Sebastes schlegeli endogenous retrovirus envelope protein percomORF, lentiviral vector and application of sebastes schlegeli endogenous retrovirus envelope protein percomORF

Abstract

The invention particularly relates to a sebastes schlegeli endogenous retrovirus envelope protein percomORF, a lentiviral vector and application of the sebastes schlegeli endogenous retrovirus envelope protein percomORF and the lentiviral vector, and belongs to the field of genetic breeding of molecular biology, and the envelope protein percomORF has an amino acid sequence as shown in SEQ ID NO.1. The invention also covers a membrane fusion protein particle constructed based on the protein, a system for in-vitro assembly of lentivirus particles, a corresponding transformant and a matched kit. By replacing conventional VSVG protein with percomORF protein from sclerobone fish, the infection and transduction efficiency of lentivirus on sclerobone fish cells can be effectively enhanced, and more efficient gene delivery is realized. Besides, the protein belongs to host endogenous Env protein and is beneficial to avoiding immunological rejection caused by introduction of foreign protein, so that the protein has good potential in in-vivo gene delivery application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic breeding of molecular biology, and particularly relates to an endogenous retrovirus envelope protein percomORF of Sebastiscus inermis, a lentivirus vector and application thereof. BACKGROUND

[0002] Gene delivery refers to the process of artificially introducing an exogenous gene into a cell or organism by means of a specific carrier or technology, which has important applications in the fields of gene therapy, functional gene verification and genome editing. According to the nature of the used carrier, gene delivery systems are generally divided into two categories: viral vector systems and non-viral vector systems. Compared with the latter, viral vector systems have a series of significant advantages, including the ability to widely infect different types of animal cells, the ability to accurately integrate the genetic information carried by them into the host genome, the ability to achieve efficient and stable expression of the target gene, and the ability to regulate the spread of the vector and inhibit the expression of viral proteins after infection. Therefore, viral vector systems have become one of the mainstream technical means for implementing gene delivery.

[0003] Among the existing viral vectors, lentivirus vectors, adenovirus vectors and adeno-associated virus vectors are widely used. Among them, lentivirus vectors have been widely used in scientific research and clinical applications due to their rapid expression, high expression level and stable integration of target genes into the host genome. Common lentivirus vectors are mainly based on human immunodeficiency virus (HIV) and their basic components include structural and functional proteins of lentivirus (such as gag, pol, rev, etc.), long terminal repeat sequences (LTR), other necessary auxiliary elements, target genes, and exogenous envelope proteins responsible for membrane fusion (env). Currently, the envelope protein is mostly the vesicular stomatitis virus glycoprotein (VSVG). Although VSVG can effectively mediate the assembly of lentivirus particles and achieve efficient transduction of most mammalian cells, it has little transduction ability for primary cells or cell lines derived from bony fish. SUMMARY

[0004] The present application provides a Sebastiscus inermis endogenous retrovirus envelope protein percomORF, a lentivirus vector containing the same and applications thereof. By modifying the Env protein of the lentivirus particle to reassemble the lentivirus particle, the target gene (EGFP) is efficiently delivered to the bony fish cells, and the overexpression of the target gene is achieved, thereby providing a new membrane protein selection and reference for the development of fish cell viral transduction systems.

[0005] The present application achieves the above-mentioned purposes through the following solutions: A Sebastiscus inermis endogenous retrovirus envelope protein percomORF, the amino acid sequence of the envelope protein percomORF is shown as SEQ ID NO. 1.

[0006] One of the technical solutions of the present application is to provide a gene encoding the envelope protein percomORF, and the nucleotide sequence of the gene is SEQ ID NO. 2. The second technical solution of the present application is to provide an envelope fusion protein plasmid comprising the gene according to the first technical solution.

[0007] The third technical solution of the present application is to provide an in vitro assembly system of a lentivirus particle, wherein the system comprises an envelope plasmid connected with the percomORF gene.

[0008] The fourth technical solution of the present application is to provide a transformant, wherein the transformant is transfected with the envelope fusion protein plasmid according to the second technical solution or the in vitro assembly system of the lentivirus particle according to the third technical solution; wherein the receptor cell of the transformant is a eukaryotic cell, preferably an animal cell, and more preferably a 293T cell.

[0009] The fifth technical solution of the present application is to provide a recombinant lentivirus vector, wherein the recombinant lentivirus vector comprises the envelope protein percomORF according to the first technical solution; or the recombinant lentivirus vector is obtained by culturing the transformant according to the fourth technical solution.

[0010] The sixth technical solution of the present application is to provide a method for preparing a recombinant lentivirus vector, wherein the recombinant lentivirus vector is obtained by culturing the transformant according to the fourth technical solution.

[0011] The seventh technical solution of the present application is to provide a kit, wherein the kit comprises the envelope fusion protein plasmid according to the second technical solution, the in vitro assembly system of the lentivirus particle according to the third technical solution, the transformant according to the fourth technical solution, and / or the recombinant lentivirus vector according to the fifth technical solution.

[0012] The eighth technical solution of the present application is to provide a use of the envelope protein percomORF according to the first technical solution in the preparation of a gene therapy drug.

[0013] Compared with the prior art, the present application has the following beneficial effects: 1) A novel slow virus envelope protein percomORF suitable for fish cell transduction is provided, which can significantly improve the transduction efficiency of various fish cells compared with the currently commonly used VSVG envelope protein, thereby realizing efficient delivery of the target gene in fish cells.

[0014] 2) The use of endogenous retroviral env gene for lentivirus recombination can effectively reduce the immune rejection response caused by exogenous env protein, providing a basis for in vivo gene delivery. At the same time, based on the mechanism of env protein entering the cell membrane by recognizing specific receptors, the constructed recombinant virus particles also have the potential to achieve targeted gene delivery. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Schematic diagram of lentivirus particle three-plasmid packaging system; Figure 2 Figure is a graph showing the transduction efficiency of lentivirus particles with percomORF as envelope protein on various teleost fish cells. a-e are Pseudosciaena crocea brain cell lines, f-j are Pseudosciaena crocea brain cell lines, and k-o are Cynoglossus semilaevis spermary cell lines. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be further explained by the following examples, but the protection scope of the present application is not limited in any form by the examples.

[0017] Based on the previous research findings of the inventors, there is an endogenous virus percomORF Using percomORF protein derived from teleost fish to replace VSVG protein can effectively improve the transduction efficiency of lentivirus particles on teleost fish cells and achieve efficient gene delivery. At the same time, as an endogenous Env protein, it overcomes the immune rejection of the host to the exogenous Env protein and also has the potential for in vivo application.

[0018] The plasmid pLVX-EGFP-IRES-Puro (addgene #128652), the plasmid psPAX2 (addgene #12260), and the plasmid pCMV-VSV-G (addgene #8454) used in the examples were purchased from Wuhan Moli Biological Technology Co., Ltd. Lipofectamine 3000 Reagent was purchased from Thermo Fisher Scientific China; The composition of ampicillin-resistant solid plate medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, ampicillin 100 mg / L; Cell freezing medium (DMEM) was purchased from Shanghai Xiaopeng Biological Technology Co., Ltd.; Fetal bovine serum (FBS) was purchased from Shanghai Xiaopeng Biological Technology Co., Ltd.; P24 ELISA kit was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; PBS buffer was purchased from Shandong Sikaijie Biological Technology Co., Ltd. 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.

[0019] Example 1 This embodiment utilizes turbot brain cell lines, turbot gill cell lines, and half-smooth tongue sole testis cell lines, employing percomORF instead of VSVG recombinant lentiviral particles for transduction. EGFP As a reporter gene, it is used to detect transduction efficiency.

[0020] 1. percomORF Construction of gene overexpression plasmids: obtained by PCR amplification percomORF The gene's ORF (open reading frame) sequence, and through homologous recombination... percomORF 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... percomORF The genes are completely identical; the specific sequence is shown in SEQ ID NO.1 in Table 1: 2. The preparation of lentiviral particles with percomORF as the envelope protein is as follows: Figure 1 As shown, the specific steps are as follows: ① The synthesized percomORF gene sequence was amplified and gel-cleaved using a high-fidelity enzyme (ABM MegaFi Fidelity 2X PCR MasterMix, brand: ABM, catalog number: G897) and primer pair 1 (ligation primer-fw: 5'-cctcgagga attctgacactATGGCCCTGATCCTGCACTTCAT-3' SEQ ID NO.3, ligation primer rv: 5'-ctgttgtgcaggatttgagTTAATTGAAATTGAAGATGAAC-3' SEQ ID NO.4; where the lowercase part is the homologous arm of the seamless clone; the uppercase part: the forward primer is ligated to the 5' end sequence of the percomORF gene, and the reverse primer is the 3' end sequence of the percomORF gene). Primer pair 2 (recovery primer-fw: 5'-agtgtcagaattcctcgagg-3' SEQ ID NO.3) was used. NO.5, the recovered primer-rv: 5'-ctcaaatcctgcacaacag-3', SEQ ID NO.6) was used to amplify the pCMV-VSV-G plasmid and then gel-cleaved for recovery; ② Use the seamless cloning kit (2x Ezmax® Ultra Universal Clone Mix, brand: TOLOBIO, part number: 24317) to mix the pCMV-VSV-G recovery product with the percomORF gene recovery product at a ratio of 1:2 by moles, and then perform ligation; transform into Trans T1 competent cells, and verify the ligation vector by plating, picking single colonies, and Sanger sequencing; use the plasmid maxi kit (one-tube universal endotoxin-free plasmid maxi kit, brand: Quanshijin, part number: EM153-01) to extract the plasmid; ③ Prepare 293T cells with a passage number of 20 or less in advance, and culture them in a 37°C, 5% CO2 incubator; when the confluence reaches 70-80%, replace the cell culture medium (DMEM) with fresh serum-free medium before transfection; ④ Prepare the transfection mixture, and the transfection system used is shown in Table 1 (using a T75 cell culture flask as an example); Table 1. Transfection system ; After preparing mixture one and mixture two, let them stand at room temperature for 5 minutes, then gently mix mixture one and mixture two, incubate at room temperature for 15 minutes, and then gently add them to 293T cells, and culture them in a 37°C, 5% CO2 incubator; 6 hours after transfection, replace the culture medium with fresh DMEM containing 10% FBS; ⑤ 48 hours after transfection, collect the supernatant virus suspension and store it in a 4°C refrigerator, replace the 293T cell culture medium 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 48 hours ago; remove the cell debris by centrifuging at 4°C, 400g for 10 minutes, and retain the supernatant; ⑥ Use the TAKARA virus concentration kit to concentrate the virus; use the Yiqiaoshenzhou P24 ELISA kit to determine the virus titer; and store the remaining virus in small tubes at -80°C for long-term storage.

[0021] 3. Preparation of recipient cells: in advance, inoculate the cell line into a 96-well plate, for example, inoculate about 10 4 cells / well of the sciaenid brain cell line, and culture them in a 24°C incubator overnight to allow the cells to adhere; 5. Transduction of percomORF lentivirus particles: remove the culture medium in the culture plate hole, add 200 μL of fresh complete L-15 culture medium; add the corresponding amount of lentivirus suspension according to the different multiplicity of infection, and mix gently; replace the fresh complete L-15 culture medium after 24 hours; the lentivirus particles with VSVG as the envelope protein are used as the positive control of the experiment, the experimental group without lentivirus particles is the negative control of the experiment, and the lentivirus particles prepared in step 2 are used as the experimental group; 6. Detection of transduction efficiency: after 48 hours of transduction, use hoechst for cell nucleus staining, detect the green fluorescent signal (FITC) and the blue fluorescent signal (DAPI) under a fluorescence microscope, and the results are shown in a-e of Figure 2 . The negative control group and the lentivirus particle transduction group with VSVG as the envelope protein do not detect obvious green fluorescent signals; when the multiplicity of infection is 10, the lentivirus particle transduction group with percomORF as the envelope protein does not detect obvious green fluorescent signals; when the multiplicity of infection reaches 30, sporadic green fluorescent signals are detected; when the multiplicity of infection reaches 100, strong and continuous green fluorescent signals are detected. The above results indicate that the lentivirus particles with percomORF as the envelope protein can efficiently transduce the Paralichthys olivaceus brain cell line at a high multiplicity of infection.

[0022] 7. Transduction of percomORF lentivirus particles in other teleost fish cells: in this example, the Paralichthys olivaceus gill cell line and the Cynoglossus semilaevis spermary cell line are used as the implementation objects, and the lentivirus particles are transduced. The preparation of the receptor cells is consistent with the Paralichthys olivaceus brain cell line, and the transduction results are shown in f-o of Figure 2 . The negative control group and the lentivirus particle transduction group with VSVG as the envelope protein do not detect obvious green fluorescent signals; when the multiplicity of infection is 10 and 30, sporadic green fluorescent signals can be detected; when the multiplicity of infection reaches 100, strong and continuous green fluorescent signals can be detected. The above results indicate that the lentivirus particles with percomORF as the envelope protein can efficiently transduce the Paralichthys olivaceus gill cell line and the Cynoglossus semilaevis spermary cell line.

[0023] Example 2 The present embodiment provides a method for delivering a target nucleic acid to a target cell, which comprises: (i) providing the lentivirus vector described in Example 1; (ii) contacting the lentiviral vector with a target cell, thereby delivering the nucleic acid of interest to the target cell. In certain embodiments, the nucleic acid is delivered to the cell when the lentivirus enters or infects the cell during step (ii). In certain embodiments, the method requires a transfection agent (e.g., a lipophilic transfection agent, such as Lipofectamine 3000). In certain embodiments, the method is performed in vitro.

[0024] In certain embodiments, the method is performed in vivo, the method comprising administering to a subject fish the lentiviral vector of Example 1, thereby delivering the nucleic acid of interest to a target cell in the fish in vivo.

[0025] In certain embodiments, the method is used to transfect the target cell.

[0026] In certain embodiments, the method is used to genetically edit the target cell.

[0027] In certain embodiments, the nucleic acid of interest comprises an exogenous gene. The exogenous gene encodes, for example, a therapeutic protein (e.g., a protein that compensates for a disease condition in the subject fish) or an antigen (such as a pathogen antigen), a gene editing tool (e.g., a Cas protein and / or gRNA of a CRISPR / Cas system), or a gene silencing tool (e.g., an shRNA).

[0028] In certain embodiments, the nucleic acid of interest encodes an mRNA molecule, optionally wherein the mRNA is an exogenous gene as described above.

[0029] In certain embodiments, the nucleic acid of interest encodes a double-stranded RNA, an antisense RNA, a microRNA, or any other RNA molecule.

[0030] In certain embodiments, the target cell surface comprises an antigen or receptor that can be targeted by a non-viral membrane-bound protein comprised by the lentiviral vector described herein.

[0031] In certain embodiments, the target cell can be any teleost cell.

[0032] Additionally, in certain embodiments, a pharmaceutical composition can be prepared using the envelope protein percomORF, comprising the lentiviral vector described in Example 1, or a target cell obtained from a method of delivering a nucleic acid of interest to a target cell using the envelope protein percomORF.

[0033] (iii) In some embodiments, by replacing the pLVX-EGFP-IRES-Puro plasmid, the packaged target nucleic acid can be optimized into a protein form. For example, using an HIV-1 Gag-mcherry plasmid, which contains a fusion expressed Gag sequence with a mcherry sequence, for expressing a fusion protein of the lentivirus structural protein Gag and mcherry, so as to realize the assembly of the mcherry protein into the virus particle.

[0034] In some embodiments, the target protein comprises an exogenous protein. The exogenous protein comprises a tag protein (such as mcherry, EGFP), a gene editing tool (a Cas protein of a CRISPR / Cas system or an RNP complex formed by a Cas9 protein and an sgRNA), or other exogenous proteins (such as a fish Oct4 protein).

[0035] Table 2 is a sequence involved in the present application .

Claims

1. A percomORF, an endogenous retroviral envelope protein from the rock scorpionfish, characterized in that, The amino acid sequence of the envelope protein percomORF is shown in SEQ ID NO.

1.

2. A membrane fusion protein particle, characterized in that, The plasmid contains the gene for the envelope protein percomORF as described in claim 1.

3. An in vitro assembly system for lentiviral particles, characterized in that, The system includes an envelope plasmid connected to the gene of the envelope protein percomORF as described in claim 2.

4. A transformant, characterized in that, The transformant is transfected with an in vitro assembly system of membrane fusion protein particles as described in claim 2 or lentiviral particles as described in claim 3; wherein the recipient cell of the transformant is a eukaryotic cell.

5. A transformant according to claim 4, characterized in that, The recipient cells were 293T cells.

6. A recombinant lentiviral vector, characterized in that, The vector comprises the envelope protein percomORF as described in claim 1; or, it is obtained by culturing the transformant as described in claim 4.

7. A reagent kit, characterized in that, The kit comprises the membrane fusion protein particle as described in claim 2, the in vitro assembly system of the lentiviral particle as described in claim 3, the transformant as described in claim 4, or the recombinant lentiviral vector as described in claim 6.

8. The application of the envelope protein percomORF as described in claim 1 in the preparation of fish transgenic vectors, gene editing vectors, and gene therapy drugs.

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