Endogenous retroviral envelope protein percomorf of sebastodes fuscescens, lentivirus vector and application thereof

Abstract

The application relates to a percom ORF (amino acid sequence as shown in SEQ ID NO. 1) of an endogenous retrovirus envelope protein of Sebastes schlegelii, a lentivirus vector and application thereof, and belongs to the genetic breeding field of molecular biology. The application also covers a membrane fusion protein plasmid constructed based on the protein, a system for in-vitro assembly of lentivirus particles, corresponding transformants and a complete kit. By replacing the conventional VSVG protein with the percom ORF protein derived from bony fish, the application can effectively enhance the infection and transduction efficiency of lentivirus on bony fish cells, and realize more efficient gene delivery. In addition, the protein belongs to the host endogenous Env protein, which can help to avoid the immune rejection reaction caused by the introduction of exogenous proteins, and thus has good potential in in-vivo gene delivery applications.

Description

Technical Field

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

[0002] Gene delivery refers to the process of artificially introducing exogenous genes into cells or organisms using specific vectors or technologies. This technology has important applications in many fields, including gene therapy, functional gene validation, and genome editing. Based on the nature of the vector used, gene delivery systems are generally divided into two main categories: viral vector systems and non-viral vector systems. Compared to the latter, viral vector systems have a series of significant advantages, including the ability to infect a wide variety of animal cells, the ability to precisely integrate the genetic information they carry into the host genome, the ability to achieve efficient and stable expression of the target gene, and the ability to regulate vector diffusion and inhibit viral protein expression after infection. Therefore, viral vector systems have become one of the mainstream technologies for gene delivery.

[0003] Among existing viral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus vectors are widely used. Lentiviral vectors, in particular, are widely used in research and clinical practice due to their advantages such as rapid expression, high expression levels, and stable integration of the target gene into the host genome. Common lentiviral vectors are mainly constructed based on human immunodeficiency virus (HIV). Their basic components include lentiviral structural and functional proteins (such as gag, pol, rev, etc.), long terminal repeats (LTRs), other necessary auxiliary elements, the target gene, and exogenous envelope proteins (env) responsible for membrane fusion. Currently, vesicular stomatitis virus glycoprotein (VSVG) is commonly used as the envelope protein. Although VSVG can effectively mediate lentiviral particle assembly and achieve efficient transduction in most mammalian cells, it has virtually no transduction ability for primary cells or cell lines derived from bony fish. Summary of the Invention

[0004] This invention addresses the aforementioned technical problems by providing a percomORF endogenous retroviral envelope protein from the rock bream, a lentiviral vector containing this protein, and its applications. By modifying the Env protein in lentiviral particles and reassembling the lentiviral particles, the target gene (EGFP) can be efficiently delivered to bony fish cells, achieving overexpression of the target gene. This provides a new 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: A percomORF, an endogenous retroviral envelope protein from the rock scorpionfish, wherein the amino acid sequence of the percomORF is shown in SEQ ID NO.1.

[0006] One of the technical solutions of the present invention is to provide a gene encoding the envelope protein percomORF, wherein the nucleotide sequence of the gene is SEQ ID NO.2. The second technical solution of the present invention is to provide a membrane fusion protein particle, which contains the gene as described in the first technical solution.

[0007] The third technical solution of the present invention provides an in vitro assembly system for lentiviral particles, the system comprising an envelope plasmid connected to the percomORF gene.

[0008] The fourth 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 second technical solution or lentiviral particles as described in the third technical solution; wherein the recipient 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 invention provides a recombinant lentiviral vector, wherein it comprises the envelope protein percomORF as described in the first technical solution; or, it is obtained by culturing the transformant as described in the fourth technical solution.

[0010] The sixth technical solution of the present invention provides a method for preparing a recombinant lentiviral vector, wherein the transformant as described in the fourth technical solution is cultured to obtain the recombinant lentiviral vector.

[0011] The seventh technical solution of the present invention is to provide a kit, wherein the kit comprises membrane fusion protein particles as described in technical solution two, an in vitro assembly system of lentiviral particles as described in technical solution three, a transformant as described in technical solution four, and / or a recombinant lentiviral vector as described in technical solution five.

[0012] The eighth technical solution of the present invention is to provide the application of the envelope protein percomORF as described in one of the technical solutions in the preparation of gene therapy drugs.

[0013] The beneficial effects of this invention compared to the prior art are as follows: 1) A novel lentiviral envelope protein, percomORF, suitable for transduction in fish cells is provided. Compared with the currently used VSVG envelope protein, this protein can significantly improve the transduction efficiency in various fish cells, thereby achieving efficient delivery of the target gene in fish cells.

[0014] 2) Lentiviral recombination using the endogenously derived retroviral env gene can effectively mitigate the immune rejection response induced by exogenous env proteins, providing a basis for in vivo gene delivery. Furthermore, based on the mechanism by which env proteins enter the cell membrane by recognizing specific receptors, the constructed recombinant viral particles also possess the potential for targeted gene delivery. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of a lentivirus particle three-plasmid packaging system; Figure 2 The graph shows the transduction efficiency of lentiviral particles with percomORF as the envelope protein in various bony fish cells; ae represents the brain cell line of turbot, fj represents the brain cell line of turbot, and ko represents the testis cell line of tongue sole. Detailed Implementation

[0016] 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.

[0017] Based on the inventor's previous research, it was found that endogenous viruses exist in the genome of Scorpionfish schlegelii. percomORF Gene delivery. Replacing the VSVG protein with the percomORF 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.

[0018] 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. 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. Cell cryopreservation medium (DMEM) was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd. The P24 ELISA kit was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. PBS buffer was purchased from Shandong Cisco Biotechnology 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 Using the Seamless Cloning Kit (2×Ezmax® Ultra Universal Clone Mix, TOLOBIO, catalog number: 24317), the pCMV-VSV-G recovered product and the percomORF 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); 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. ④ Prepare the transfection mixture. The transfection system used is shown in Table 1 (taking T75 cell culture flasks as an example). Table 1. Transfection System ; 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. 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. 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.

[0021] 3. Preparation of recipient cells: Cells are passaged into 96-well plates in advance. Taking the turbot brain cell line as an example, the seeding time is approximately 10... 4 Cells per well were incubated overnight at 24°C until cells adhered to the cell wall. 5. Transduction of lentiviral particles with percomORF 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. 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 2 As 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 percomORF 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 percomORF as the envelope protein can efficiently transduce turbot brain cell lines at high MOIs.

[0022] 7. Transduction of lentiviral particles with percomORF as the envelope protein in other bony fish cells: In this example, both turbot gill cell lines and tongue sole testis cell lines were used for lentiviral particle transduction. Recipient cell preparation was consistent with that of the turbot brain cell line. The transduction results are as follows: Figure 2 As shown in the figure, 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 signals were detected at MOI of 100. These results indicate that lentiviral particles with percomORF as the envelope protein can efficiently transduce turbot gill cell lines and tongue sole testis cell lines.

[0023] Example 2 This embodiment provides a method for delivering target nucleic acid to target cells, comprising: (i) Provide the lentiviral vector as described in Example 1; (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.

[0024] 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.

[0025] In some embodiments, the method is used to genetically modify the target cells.

[0026] In some embodiments, the method is used to perform gene editing on the target cells.

[0027] 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).

[0028] In some embodiments, the target nucleic acid encodes an mRNA molecule, optionally wherein the mRNA is the aforementioned exogenous gene.

[0029] In some implementations, the target nucleic acid encodes double-stranded RNA, antisense RNA, microRNA, or any other RNA molecule.

[0030] 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.

[0031] In some implementations, the target cell can be any bony fish cell.

[0032] In addition, in some embodiments, a pharmaceutical composition can be prepared using the envelope protein percomORF, 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 percomORF.

[0033] (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.

[0034] 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).

[0035] Table 2 shows the sequences involved in this invention. .

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, wherein the fish are bony fish.

Images