CN114381437A - Method for producing rabies virus pseudovirus system by using stable cell line capable of inducing expression of rabies virus protein - Google Patents
Method for producing rabies virus pseudovirus system by using stable cell line capable of inducing expression of rabies virus protein Download PDFInfo
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Abstract
A method for producing a rabies virus pseudovirus system using a stable cell line inducible for expression of rabies virus proteins, comprising: constructing a rabies virus GP protein expression recombinant plasmid; transfecting the recombinant plasmid into HEK293T cells through a lentivirus three-plasmid or four-plasmid system to carry out lentivirus packaging to obtain lentivirus Lenti-TRE-RABV; screening lentivirus infected host cells carrying the Tet-ON gene to obtain a cell line expressing the Tet-ON gene; a cell line which can induce and express rabies virus GP protein is obtained after a cell line which expresses the Tet-ON gene is transfected by Lenti-TRE-RABV and screened; packaging and producing the rabies virus pseudovirus. The pseudovirus of rabies virus produced by the invention has a structure similar to that of natural rabies virus, has the characteristics of small difference between virus batches, good stability, high virus titer, large-scale production capacity and the like, and can be applied to the fields of rabies virus neutralizing antibody detection and evaluation, vaccine research and development, medicament screening and the like.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for producing a rabies virus pseudovirus system by utilizing a stable cell line capable of inducing expression of rabies virus protein.
Background
Rabies (Rabies) is a zoonotic infectious disease caused by the Rabies virus (RABV), which is transmitted mainly by the infected animal through saliva in the way of a bite. Rabies has a high mortality rate, and human beings have almost 100% mortality rate once infected with rabies virus if they are not effectively vaccinated before or after exposure. According to the statistics of the world health organization, the rabies epidemic area covers 33 hundred million, most of rabies cases occur in Asia worldwide and account for 59.6 percent of rabies deaths worldwide, wherein more than 40 percent of cases are children under 15 years old.
The Rabies virus (rabis virus, RABV) belongs to the order Mononegavirales (Mononegavirales) Rhabdoviridae (Rhabdoviridae) genus Rabies virus (Lyssavirus). The rabies virus particles are bullet-shaped, are 100-300nm long and have the diameter of about 75 nm. The viral genome is about 12kb in length and is an unfragmented single-stranded negative-strand RNA, and 5 structural proteins, including Nucleoprotein (N), Phosphoprotein (P), Matrix protein (M), Glycoprotein (GP) and RNA-dependent RNA polymerase (RNA dependent RNA polymerase or Large protein, L) are encoded in sequence from 3 'to 5'. The virus particles are composed of two parts of Envelope (Envelope) and Nucleocapsid (Nucleocapsid), and the genome RNA and N, P, L protein which is tightly coiled on the outer layer jointly form the Nucleocapsid with the functions of transcription and translation; the surface of lipid membrane on the outer layer of the particle is embedded with fiber process (Spike) formed by GP protein and trimer, which is the part for virus neutralizing antigen and combining with host receptor, and M protein is located between the inner side of the shell and the nucleocapsid to connect the inner part and the outer part.
Rabies vaccine and immunoglobulin (RIG) are applied to treatment after rabies exposure, the treatment is an effective measure for preventing rabies, and a Rapid Fluorescent Focus Inhibition Test (RFFIT) for detecting the treatment effect after rabies exposure, which is recommended by the World Health Organization (WHO), can find problems in time and take remedial measures. The RFFIT method is also one of standard methods for detecting rabies virus neutralizing antibodies specified in the current pharmacopoeia of China. While RFFIT is considered the gold standard for assessing rabies neutralizing antibody titers, this method requires expensive FITC-labeled rabies virus antibodies (high quality antibody supply is not in demand), has a long experimental period, involves observation under a manual microscope, counts, and can only be performed in a biosafety level 3 (BSL-3) laboratory due to the use of highly pathogenic rabies virus for the experiments.
In order to overcome the defects of high cost, low flux, poor safety and the like of the RFFIT standard method, researchers have developed a pseudovirus method neutralizing antibody for detection to replace RFFIT so as to meet the requirements of practical application. Pseudoviruses have been used extensively in pathogen biology research, particularly for viruses requiring laboratories of high biosafety levels. However, most of the rabies virus pseudoviruses adopt a lentivirus system at present and cannot well reflect the rhabdovirus characteristics. Meanwhile, pseudoviruses using lentiviruses as vectors have the problems of large batch difference, low virus titer, poor stability and the like, and the effective application of the rabies virus pseudoviruses in neutralizing antibody detection and drug screening is also disturbed.
The invention provides a method for producing a rabies virus pseudovirus system by combining a stable cell line capable of inducing and expressing rabies virus protein with a replication-defective VSV virus vector (dVSV delta G-EGFP), and solves the technical problem.
Disclosure of Invention
A method for producing a rabies virus pseudovirus system by using a stable cell line capable of inducing expression of rabies virus proteins comprises the following steps:
s1, constructing rabies virus GP protein expression recombinant plasmids;
the rabies virus GP gene (SEQ ID NO:1) is amplified through gene synthesis and a specific primer, and the GP gene is inserted into a multiple cloning site of a plasmid intermediate vector; and recombining the rabies virus GP gene on the plasmid vector to an expression vector by the plasmid vector through a recombination technology to obtain a recombinant plasmid FG-TRE-DEST-RABV.
Further, the intermediate vector is pEntry;
further, the expression vector is FG-TRE-DEST-zf-ppw.
S2, packaging rabies virus GP protein expression lentivirus particles;
packaging of lentiviral lentivirus particles expressing the rabies virus GP protein is accomplished using a lentivirus three-plasmid system, a lentivirus four-plasmid system. For example, the constructed recombinant plasmid of FG-TRE-DEST-RABV is mixed with helper plasmids PLS3, PLS4 and PLS5, transfected into 293T cells by a transfection reagent for lentivirus packaging, and the supernatant is collected to obtain the lentivirus particle Lenti-TRE-RABV expressing rabies virus GP protein.
Further, the transfection reagent is PEI (polyethyleneimine 40K).
S3, constructing a cell line expressing the Tet-ON gene;
after infecting host cells with lentiviruses carrying the Tet-ON genes, performing drug screening by using BLASTICIdin, wherein the screening period is 5-7 days, and obtaining a cell line expressing the Tet-ON genes.
Further, the host cell is a BHK-21 cell, an HEK293T cell or an HEK293 cell, and cell lines HEK293T-Tet-ON, HEK293-Tet-ON and BHK-21-Tet-ON are obtained respectively.
Further, the concentration of the BLASTICIdin is 10-30 mu g/ml.
S4, establishing a cell line capable of inducing and expressing rabies virus GP protein;
and (3) transfecting the cell line (HEK293T-Tet-ON cell, HEK293-Tet-ON cell or BHK-21-Tet-ON cell) in the S3 step by using Lenti-TRE-RABV, adding puromycin to carry out drug screening, removing the drug after the screening period is 10-20 days, and carrying out maintenance culture to respectively obtain the cell line capable of inducing and expressing the rabies virus GP protein.
Furthermore, HEK293T 293T-Tet-ON cells, HEK293-Tet-ON cells or BHK-21-Tet-ON cells are transfected by Lenti-TREB-RABV to respectively obtain HEK 293T-indicator RABV GP, HEK 293T-indicator RABV GP and BHK-21 Tet-indicator RABV GP cell lines.
Further, puromycin concentration is 1-10. mu.g/ml.
S5, packaging and producing the rabies virus pseudoviruses.
After a cell line capable of inducing and expressing rabies virus GP protein is induced by DOX for 12-36 hours, dVSV delta G-EGFP seed pseudovirus is added, wherein the dVSV delta G-EGFP is derived from an invention patent with the publication number of CN112760297A (the patent name is: a coronavirus pseudovirus packaging system and a packaging method thereof, and the application of coronavirus pseudovirus in the evaluation of killing efficacy), a reporter gene of the EGFP is integrated between an M gene and an L gene of the dVSV in the dVSV delta G-EGFP, and supernatant is collected after 12-36 hours of culture, so that the rabies virus pseudovirus can be obtained.
Furthermore, the MOI of the dVSV delta G-EGFP seed pseudovirus is 0.1-10.
Furthermore, the concentration of DOX is 10-2000 ng/ml.
In addition, the invention discloses a method for detecting rabies virus neutralizing antibody by using rabies virus pseudovirus, which comprises the following steps:
diluting a sample to be detected in a gradient manner, incubating the sample with the rabies virus pseudovirus for a period of time, infecting cells, detecting the cells by using a multi-well plate cell imager, and measuring and calculating the neutralizing antibody and/or titer of the rabies virus in the sample by detecting the number of the cells with green fluorescent reporter protein signals.
Compared with the prior art, the invention has the following beneficial effects:
(1) the pseudovirus of rabies virus prepared by the invention is produced based on replication-defective VSV virus, both VSV virus and rabies virus belong to rhabdovirus, the structures are more similar, and the immunogenicity of the pseudovirus can be improved.
(2) The pseudovirus constructed by the method has the advantages of strong stability, wide host tropism and the like, and can be widely applied to aspects of screening of virus host cells, research and development of vaccines, detection and research of neutralizing antibodies and the like.
(3) The pseudovirus has high virus titer, is easy for large-scale production, and has non-replicability, no gene insertion risk and higher safety compared with the lentivirus pseudovirus.
(4) The invention can produce the pseudovirus only by adding the inducer DOX and the seed pseudovirus on the basis of constructing the cell line, does not need a lentivirus packaging system, and can greatly shorten the production period of the rabies virus pseudovirus.
Drawings
The embodiments are further described with reference to the accompanying drawings, in which:
FIG. 1 is a map of FG-TRE-DEST-RABV expression plasmid constructed in accordance with the present invention;
FIG. 2 is a graph of the assay for the induction of GP protein expression by inducible cells at different DOX concentrations according to the present invention;
FIG. 3 is a graph of the titer of pseudoviruses of rabies virus produced under induction with different concentrations of DOX according to the present invention;
FIG. 4 is a study of the infectivity of rabies virus pseudoviruses produced by the present invention on different cell lines;
FIG. 5 shows the application of the pseudovirus produced by the present invention in rabies vaccine vaccination induction neutralizing antibody detection;
the following detailed description will further illustrate the invention in conjunction with the above-described figures.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
1. Construction of rabies virus GP protein expressing recombinant plasmid
(1) PCR amplification of target genes
Synthesizing a target sequence according to the RABV gene sequence, and simultaneously designing the following primers:
RABV-Notl-F1：5’-ATAAGAATGCGGCCGCAATGGTTCCTCAGGCTCTCCTGT-3’(SEQ ID NO:2)
RABV-Ascl-R1：5’-TTGGCGCGCCCTCACAGTCCGGTCTCACCCCCGCTC-3’(SEQ ID NO:3)
an amplification system: 5 XQ 5 Reaction buffer 10. mu.l, forward primer and reverse primer 2.5. mu.l each, artificially synthesized template 1. mu.l, dNTPs 4. mu.l, Q5 Hot Start High-Fidelity DNA polymerase0.5. mu.l, ddH2O 29.5.5. mu.l, constituting a 50. mu.l Reaction system;
optimized amplification conditions: pre-denaturation at 98 ℃ for 30 sec; denaturation at 98 ℃ for 5-10sec, annealing at 50-72 ℃ for 10-30sec, extension at 72 ℃ for 30sec, 35 cycles; finally, extending for 2min at 72 ℃;
(2) target fragment PCR product and plasmid vector pEntry enzyme digestion and recovery
The PCR product and pEntry vector were digested with restriction enzymes ASC I and Not I.
The optimized enzyme digestion system is as follows: PCR product/plasmid 10. mu.l, restriction enzymes 1. mu.l each, 10 XNEB buffer (CutSmart) 5. mu.l, ddH2O33. mu.l, and the enzyme digestion is carried out for 1-4h at 37 ℃.
After completion of the digestion, the digestion product of the objective fragment was recovered by Purification with reference to the TIANquick Midi Purification kit (TIANGEN) instructions, and the DNA concentration was measured.
(3) Connection of target gene and pEntry vector
And (3) connecting the PCR amplification product GP gene after the enzyme digestion recovery in the step (2) with a pEntry vector to construct a pEntry-GP plasmid.
The optimized connection system is as follows: solution l (2X) 5. mu.l, pEntry 1. mu.l, target gene 1. mu.l, ddH2Mu.l of O3 was added to constitute a 10. mu.l reaction system, and the reaction was carried out overnight at 16 ℃.
(4) Plasmid transformation and Positive clone selection
And (3) taking 9 mu l of the connection product in the step (3), adding 1 mu l of Solution ll, and uniformly mixing for later use. Transformation was performed according to the E.coli DH 5. alpha. compent Cells (TaKaRa) instructions, and spread on LB (Kana) plates, and when the bacterial solution was just dried, the plates were covered and placed in a 37 ℃ incubator for 10-24 h.
Positive clones were selected and transferred to 6mL shake tubes and shake-cultured at 37 ℃ and 250rpm for 16-18 h. The pEntry-RABV plasmid is separated by an alkaline lysis extraction method.
(5) FG-TRE-DEST-RABV was prepared from pEntry-RABV plasmid by LR reaction
The RABV fragment of interest on pEntry was recombined onto FG-TRE-DEST-zf-ppw vector by LR Cloning.
The optimized recombination system comprises the following steps: pEntry-RABV plasmid 1. mu.l, FG-TRE-DEST-zf-ppw 1. mu.l, LR clone enzyme mix 0.5. mu.l, reacted at 25 ℃ for 2 h.
(6) Transformation of recombinant products and Positive clone selection
Transformation was performed according to the E.coli DH 5. alpha. compent Cells (TaKaRa) instructions, and spread on LB (KANA) plates, and when the bacterial solution was just dried, the plates were covered and placed in a 37 ℃ incubator for 10-24 h.
The positive clones were selected and transferred to 6ml shake culture tubes and shake cultured at 37 ℃ and 250rpm for 10-24 h. FG-TRE-DEST-RABV plasmid is separated by alkaline lysis extraction.
2. Packaging lentiviral particles expressing rabies virus GP protein;
the helper plasmids PLS3, PLS4, PLS5 and the main plasmid FH-TRE-DEST-RABV (puromycin resistance) were mixed and incubated, and PEI (polyethyleneimine 40K) was added in an amount 3 times the total DNA amount, and the mixed solvent was Opti-MEM serum-free medium. And (3) transfecting the plasmid in vitro into HEK293T cells for lentivirus packaging, carrying out liquid change after 2-8 h of transfection, and collecting supernatant after 24-48h of liquid change to obtain lentivirus Lenti-TRE-RABV.
3. Construction of HEK293T-Tet-ON cells
After HEK293T cells are infected by lentivirus carrying Tet-ON genes, drug screening is carried out by using 10-30 mu g/ml of blisticin, the screening period is 10-20 days, and finally a cell line HEK293T-Tet-ON expressing the Tet-ON genes is obtained.
4. Establishment of cell line capable of inducing expression of rabies virus GP protein
And (2) recovering and culturing HEK293T-Tet-ON cells, adding lentivirus Lenti-TRE-RABV for infection for 24h, adding puromycin with the concentration of 1-10 mu g/ml for drug screening, wherein the screening period is 2 weeks, removing the drug, and performing maintenance culture to obtain an inducible cell line HEK293T Tet-indelible RABV GP.
5. Packaging production of rabies virus pseudoviruses
After cells HEK293T Tet-indelible RABV GP is inoculated in a T25 bottle for 24h, DOX (the final concentration is 10-1000ng/ml) is added to induce expression for 24h, dVSV delta G-EGFP seed pseudovirus (MOI is 0.1-5) is added to culture for 24-48h, and then supernatant is collected. The cell culture supernatant was centrifuged at 2000g for 5min to remove precipitated substances, and the virus supernatant was filtered at 0.22 μm to obtain rabies virus pseudovirus.
6. Rabies virus neutralizing antibody detection
Sample dilution: serum samples and positive references were diluted in PBS in a 96-well plate with a gradient.
Sample adding: adding rabies virus pseudovirus into each hole, and putting the sample and virus mixed solution into a 5% CO2 cell culture box at 37 ℃ for CO-incubation for 1-2 h.
Viral infection: adding the cells to be infected into a 96-well cell culture plate, wherein the inoculation amount is 2-4 multiplied by 104Individual cells/well.
Infection detection: and (3) calculating the rabies virus neutralizing antibody and/or the titer thereof in the sample by detecting the count of the number of green fluorescent protein positive cells after the cells are infected with the pseudovirus for 24-48 h.
Examples
The following examples are further illustrative of the present invention and are not to be construed as limiting thereof.
Example 1: effect of different dose of DOX on HEK293T Tet-induced rabies virus GP protein expression induced by GP cell line
Different doses of DOX were used to induce HEK293T Tet-indicbile RABV GP cell line, which was analyzed by Western blotting. The result is shown in figure 2, the negative control cell HEK293T has no rabies virus GP protein expression, and the DOX induced expression of the experimental group cell is gradually enhanced along with the increase of DOX concentration, which shows that the invention successfully constructs a stably expressed HEK293T Tet-indicator RABV GP cell line which can be induced and regulated by DOX.
Example 2: production of pseudoviruses of rabies virus at different doses of DOX
The inducible cell line constructed by the invention is used for producing pseudoviruses, HEK293T Tet-indelible RABV GP cell lines are induced and expressed by DOX with different concentrations, then dVSV delta G-EGFP seed pseudoviruses are added, and virus supernatants are collected after the culture for a period of time. The virus was detected by HEK293T, and the results are shown in FIG. 3, when the DOX concentration reached 100-1000ng/ml, rabies virus pseudovirus with high concentration could be obtained.
Example 3: infection of different cell lines by rabies virus pseudoviruses
The rabies virus pseudoviruses prepared by the invention are respectively used for infecting different cell lines (HEK293T, HEK293, Vero and BHK-21), the imaging result of the EGFP fluorescent protein carried by the pseudoviruses expressed by the infected cells is shown in figure 4, and the result shows that the rabies virus pseudoviruses have the infection effect on different cells and the application range is wide.
Example 4: stability of rabies virus pseudovirus after repeated freeze thawing in refrigerator at-80 DEG C
The pseudovirus of rabies virus prepared by the invention is frozen and thawed repeatedly for 3 times by a refrigerator with minus 80 ℃, and then the HEK293T is used for detecting the virus, the virus titer has no obvious change, and the result is shown in table 1, which indicates that the pseudovirus produced by the invention has stable virus particle structure.
TABLE 1 Virus titer results of rabies virus pseudoviruses after repeated storage and thawing in a-80 deg.C refrigerator
Example 5: storage stability of pseudorabies virus
The pseudovirus of rabies virus prepared by the invention is used for storage stability detection, and as shown in table 2, the virus titer has no obvious change when the virus is stored in a refrigerator at 4 ℃ and minus 80 ℃ for 14 days.
TABLE 2 Long term storage of rabies pseudovirus Virus titer results
Example 6: application of rabies virus pseudovirus in detection of neutralizing antibody
The human serum neutralizing antibody titer after the rabies virus pseudovirus is inoculated is detected by using a rabies virus neutralizing test, and the detection result shows that (as shown in figure 5), the pseudovirus produced by the invention can replace the natural rabies virus to carry out the serum neutralizing antibody titer detection.
The invention utilizes the stable cell line which can induce and express the rabies virus protein to produce the rabies virus pseudovirus system, has short pseudovirus production period, high virus titer, small batch difference, good safety and easy large-scale production, and can be widely applied to the fields of rabies neutralizing antibody detection, evaluation, new-generation vaccine research and development, medicament screening and the like.
In conclusion, the invention has important value and application prospect.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Rui Feng kang biomedical science and technology (Zhejiang) Co., Ltd
<120> method for producing rabies virus pseudovirus system by using stable cell line capable of inducing expression of rabies virus protein and application
<130> 2021.12.27
<141> 2021-12-30
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ctcagctgcc caaacaattt ggtagtggag gacgaaggat gcaccaacct gtcagggttc 180
tcctacatgg aacttaaagt tggatacatc tcagccataa aaatgaacgg gttcacttgc 240
acaggcgttg tgacggaggc tgaaacctac actaacttcg ttggttatgt cacaaccacg 300
ttcaaaagaa agcatttccg cccaacacca gatgcatgta gagccgcgta caactggaag 360
atggccggtg accccagata tgaagagtct ctacacaatc cgtaccctga ctaccactgg 420
cttcgaactg taaaaaccac caaggagtct ctcgttatca tatctccaag tgtggcagat 480
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gtagcggtgt cttctaccta ctgctccact aaccacgatt acaccatttg gatgcccgag 600
aatccgagac tagggatgtc ttgtgacatt tttaccaata gtagagggaa gagagcatcc 660
aaagggagtg agacttgcgg ctttgtagat gaaagaggcc tatataagtc tttaaaagga 720
gcatgcaaac tcaagttatg tggagttcta ggacttagac ttatggatgg aacatgggtc 780
gcgatgcaaa catcaaatga aaccaaatgg tgccctcccg gtcagttggt gaatttgcac 840
gactttcgct cagacgaaat tgagcacctt gttgtagagg agttggtcaa gaagagagag 900
gagtgtctgg atgcactaga gtccatcatg accaccaagt cagtgagttt cagacgtctc 960
agtcatttaa gaaaacttgt ccctgggttt ggaaaagcat ataccatatt caacaagacc 1020
ttgatggaag ccgatgctca ctacaagtca gtcagaactt ggaatgagat catcccttca 1080
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ggtataatat taggacctga cggcaatgtc ttaatcccag agatgcaatc atccctcctc 1200
cagcaacata tggagttgtt ggtatcctcg gttatccccc ttatgcaccc cctggcagac 1260
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Claims (10)
1. A method for producing a rabies virus pseudovirus system by using a stable cell line capable of inducing expression of rabies virus proteins comprises the following steps:
s1, constructing rabies virus GP protein expression recombinant plasmids;
s2, packaging rabies virus GP protein expression lentivirus particles;
s3, constructing a cell line expressing the Tet-ON gene;
s4, establishing a cell line capable of inducing and expressing rabies virus GP protein;
s5, packaging and producing the rabies virus pseudoviruses.
2. The method of claim 1, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: in the step S1, rabies virus GP genes are amplified through gene synthesis and specific primers and are inserted into multiple cloning sites of a plasmid intermediate vector; and recombining the rabies virus GP gene on the plasmid vector to an expression vector by the plasmid vector through a recombination technology to obtain a recombinant expression plasmid FG-TRE-DEST-RABV.
3. The method of claim 2, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: the intermediate vector is pEntry, and the expression vector is FG-TRE-DEST-zf-ppw.
4. The method of claim 1, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: in step S2, packaging of lentiviral particles expressing rabies GP protein is accomplished using a lentiviral three-plasmid system or a lentiviral four-plasmid system.
5. The method of claim 4, wherein the stable cell line is induced to express rabies virus protein, and the method comprises the following steps: and mixing the constructed recombinant plasmid of FG-TRE-DEST-RABV with helper plasmids PLS3, PLS4 and PLS5, transfecting into 293T cells by using a transfection reagent for lentivirus packaging, and collecting supernatant to obtain the lentivirus particles Lenti-TRE-RABV expressing rabies virus GP protein.
6. The method of claim 1, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: in the step S3, after infecting host cells with lentiviruses carrying Tet-ON genes, performing drug screening by using BLASTICIdin, wherein the screening period is 10-20 days, and obtaining a cell line expressing the Tet-ON genes.
7. The method of claim 6, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: the host cell is a BHK-21 cell, an HEK293T cell or an HEK293 cell, cell lines HEK293T-Tet-ON, HEK293-Tet-ON and BHK-21-Tet-ON are respectively obtained, and the concentration of the BLASTICIdin is 3-5 mu g/ml.
8. The method of claim 7, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the steps of: s4, transfecting Lenti-TRE-RABV lentivirus into HEK293T-Tet-ON cells, HEK293-Tet-ON cells or BHK-21-Tet-ON cells, adding puromycin to carry out drug screening for 5-10 days, removing the drug, and carrying out maintenance culture to respectively obtain cell lines HEK293T Tet-indicator RABV GP, HEK293 Tet-indicator RABV GP and BHK-21 Tet-indicator RABV cell lines capable of inducing expression of rabies virus GP proteins.
9. The method of claim 1, wherein the stable cell line is induced to express rabies virus proteins, and the method comprises the following steps: and in the step S5, a cell line capable of inducing expression of rabies virus GP protein is induced by DOX for 12-36 hours, dVSV delta G-EGFP seed pseudovirus is added, and after cultivation for a period of time, supernatant is collected, so that rabies virus pseudovirus can be harvested.
10. A method for detecting rabies virus neutralizing antibody detection, comprising the steps of:
diluting a sample to be tested in a gradient manner, incubating the sample with the rabies virus pseudovirus produced by the method for producing the rabies virus pseudovirus system disclosed by any one of claims 1-9 for a period of time, infecting cells, detecting the cells by using a multi-well plate cell imager, and measuring the number of the cells with a fluorescent reporter protein signal so as to measure and calculate the titer of the neutralizing antibody and/or the titer of the rabies virus in the sample.
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| CN115948343A (en) * | 2023-01-30 | 2023-04-11 | 吉林大学 | Steady transfer cell strain for expressing rabies virus glycoprotein and construction method and application thereof |
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| US20150182617A1 (en) * | 2012-07-25 | 2015-07-02 | Theravectys | Glycoproteins for pseudotyping lentivectors |
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