiviral element LTR, other auxiliary elements and the target gene, as well as the outer membrane envelope protein env, which has fusion function. Currently, lentiviruses typically use the vesicular stomatitis virus envelope protein VSVG. Although the VSVG protein can efficiently recognize lentiviral elements and assemble into lentiviral particles, achieving efficient transduction for almost all mammalian cell types, it is almost impossible to transduce lentiviral particles into primary cells or cell lines derived from bony fish. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing a lentiviral vector containing the endogenous retroviral protein P_env from the rockfish *Scorpionichthys schlegelii*, and its applications. By modifying the Env protein in lentiviral particles and reassembling the particles, the target gene (EGFP) can be efficiently delivered to bony fish cells, achieving overexpression of the target gene. This provides a novel membrane protein selection and reference for the development of virus transduction systems in fish cells.

[0005] The present invention achieves the above objectives through the following methods:

[0006] A transgenic retroviral envelope protein P_env from the rock scorpionfish, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments, an SP signal peptide sequence is added to the 5' end of the ORF encoding the envelope protein P_env gene, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The second technical solution of the present invention is to provide a gene encoding the envelope protein P_env, the nucleotide sequence of which is SEQ ID NO.2.

[0009] As one preferred embodiment, a gene encoding an envelope protein P_env linked to an SP signal peptide is provided, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] The third technical solution of the present invention is to provide a membrane fusion protein particle, which contains the gene as described in the second technical solution.

[0011] The fourth technical solution of the present invention provides an in vitro assembly system for lentiviral particles, the system comprising an envelope plasmid connected to the P_env gene.

[0012] The fifth technical solution of the present invention provides a transformant, wherein the transformant is transfected with an in vitro assembly system of membrane fusion protein particles as described in the third technical solution or lentiviral particles as described in the fourth technical solution; wherein the recipient cell of the transformant is a eukaryotic cell, preferably an animal cell, and more preferably a 293T cell.

[0013] The sixth technical solution of the present invention provides a recombinant lentiviral vector, wherein it contains the envelope protein P_env as described in the first technical solution; or, it is obtained by culturing the transformant as described in the fifth technical solution.

[0014] The seventh technical solution of the present invention provides a method for preparing a recombinant lentiviral vector, wherein the transformant as described in the fifth technical solution is cultured to obtain the recombinant lentiviral vector.

[0015] The eighth technical solution of the present invention provides a kit, wherein the kit comprises membrane fusion protein particles as described in the third technical solution, an in vitro assembly system of lentiviral particles as described in the fourth technical solution, a transformant as described in the fifth technical solution, and / or a recombinant lentiviral vector as described in the sixth technical solution.

[0016] The ninth technical solution of the present invention is to provide the application of the envelope protein P_env as described in one of the technical solutions in the preparation of gene therapy drugs.

[0017] The beneficial effects of this invention compared to the prior art are as follows:

[0018] 1) A novel membrane protein of lentiviral particles that can be used for transduction in fish cells is provided. Compared with the VSVG envelope protein used in traditional viral particles, this novel protein can efficiently transduce a variety of fish cells and deliver the target gene to the fish cells.

[0019] 2) Utilizing retroviruses that have already been endogenized env Recombining genes with lentiviral particles can effectively reduce exogenous [genes / genes]. env The immune rejection response caused by genes has the potential for in vivo application; at the same time, utilizing env Gene delivery to the membrane requires the ability to recognize receptors, and recombinant viral particles also have the potential to target and deliver genes. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of a lentivirus particle three-plasmid packaging system;

[0021] Figure 2 The graph shows the transduction efficiency of lentiviral particles with P_env as the envelope protein in various bony fish cells; ae represents primary ovarian cells of Scorpae schlegelii, fj represents testicular cells of Tongue sole, ko represents brain cells of turbot, and pt represents gill cells of turbot. Detailed Implementation

[0022] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0023] Based on the inventor's previous research, it was found that endogenous viruses exist in the genome of Scorpionfish schlegelii. P_env Gene delivery. Replacing the VSVG protein with the P_env protein derived from bony fish will effectively improve the transduction efficiency of lentiviral particles in bony fish cells, achieving highly efficient gene delivery. Simultaneously, as an endogenous Env protein, it overcomes the host's immune rejection of exogenous Env proteins and also has the potential for in vivo application.

[0024] The plasmids pLVX-EGFP-IRES-Puro (addgene#128652), psPAX2 (addgene#12260), and pCMV-VSV-G (addgene#8454) used in the examples were all purchased from Wuhan Miaoling Biotechnology Co., Ltd. Lipofectamine 3000 Reagent was purchased from Thermo Fisher Scientific China.

[0025] The components of the solid plate medium for ampicillin resistance are: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, and ampicillin 100 mg / L.

[0026] Cell cryopreservation medium (DMEM) was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0027] Fetal bovine serum (FBS) was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0028] The P24 ELISA kit was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0029] PBS buffer was purchased from Shandong Cisco Biotechnology Co., Ltd.

[0030] The complete L-15 medium contains: 20% FBS, 1% antibiotics, 1% non-essential amino acids, 1% sodium pyruvate, and 1% glutamine. All reagents mentioned above were purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.

[0031] Example 1

[0032] This embodiment utilizes primary ovarian cells from *Scorpionichthys schlegelii*, testicular cells from *Cyprinus semismoothienos*, brain cells from *Flounder*, and gill cells from *Flounder*. P_env is used instead of VSVG recombinant lentiviral particles for transduction. EGFP As a reporter gene, it is used to detect transduction efficiency.

[0033] 1. P_env Construction of gene overexpression plasmids: obtained by PCR amplification P_env The gene's ORF (open reading frame) sequence, and through homologous recombination... P_env The gene's ORF was recombined into the pCMV3 plasmid, transformed into *E. coli* strain Dh5α, and plated on ampicillin-resistant solid agar plates. After 12 hours of growth, single colonies were picked for Sanger sequencing verification. The verification results confirmed that the sequencing sequence was consistent with... P_env The genes are completely identical, and the specific sequence is shown in SEQ ID NO.1 in Table 1.

[0034] 2. The preparation of lentiviral particles with P_env as the envelope protein is as follows: Figure 1 As shown, the specific steps are as follows:

[0035] ① The synthesized P_env gene sequence containing the SP sequence was amplified and gel-cleaved using a high-fidelity enzyme (ABM MegaFi Fidelity 2X PCR MasterMix, brand: ABM, catalog number: G897). Primer pair 1 (ligation primer-fw: 5'-cctcgagga attctgacactATGGGCTGGTCCTGTATCATCCTG-3' SEQ ID NO.5, ligation primer rv: 5'-ctgttgtgcaggatttgagTCAGGGCGTTTTTTTCTCTATAA-3', SEQ ID NO.6; where the lowercase part is the homologous arm of the seamless clone; the uppercase part: the forward primer is the SP sequence ligated to the 5' end of the P_env gene, and the reverse primer is the P_env gene sequence.) was then used to amplify and gel-cleaved the P_env gene sequence. Primer pair 2 (recovery primer-fw: 5'-agtgtcagaattcctcgagg-3' SEQ ID NO.5) was used to amplify and gel-cleaved the P_env gene sequence containing the SP sequence. NO.7, the recovered primer-rv: 5'-ctcaaatcctgcacaacag-3', SEQ ID NO.8) was used to amplify the pCMV-VSV-G plasmid and then recovered by gel cutting;

[0036] ② Using the Seamless Cloning Kit (2×Ezmax® Ultra Universal Clone Mix, TOLOBIO, catalog number: 24317), the pCMV-VSV-G recovered product and the P_env gene recovered product were mixed at a molar ratio of 1:2 and ligated; the mixture was then transformed into Trans T1 competent cells, and the ligation vector was validated by plating, picking single clones, and Sanger sequencing; plasmid extraction was performed using the Plasmid Extraction Kit (One-tube Universal Endotoxin-Free Plasmid Extraction Kit, TransGen, catalog number: EM153-01);

[0037] ③ Prepare 293T cells with passage number within 20 in advance, and culture them in a 37℃, 5% CO2 incubator. When the confluence reaches 70%~80%, transfection can be performed. Replace with fresh serum-free cell cryopreservation medium (DMEM) before transfection.

[0038] ④ Prepare the transfection mixture. The transfection system used is shown in Table 1 (taking T75 cell culture flasks as an example).

[0039] Table 1. Transfection System

[0040] ;

[0041] After preparing Mixture 1 and Mixture 2, let them stand at room temperature for 5 minutes. Then, gently mix Mixture 1 and Mixture 2 and incubate at room temperature for 15 minutes. Gently add the mixture to 293T cells and incubate at 37°C in a 5% CO2 incubator. 6 hours after transfection, replace the culture medium with fresh DMEM containing 10% FBS.

[0042] ⑤ 48 hours after transfection, collect the supernatant virus suspension and store it temporarily at 4°C. Replace the 293T cells with fresh DMEM containing 10% fetal bovine serum (FBS). 72 hours after transfection, collect the virus suspension again and mix it with the virus suspension collected at 48 hours. Centrifuge at 4°C, 400g for 10 minutes to remove cell debris and retain the supernatant.

[0043] ⑥ Concentrate the virus using the TAKARA Virus Concentration Kit; determine the virus titer using the Sinopharm P24 ELISA Kit; aliquot the remaining virus into tubes and store at -80℃ for long-term storage.

[0044] 3. Culture of primary cells from the ovaries of *Scorpionichthys schlegelii*: Ovarian tissue from female *Scorpionichthys schlegelii* was washed repeatedly at least 6 times in PBS containing 5% penicillin and antibiotics. The tissue was then transferred to L-15 medium containing 1% penicillin and antibiotics, minced, and digested with collagenase and trypsin for 1 hour. The digestion reaction was terminated with FBS, and the tissue fragments were collected by centrifugation. The fragments were then evenly spread into T25 cell culture flasks and incubated upside down overnight. Complete L-15 medium containing 20% ​​FBS, 1% penicillin and antibiotics, 1% non-essential amino acids, 1% sodium pyruvate, and 1% glutamine was added, and the flasks were incubated at 24°C. Once the primary cells reached confluence, they were passaged for experiments.

[0045] 4. Preparation of recipient cells: Primary cells are passaged into 96-well plates in advance. Taking primary Scorpionichthys ovarian cells as an example, the seeding time is approximately 10 wells. 4 Cells were cultured overnight at 24°C in an incubator, one cell per well, until the cells adhered to the culture vessel.

[0046] 5. Transduction of lentiviral particles with P_env as the envelope protein: Aspirate the culture medium from the wells of the culture plate and add 200 μL of fresh complete L-15 medium; add the corresponding amount of lentiviral suspension according to different multiplicity of infection and mix gently; replace with fresh complete L-15 medium after 24 hours; lentiviral particles with VSVG as the envelope protein are used as the positive control in this experiment, and the experimental group without lentiviral particle transduction is used as the negative control in this experiment. The lentiviral particles prepared in step 2 above are used as the experimental group.

[0047] 6. Detection of transduction efficiency: 48 hours after transduction, cell nuclei were stained with Hoechst, and green fluorescence signal (FITC) and blue fluorescence signal (DAPI) were detected under a fluorescence microscope. The results are as follows: Figure 2As shown in Figure ae, no obvious green fluorescence signal was detected in the negative control group and the lentiviral particle transduction group with VSVG as the envelope protein. At a multiplicity of infection (MOI) of 10, no obvious green fluorescence signal was detected in the lentiviral particle transduction group with P_env as the envelope protein. At an MOI of 30, sporadic green fluorescence signals were detected. At an MOI of 100, strong and continuous green fluorescence signals were detected. These results indicate that lentiviral particles with P_env as the envelope protein can efficiently transduce primary ovarian cells of *Scorpene schlegelii* at high MOIs.

[0048] 7. Transduction of lentiviral particles with P_env as the envelope protein in other bony fish cells: In this example, lentiviral particle transduction was performed using half-smooth tongue sole testis cell lines, turbot brain cell lines, and turbot gill cell lines. Recipient cell preparation was consistent with that of primary Scorpionfish cells. The transduction results are as follows: Figure 2 As shown in ft. No obvious green fluorescence signal was detected in the negative control group and the lentiviral particle transduction group with VSVG as the envelope protein; sporadic green fluorescence signals were detected at multiplicity of infection (MOI) of 10 and 30; and strong and continuous green fluorescence signal was detected at MOI of 100. These results indicate that lentiviral particles with P_env as the envelope protein can efficiently transduce the testis cell line, brain cell line, and gill cell line of *Scopterocarpus natans*.

[0049] Example 2

[0050] This embodiment provides a method for delivering target nucleic acid to target cells, comprising:

[0051] (i) Provide the lentiviral vector as described in Example 1;

[0052] (ii) The lentiviral vector is brought into contact with target cells to deliver the target nucleic acid to the target cells. In some embodiments, the nucleic acid is delivered to the cells when the lentivirus enters or infects the cells during step (ii). In some embodiments, the method requires a transfection agent (e.g., a lipophilic transfection agent, such as Lipofectamine 3000). In some embodiments, the method is performed in vitro.

[0053] In some embodiments, the method is carried out in vivo and includes administering the lentiviral vector of Example 1 to test fish, thereby delivering the target nucleic acid to target cells in the fish.

[0054] In some embodiments, the method is used to genetically modify the target cells.

[0055] In some embodiments, the method is used to perform gene editing on the target cells.

[0056] In some embodiments, the target nucleic acid comprises a foreign gene. The foreign gene encodes, for example, a therapeutic protein (e.g., a protein that compensates for a disease condition in the test fish) or an antigen (such as a pathogen antigen), a gene editing tool (e.g., Cas proteins and / or gRNAs of the CRISPR / Cas system), or a gene silencing tool (e.g., shRNA).

[0057] In some embodiments, the target nucleic acid encodes an mRNA molecule, optionally wherein the mRNA is the aforementioned exogenous gene.

[0058] In some implementations, the target nucleic acid encodes double-stranded RNA, antisense RNA, microRNA, or any other RNA molecule.

[0059] In some embodiments, the target cell surface contains antigens or receptors that can be targeted by non-viral membrane-binding proteins contained in the lentiviral vector described herein.

[0060] In some implementations, the target cell can be any bony fish cell.

[0061] In addition, in some embodiments, a pharmaceutical composition can be prepared using the envelope protein P_env, comprising the lentiviral vector described in Example 1, or target cells obtained by the method of delivering target nucleic acids to target cells using the envelope protein P_env.

[0062] (iii) In some embodiments, the target nucleic acid being packaged can be optimized into a protein form by replacing the pLVX-EGFP-IRES-Puro plasmid. For example, the HIV-1 Gag-mcherry plasmid containing fusion-expressed Gag and mcherry sequences can be used to express a fusion protein of the lentiviral structural proteins Gag and mcherry, thereby enabling the assembly of the mcherry protein into the viral particle.

[0063] In some embodiments, the target protein comprises a foreign protein. The foreign protein may include a tag protein (e.g., mcherry, EGFP), a gene editing tool (the Cas protein of the CRISPR / Cas system or the RNP complex formed by the Cas9 protein and sgRNA), or other foreign proteins (e.g., fish Oct4 protein).

[0064] Table 1 shows the sequences involved in this invention.

[0065] ;

[0066] .

Claims

1. A Sebastes schlegelii endogenous retroviral envelope protein P_env, characterized in that, The amino acid sequence of the envelope protein P_env is shown as SEQ ID NO.

1.

2. The Quillback (Carpiodes cyprioides) endogenous retrovirus envelope protein P_env of claim 1, wherein, The nucleotide sequence of the 5' end of the ORF of the gene encoding the envelope protein P_env to which the SP signal peptide is added, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.

4.

3. A gene encoding the envelope protein P_env of claim 2, characterized in that, The gene of the envelope protein P_env is connected with the SP signal peptide, and the nucleotide sequence of the gene of the envelope protein P_env connected with the SP signal peptide is shown as SEQ ID NO.

3.

4. A plasmid for a membrane fusion protein, characterized by comprising the nucleotide sequence of SEQ ID NO:

1. The plasmid comprises the gene according to claim 3.

5. An in vitro assembly system of lentiviral particles, characterized in that, The system comprises the envelope plasmid connected with the gene according to claim 3.

6. A transformant characterized in that, The transformant is transfected with the membrane fusion protein plasmid according to claim 4 or the in vitro assembly system of the lentivirus particle according to claim 5; wherein the receptor cell of the transformant is a eukaryotic cell.

7. A transformant according to claim 6, characterized in that, The receptor cell is a 293T cell.

8. A recombinant lentiviral vector, characterized in that, It comprises the envelope protein P_env according to claim 1; or, it is obtained by culturing the transformant according to claim 6.

9. A kit characterized in that, The kit comprises the membrane fusion protein plasmid according to claim 4, the in vitro assembly system of the lentivirus particle according to claim 5, the transformant according to claim 7 or the recombinant lentivirus vector according to claim 8.

10. The envelope protein P_env according to claim 1 is used in the preparation of a fish transgenic carrier, a gene editing carrier or a gene therapy drug.

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