Preparation method of pseudovirus particles

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of pseudovirus particles. According to the invention, a part of an extracellular structural domain of rabies virus glycoprotein G and a whole transmembrane structural domain are fused with VSV-G to obtain a new enveloped plasmid, and the enveloped plasmid is found to be capable of remarkably improving the packaging efficiency and stability of rabies virus pseudoviruses. The obtained pseudovirus particles can be further applied to biological activity detection of antiviral drugs and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for preparing pseudovirus particles. Background Art

[0002] Rabies virus is a highly lethal virus, and its vaccine development and virus research are of great significance to public health. However, since live rabies virus is highly pathogenic and contagious and has high requirements for the operating environment and personnel, it brings huge risks to laboratory operations. Therefore, in the production, development and quality control of vaccines and antiviral drugs, the use of pseudovirus standard substances for condition optimization, efficacy research, detection method verification, and traceability of detection results has become an ideal choice.

[0003] Therefore, the preparation of pseudovirion particles that have rabies virus antigenicity but are non-infectious has become a safe and effective research method. However, the common pseudovirion preparation methods in the prior art have problems such as low packaging efficiency and poor stability. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preparing pseudoviral particles, which comprises the following steps: (1) Constructing a fusion expression vector of VSV-G and rabies virus glycoprotein G fragment; (2) Clone the rabies virus-specific target gene sequence by PCR amplification; (3) using homologous recombination to connect the gene sequence obtained by the above cloning to a lentiviral expression vector to obtain a recombinant expression plasmid; (4) co-transfecting the obtained fusion expression vector, recombinant expression plasmid and pseudovirus packaging plasmid into cells and packaging pseudovirus particles to obtain pseudovirus particles secreted in the supernatant that package the rabies virus-specific target gene sequence and have an outer membrane protein that is a fusion outer membrane protein of VSV-G and rabies virus glycoprotein G fragment; (5) Collect the supernatant of the cell culture medium from the previous step, and obtain pseudovirus particles after centrifugation and filtration.

[0005] Furthermore, the step (1) comprises: firstly, enzymatically removing the VSV gene on the pMD2.G vector, and then cloning the nucleic acid sequence (sequence as shown in SEQ ID NO: 1) comprising VSV-G encoding DNA without the stop codon and glycoprotein G fragment encoding DNA containing the stop codon into the pMD2.G vector after the enzymatic cutting, so as to obtain a VSV-G and glycoprotein G fragment fusion expression vector.

[0006] Furthermore, the step (1) comprises: 1.1: digesting the VSV gene on the pMD2.G vector and recovering the large vector DNA fragment; 1.2: treating the large vector DNA fragment recovered in step 1.1 to prevent the vector from self-ligating; 1.3: synthesizing a DNA sequence comprising VSV-G encoding DNA with the stop codon removed and glycoprotein G fragment DNA containing the stop codon, and introducing restriction sites at both ends; 1.4: treating the DNA sequence synthesized in step 1.3 with an endonuclease and recovering it; 1.5: mixing the large vector DNA fragment treated in step 1.2 with the DNA sequence recovered in step 1.4, and performing enzyme ligation to obtain a ligation product; 1.6: transforming the ligation product obtained in step 1.5, and screening positive clones to obtain a VSV-G and glycoprotein G fusion expression vector.

[0007] Furthermore, in step 1.2, the large vector DNA fragment recovered in step 1.1 is treated with alkaline phosphatase.

[0008] Furthermore, in step 1.5, T4 ligase is used for enzymatic ligation.

[0009] Furthermore, the rabies virus glycoprotein G fragment is amino acids 400-468 of rabies virus glycoprotein G.

[0010] Furthermore, in step (2), the rabies virus-specific target gene sequence is the LN34 gene sequence.

[0011] Furthermore, in step (3), the vector plasmid is FV115, and the framework structure of the vector plasmid is CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin.

[0012] Furthermore, the pseudovirus packaging plasmid is selected from pCMV-dR8.91, psPAX2, pMDLg / pRRE, pLP1 or pNL4-3.

[0013] Furthermore, the cells in step (4) are HEK293T cells.

[0014] The present invention also provides a pseudovirus particle obtained by the preparation method described herein.

[0015] Furthermore, the outer membrane protein of the pseudovirus particle is a fusion outer membrane protein of VSV-G and a rabies virus glycoprotein G fragment, and the core gene is a rabies virus-specific target gene sequence.

[0016] Advantageous Effects of the Invention The present invention fuses part of the extracellular domain and the entire transmembrane domain of rabies virus glycoprotein G with VSV-G to obtain a new envelope plasmid, which is found to significantly improve the packaging efficiency and stability of rabies virus pseudovirus. The obtained pseudovirus particles can be further used in applications such as antiviral drug biological activity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A diagram of the structure of the FV115 vector plasmid is shown.

[0018] Figure 2 A schematic diagram of the pMD2.G plasmid used to construct the VSV-G-glycoprotein G envelope protein expression vector is shown.

[0019] Figure 3 The graph shows the test results of the virus titer of the pseudovirion particles prepared in Example 1 and Comparative Examples 1-4. DETAILED DESCRIPTION

[0020] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0021] Embodiment 1:

[0022] A method for preparing rabies virus pseudoviral particles comprises the following steps: (1) Construction of a fusion expression vector of VSV-G and rabies virus glycoprotein G fragment 1.1: Use EcoR I restriction endonuclease to digest the VSV gene on the pMD2.G vector, and recover the large fragment DNA of the vector by agarose gel electrophoresis; 1.2: Use alkaline phosphatase to treat the large fragment DNA of the vector recovered in step 1.1 to prevent the vector from self-ligating; 1.3: Synthesize a DNA sequence comprising VSV-G coding DNA without a stop codon and a DNA coding for amino acids 400-468 of glycoprotein G containing a stop codon (the sequence is shown in SEQ ID NO: 1), and introduce EcoR I restriction sites at both ends; 1.4: Treat with EcoR I restriction endonuclease and recover the DNA sequence synthesized in step 1.3 by agarose gel electrophoresis; 1.5: Mix the large vector DNA fragment treated in step 1.2 with the DNA sequence recovered in step 1.4, and perform enzyme ligation using T4 ligase to obtain a ligation product; 1.6: The ligation product obtained in step 1.5 was transformed into E. coli DH5α, and positive clones were screened to obtain a VSV-G and glycoprotein G fusion expression vector, which was subjected to DNA sequencing to confirm successful cloning.

[0023] (2) The LN34 gene of rabies virus (RV) was synthesized by chemical synthesis, and restriction sites and protective bases of XbaI and NotI were inserted upstream and downstream; the primer sequences are as follows: Upstream primer: 5'-AGGCATGGAACTGACAAGAG-3', as shown in SEQ ID NO.2; Downstream primer: 5'-AGTGCTTTGCCCGGATATT-3', as shown in SEQ ID NO.3; (3) The CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin vector plasmid (FV115) and the LN34 target gene fragment were double-digested and then ligated by enzyme to obtain an enzyme ligation product; (4) Using Amp as the prokaryotic amplification selection marker and Puromycin as the eukaryotic selection marker, the enzyme-linked product was transformed into the competent E. coli DH5α and inoculated into 5 mL of LB medium without resistance, and the bacteria were shaken at 200 rpm for 4 h. Then, 100 μL of the bacterial solution was diluted and spread on an LB plate containing Amp resistance, and incubated at 37°C overnight to select positive clones; (5) The positive clones were expanded and cultured in LB medium containing Amp resistance. The plasmids were extracted using the alkaline lysis method, and the inserted fragments were sequenced and verified. The results were consistent with those in the NCBI database. The primers used for the sequencing are as follows: Forward primer: 5'-CGCAAATGGGCGGTAGGCGTG-3', as shown in SEQ ID NO.4; Reverse primer: 5'-AGTCCCGTCCTAAAATGTC-3', as shown in SEQ ID NO.5; (6) The obtained LN34 gene vector plasmid, the VSV-G-glycoprotein G fusion expression vector obtained in (1) and the auxiliary plasmid psPAX2 were co-transfected into cells to prepare pseudoviral particles; the specific transfection method is as follows: a. After HEK293T cells were revived, they were cultured for 2 generations in DMEM medium containing 10% FBS until the cells were stable, then the cells were digested with trypsin and placed in a culture dish and cultured until the cell confluence reached 70% for transfection; b. 4 h before transfection, remove the serum-containing medium, wash the cells with sterile PBS, and then replace with Opti-ME M medium to starve the cells; c. Take 10 μg of vector plasmid, 5 μg of psPAX2 plasmid, and 5 μg of VSV-G-glycoprotein G fusion expression vector plasmid, adjust the volume to 500 μL with Opti-MEM medium, and let stand at room temperature for 5 minutes to obtain a plasmid mixture; d. Mix 50 μL of PEI (polyethyleneimine) transfection reagent with 450 μL of Opti-MEM medium, let stand at room temperature for 5 minutes, add the plasmid mixture to the PEI diluent, mix by inversion, let stand at room temperature for 10 minutes, then add the cells and shake the culture dish to ensure even distribution; e. 8 h after transfection, remove the culture medium and replace it with fresh serum-containing culture medium; f. Observe the expression of green fluorescent protein under an inverted microscope 48 hours after transfection.

[0024] After successful transfection, the culture medium was collected, filtered through a 0.22 μm filter membrane and collected in a sterile ultracentrifuge tube, centrifuged at 80,000 g for 4 h, the supernatant was discarded, the precipitate was resuspended with Virus store buffer, and stored in aliquots at -80°C to obtain the pseudovirus standard substance.

[0025] Comparative Example 1 The same as Example 1, the only difference is that in step 1.3, a DNA sequence comprising VSV-G encoding DNA without a stop codon and a DNA encoding amino acids 300-468 of glycoprotein G containing a stop codon is synthesized.

[0026] Comparative Example 2 The same as Example 1, the only difference is that in step 1.3, a DNA sequence comprising VSV-G encoding DNA with the stop codon removed and glycoprotein G amino acid 431-452 encoding DNA containing the stop codon is synthesized.

[0027] Comparative Example 3 The same as Example 1, the only difference is that in step 1.3, a DNA sequence comprising VSV-G encoding DNA with the stop codon removed and glycoprotein G amino acid 431-468 encoding DNA containing the stop codon is synthesized.

[0028] Comparative Example 4 The same as Example 1, the only difference is that the pMD2G plasmid is used instead of the VSV-G-glycoprotein G fusion expression vector plasmid.

[0029] Test Example 1: The packaging efficiency of Example 1 and Comparative Examples 1-4 was tested, and the testing method was as follows: (1) The cultured 293T cells (inoculated with rabies virus) were inoculated into a 96-well plate, with approximately 1×10 cells per well. 3cells, 50 μL / well, each cell type was inoculated in 18 wells, of which 15 wells were used for inoculation of gradient diluted virus solution, and 5 wells were inoculated for each dilution multiple; the remaining 3 wells were used as blanks for counting before virus inoculation; (2) After 24 hours, observe the experimental cells under a microscope to ensure that the cells are full, evenly distributed, and free of contamination. Digest the cells in the three blank wells with trypsin and count them separately. The average result is 1×10 4 Pieces / hole; (3) Prepare gradient virus samples by doubling dilution: Take one EP tube, add 900 μL of DMEM medium containing 2% FBS, take 100 μL of the virus stock solution and add it to mix; then take 100 μL of the dilution solution and add it to the second EP tube, and mix it by pipetting; (4) Dilute virus and add sample: Pipette 100 μL of virus and slowly add it into the 96-well plate containing cells; (5) After 48 h of culture, fluorescence microscopy was performed to take photos; The results are as follows Figure 3 As shown, it can be seen that the packaging efficiency from high to low is Example 1, Comparative Example 3, Comparative Example 2, Comparative Example 4, and Comparative Example 1.

[0030] Test Example 2: The stability of the pseudovirion particles prepared in Example 1 and Comparative Examples 1-4 was tested, and the test method was as follows: the nucleic acid values ​​of randomly selected samples (3 tubes) were monitored at six time points at 0 days, 7 days, 14 days, 21 days, 28 days, and 35 days at -20°C; the nucleic acid values ​​of randomly selected samples (3 tubes) were monitored at five time points at 0h, 1h, 2h, 3h, 4h, and 5h at 25°C. The test results are shown in Tables 1 and 2.

[0031] Table 1: Mean values ​​of nucleic acid amounts measured at different time points at -20°C (LN34 gene(×10 4 copies / ml) Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Example 1 1556.51 1541.34 1595.57 1577.83 1557.22 1537.05 Comparative Example 1 67.23 69.31 67.55 67.16 68.42 67.03 Comparative Example 2 625.84 618.72 606.93 584.86 552.90 475.35 Comparative Example 3 753.93 746.49 736.19 743.92 738.77 668.24 Comparative Example 4 422.60 437.20 427.47 411.16 416.02 294.13 Table 2: Mean values ​​of nucleic acid amounts measured at different time points at 25°C (LN34 gene(×10 4 copies / ml) 0h 1h 2h 3h 4h Example 1 1524.39 1508.44 1502.15 1498.43 1321.89 Comparative Example 1 67.51 67.88 67.04 66.18 63.25 Comparative Example 2 635.74 636.82 624.53 610.79 433.28 Comparative Example 3 750.79 738.48 749.17 738.78 526.82 Comparative Example 4 432.63 435.19 426.04 411.47 190.56 The results show that the stability from high to low is Comparative Example 1, Example 1, Comparative Example 3, Comparative Example 2, and Comparative Example 4.

[0032] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for preparing pseudoviral particles, characterized in that: The following steps are involved: (1) Constructing a fusion expression vector of VSV-G and rabies virus glycoprotein G fragment; (2) Clone the rabies virus-specific target gene sequence by PCR amplification; (3) using homologous recombination to connect the gene sequence obtained by the above cloning to a lentiviral expression vector to obtain a recombinant expression plasmid; (4) co-transfecting the obtained fusion expression vector, recombinant expression plasmid and pseudovirus packaging plasmid into cells and packaging pseudovirus particles to obtain pseudovirus particles secreted in the supernatant that package the rabies virus-specific target gene sequence and have an outer membrane protein that is a fusion outer membrane protein of VSV-G and rabies virus glycoprotein G fragment; (5) Collect the supernatant of the cell culture medium from the previous step, and obtain pseudovirus particles after centrifugation and filtration.

2. The preparation method according to claim 1, characterized in that: The step (1) comprises: firstly, enzymatically cutting and removing the VSV gene on the pMD2.G vector, and then cloning the nucleic acid sequence comprising the VSV-G encoding DNA without the stop codon and the glycoprotein G fragment encoding DNA containing the stop codon into the enzymatically cut pMD2.G vector to obtain a VSV-G and glycoprotein G fragment fusion expression vector.

3. The preparation method according to claim 1, characterized in that: The rabies virus glycoprotein G fragment is amino acids 400-468 of rabies virus glycoprotein G.

4. The preparation method according to claim 1, characterized in that: In step (2), the rabies virus-specific target gene sequence is the LN34 gene sequence.

5. The preparation method according to claim 1, characterized in that: In step (3), the vector plasmid is FV115, and the framework structure of the vector plasmid is CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin.

6. The preparation method according to claim 1, characterized in that: The pseudovirus packaging plasmid is selected from pCMV-dR8.91, psPAX2, pMDLg / pRRE, pLP1 or pNL4-3.

7. The preparation method according to claim 1, characterized in that: The cells in step (4) are HEK293T cells.

8. A pseudoviral particle, characterized in that Obtained by the preparation method according to any one of claims 1 to 7.

9. The pseudovirion according to claim 8, characterized in that The outer membrane protein of the pseudovirus particle is a fusion outer membrane protein of VSV-G and a rabies virus glycoprotein G fragment, and the core gene is a rabies virus-specific target gene sequence.

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