CN106755110B - Preparation method of sensitive cell subcloned Vero/Slam/V for enhancing PPRV replication - Google Patents
Preparation method of sensitive cell subcloned Vero/Slam/V for enhancing PPRV replication Download PDFInfo
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Abstract
A preparation method of sensitive cell subclone Vero/Slam/V for enhancing PPRV replication comprises a goat lymphocyte signal activation factor Slam/V primer sequence, construction of a Slam/V lentivirus expression vector, acquisition of Slam/V replication-defective lentivirus, infection of Slam/V lentivirus on target cell Vero, screening and verification of sensitive cell subclone Vero/Slam/V, verification of enhanced peste des petits ruminants virus susceptibility and the like. The invention comprises a positive cell subclone with PPRV replication capacity improving function, the positive cell subclone has good biological characteristics and genetic stability, and the stably expressed Slam/V has the effect of obviously enhancing PPRV replication.
Description
Technical Field
The invention relates to a preparation technology of sensitive cell subclone in the field of biology, in particular to a preparation method of sensitive cell subclone Vero/Slam/V for enhancing PPRV replication.
Background
Peste des petits ruminants (PPR) is caused by Peste des petits ruminants virus (PPRV), and is clinically characterized by hyperpyrexia (above 40 ℃), conjunctivitis, eye, nasal mucus or purulent secretion, oral ulcer, cough and foul diarrhea, and pneumonia and hemorrhagic enteritis are acute, febrile and high-contact infectious diseases with main pathological changes. The disease belongs to the family paramyxoviridae (Paramyxo-viridae) Morbillivirus (Morbillivirus), and also to the genus Rinderpest Virus (RPV), Canine Distemper Virus (CDV), seal distemper Virus (PDV), Dolphin Distemper Virus (DDV) and Measles Virus (Measles Virus, MV). The virus isolation and identification are the basis of the biological characteristic research of the current epidemic PPRV strain, but PPRV is not easy to adapt on the common cell lines such as Vero, BHK-21, PK-15 and the like, or does not generate typical Cytopathy (CPE), or the virus is lost after passage for several times, the isolation rate is low, and the research on the isolation, the biological characteristic and the pathogenic mechanism of the PPRV wild strain is influenced and limited. Therefore, the method establishes sensitive cells which are easy to culture and proliferate the PPRV, and has important application value for developing deep research on the PPRV and producing vaccine viruses.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect that high-titer peste des petits ruminants virus is difficult to separate in the prior art, and constructs a preparation method of sensitive cell subclone Vero/Slam/V for enhancing PPRV replication, which is stably integrated by mediating a lentiviral vector targeting a specific cell receptor of the peste des petits ruminants virus.
In order to solve the problems, the invention adopts the following technical scheme:
a preparation method of sensitive cell subclone Vero/Slam/V for enhancing PPRV replication comprises the steps of constructing a goat signal lymphocyte activating molecule Slam/V primer sequence, obtaining Slam/V replication-defective lentivirus-like particles by constructing a goat Slam/V lentivirus expression vector, infecting Vero target cells with the obtained Slam/V replication-defective lentivirus-like particles, screening Vero positive cell subclones stably expressing Slam/V, and verifying the Slam activity, genetic stability and the propagation effect on PPRV replication of the Vero/Slam/V subclones positive cells.
The sequences SEQ ID NO 1 to SEQ ID NO 2 include:
SEQ ID NO.1:GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2:GGGGACAACTTTTGTATACAAAGTTGTGGCATGCTGAGGGCCAAGAGTGAG。
the construction of the Slam/V lentivirus expression vector comprises Slam/V and pDONRTMThe 221 donor vector is subjected to B, P site recombination to obtain an entry vector pDONR/Slam/V, and pDONR/Slam/V and pLenti6.2/V5-DESTTM 
The Vector expression Vector is subjected to L, R site recombination to obtain an expression clone pDEST/Slam/V.
The Slam/V complete lentivirus is obtained by co-transfecting a pDEST/Slam/V plasmid and a packaging mixture pLP1, pLP2 and VSV-G into a human embryonic kidney cell line 293-FT, and harvesting cell supernatant to obtain infectious Slam/V replication-defective lentivirus-like particles.
And subcloning the Vero/Slam/V positive sensitive cells, infecting the Vero cells by the obtained Slam/V slow virus-like particles, and performing resistance screening by adopting blasticidin to obtain Vero positive cell subclones for stably expressing Slam/V.
The verification method for improving the replication effect of the peste des petits ruminants virus by the Vero/Slam/V positive cell subclone adopts the technologies such as target gene amplification, indirect immunofluorescence, western-blot immunoblotting detection technology, cytopathic effect and the like to respectively verify integration and transcription of a Slam/V genome, expression of Slam/V protein, reaction activity and the proliferation effect of PPRV on the expression of the Slam/V positive cell subclone.
The preparation and verification method of the Vero/slam/V functional region sensitive cell subclone comprises the following specific steps:
a. designing and synthesizing a primer aiming at the Slam/V functional region, wherein the primer is provided with a B site homologous arm matched with a carrier P site;
b. separating peripheral blood lymphocytes of the goat and extracting total RNA of the peripheral blood lymphocytes;
c. amplifying Slam/V functional region and combining with pDONRTM221 Donor vectors utilize BP
II Enzyme Mix Enzyme carries out B, P site homologous recombination, and the recombinant is transformed into One
Mach1TMT1RSelecting positive clones from competent cells of chemical laboratory cells, sequencing the positive clones to check the fidelity of the sequence, and obtaining an entry vector pDONR/Slam/V;
d. mixing the above pDONR/Slam/V and pLenti6.2/V5-DESTTM 
Vector expression Vector by LR
II plus Enzyme Mix Enzyme carries out L, R site homologous recombination to obtain an expression skeleton pDEST/Slam/V;
e. co-transfecting 293-FT cells with the obtained expression skeleton pDEST/Slam/V plasmid, Packaging Mix pLP1, pLP2 and VSV-G to obtain Slam/V replication-defective lentivirus-like particles;
f. infecting Vero cells with the Slam/V replication-defective lentivirus-like particles obtained above;
screening resistance of BLASTICIdin to obtain Vero positive cell subclones expressing Slam/V functional regions;
h. and respectively verifying integration and transcription of slam/V genome, expression of slam/V protein, reaction activity and proliferation effect of PPRV on expression of slam/V positive cell subclones by using target gene amplification, indirect immunofluorescence, western-blot immunoblot detection technology, cytopathic effect and other technologies.
Lymphocyte activation mechanism (SLAM, also known as CD150) is a PPRV-infected cell receptor with two characteristic domains of the immunoglobulin superfamily: a V domain at the N-terminus and a C2 domain at the C-terminus, wherein the V domain is a functional region that binds to a viral protein and comprises a critical amino acid site that specifically binds to a viral protein. Thus, receptors expressing Slam/V domains do improve the sensitivity of cell-isolated viruses, but there are currently no sensitive cells in China that can improve Peste des petits ruminants isolation. Therefore, the Slam/V structural domain is selected as a target to establish PPRV sensitive cells.
In view of the above, the invention stably integrates the goat Slam/V functional region gene on Vero cell genome chromosome by means of a lentivirus expression system and adopting a Gateway technology, obtains stable cell subclone permanently expressing the Slam/V functional region by blasticidin blastcidin resistance screening, provides a reliable tool for separation and identification of peste des petits ruminants virus, lays a foundation for research on PPRV infection process, pathogenesis and the like, and has important application value for developing deep research on PPRV and vaccine virus production. The concrete embodiment is as follows:
firstly, a Gateway technology consisting of BP and LR reactions is used for quickly, accurately and efficiently constructing a pDEST/Slam/V expression vector. The technology greatly simplifies the steps of gene cloning and subcloning, ensures the correct direction and reading frame, and is convenient for the analysis of functional genes and the expression of proteins. The target DNA fragments can be transferred between vectors by site-specific recombination.
And secondly, stably integrating the Slam gene on a Vero cell genome chromosome by using a lentivirus expression system for the Vero/Slam/V sensitive cell subcloning, so that the Vero/Slam/V sensitive cell subcloning has good genetic stability, namely passage is not lost, transient transfection has certain effect but only stays in a verification stage, and a positive cell subcloning which really has the function of stably enhancing PPRV replication is not established.
And thirdly, compared with a normal Vero cell, the constructed subclone of the Vero/Slam/V sensitive cell has slightly changed morphology, and the biological characteristics of the subclone are not abnormal through a series of experiments such as a cell growth curve, a cytotoxicity experiment and the like, so that the Vero/Slam/V sensitive cell subclone can be completely used for small-batch or large-scale production and use no matter scientific research or vaccine production.
Fourthly, compared with normal Vero cells, the constructed Vero/Slam/V sensitive cell subclone can obviously improve the PPRV separation rate, the lesion time of the Vero/Slam/V sensitive cell subclone caused by PPRV is obviously shortened compared with the lesion time of the normal Vero cells, and the copy number of the Vero/Slam/V sensitive cell subclone isolated virus is improved by more than 100 times than that of the normal Vero cells.
The Vero/slam/V sensitive cell subclone prepared by the lentivirus expression system shows good activity no matter the verification is carried out on the gene level, the transcription level or the protein level. The growth characteristics of Vero/slam/V and normal Vero have no significant difference, which indicates that the expressed protein has no obvious toxicity to cells. Compared with a normal Vero cell, the Vero/slam/V has the advantages that the cytopathic time is obviously shortened after PPRV is inoculated, and the slam/V expressed by the Vero/slam/V sensitive cell subclone can obviously improve the separation rate of the PPRV.
Drawings
FIG. 1 is a nucleic acid electrophoresis diagram of integration of slam/V gene and its genetic stability in Vero/slam/V functional domain sensitive cells verified by genome amplification;
FIG. 2 is a fluorescence plot of indirect immunofluorescence verifying slam gene expression in Vero/slam/V domain-sensitive cells from the protein level;
FIG. 3 is a western-blot result protein electrophoresis chart of activity analysis after Vero/slam/V functional domain sensitive cells express slam/V functional domains;
FIG. 4 is a diagram of cells susceptible to PPRV to Vero/slam/V domain-sensitive cells;
FIG. 5 is a bar graph of real-time RT-PCR to verify the effect of PPRV proliferation on vero/slam/V zone-sensitive cells.
In FIG. 1, A is amplification slam/V gene after screening and amplification culture of Vero/slam/V functional region cells; b is an amplification slam/V gene after Vero/slam/V functional region cells pass for 30 generations; m is DL2000 molecular weight Marker;
in FIG. 2, A is normal vero cells; b is vero/slam/V functional zone cells; c is the expression of the slam/V gene in vero cells detected by immunofluorescence; d is the expression of the immune fluorescence detection slam/V gene in cells of the vero/slam/V functional region;
in FIG. 4, A is vero cytopathic effect caused by PPRV; b is vero/slam/V functional region cytopathic effect caused by PPRV.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1
1. Designing and synthesizing a primer aiming at a signal lymphocyte activating molecule Slam/V functional region of a goat, wherein the primer is matched with a carrier P site and can carry out homologous recombination, namely the primer is provided with a B site homology arm, and the sequence is as follows:
SEQ ID NO.1:GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2:GGGGACAACTTTTGTATACAAAGTTGTGGCATGCTGAGGGCCAAGAGTGAG。
2. preparation of goat signal lymphocyte activating molecule RNA template
Collecting and separating peripheral blood lymphocytes of the healthy goats, and extracting the total RNA of the peripheral blood lymphocytes.
3. Preparation of DNA of Slam/V functional region
Amplifying the Slam/V functional region and establishing a 50ul amplification system as follows:
and (3) uniformly mixing the above clicks, and carrying out RT-PCR amplification under the reaction conditions of:
4. Slam/V entry vector construction
The PCR product was recovered and purified by gel chromatography and reacted with pDONRTM221 Donor vectors utilize BP
II, carrying out B, P homologous recombination on Enzyme Mix Enzyme, wherein the BP recombination reaction system is as follows:
click and mix evenly, carry on the recombination reaction at 25 deg.C, after 2h, add protease k into the system, after reaction for 10min at 37 deg.C, the product transforms into One
Mach1TMT1RChemical ly competition Cells, selecting positive clones, sequencing to check the sequence fidelity, and obtaining an entry vector named pDONR/slam/V.
5. Construction of pDONOOR/slam/V expression vector
The pDONR/slam/V functional region and pLenti6.2/V5-DEST are combinedTM 
Vector expression Vector by LR
II plus Enzyme Mix Enzyme carries out L, R site homologous recombination, and the LR recombination reaction system is as follows:
click and mix evenly, carry on the recombination reaction at 25 deg.C, after 16h, add protease k into the system, react for 10min at 37 deg.C, the product is put into 50uL ice-melted One
Mach1TMT1RChemically Uniformly mixing the Competent Cells of the component Cells, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 60s (without shaking), carrying out ice bath for 2-3 min, adding 450uL of preheated SOC culture medium, sealing a sealing film, uniformly mixing, carrying out centrifugation at 37 ℃ for 2min at a horizontal rotating speed of 225rpm, shaking for 1h and 4000rpm, removing about 350uL by suction, coating the residual 100uL of bacterial liquid on an ampicillin-resistant LB (lysogeny broth) plate, absorbing the liquid by an erecting plate for 10min, and carrying out inverted culture at 37 ℃ for 14h until a single colony appears. Randomly picking a plurality of single colonies growing on LB (ampicillin resistance), extracting plasmids in small quantity after enrichment culture, verifying the fidelity of the plasmids by sequencing which is identified as positive by PCR, and verifying the expression skeleton which is suitable to be obtained, namely pDEST/slam/V.
6. Acquisition of slam/V replication-deficient Lentiviral-like particles
pDEST/slam/V functional regions and optimized ViraPowerTMPackaging Mix (pLP1, pLP2 and VSV-G) cotransfected human embryonic kidney cells 293-FT (transfection by liposome 3000 method) and harvested at 48h as replication-incompetent slam/V lentivirus supernatant. Centrifuging at 4 ℃ and 3500rpm for 5min to remove cell debris, and collecting the supernatant, i.e., the slam/V replication-defective lentivirus-like particles, and storing at-70 ℃ for later use.
7. Vero cell infection by slam/V replication-defective lentivirus-like particles and screening for positive cell subclones
And (3) absorbing the Vero cell culture solution, adding 3ml of slam/V replication-defective slow virus solution, incubating for 2h at 37 ℃, supplementing 7ml of normal cell culture solution, and replacing fresh DMEM culture solution containing blastcidin after 2d to perform resistance screening. When a blast resistant cell clone appeared, trypsinization was transferred to a new flask and resistance screening continued after the cells were full. And (3) carrying out limited dilution until the cells do not die any more, finding cell subcloned islands, carrying out enlarged culture and continuous passage, and carrying out a series of subsequent verification and analysis.
8. Verification of slam/V Gene integration and its genetic stability in Vero/slam/V cell subclones from Gene level by genomic amplification
Taking a bottle of Vero/slam/V functional region cells subjected to amplification culture and continuous transmission for 30 generations, sucking and discarding culture solution, digesting with pancreatin, resuspending with 500ul PBS, extracting genomic DNA by using a genomic DNA extraction kit, and performing amplification by using SEQ ID NO.1 and SEQ ID NO.2 as primers, wherein an amplification system and amplification conditions are as described above.
9. Results
As shown in figure 1, genes of the slam/V functional region are successfully amplified in the Vero/slam/V functional region and 30 generations of cells, the size of the fragment is consistent with the expectation and is 429bp, and sequencing verification is proper, so that the slam/V functional region is successfully integrated on a Vero cell genome chromosome, and is not lost after continuous 30 generations, and the Vero/slam/V functional region has good genetic stability.
Example 2
1-7 the procedure is as in example 1.
8. Verification of slam/V Gene expression in Vero/slam/V functional Domain cells from protein levels by Indirect immunofluorescence
The positive cells of the positive vero/slam/V functional region screened in the step 7 of the embodiment 1 are subcloned and inoculated in a 6-well cell culture plate with a flyer added in advance, the flyer is taken out after 48h of culture, the cell surface is lightly washed with PBS buffer solution (pH7.6) for 2 times, precooled 80% acetone is added after liquid is drained, the cell surface is fixed in a refrigerator at the temperature of-20 ℃ for 25min, the acetone is sucked and removed, and a single-layer cell is washed with PBST buffer solution (pH7.6) for 3 times. Blocking with PBS containing 3-5% BSA at room temperature for 45 min. The blocking solution was discarded and the cells were washed 3 times with PBS. Goat anti-Slam primary antibody (St. Cruis Biotechnology Co., Ltd.) diluted 1:200 in PBS was added to each well, allowed to act in a 37 ℃ wet box for 1h, the primary antibody was discarded, and the monolayer of cells was washed 3 times with PBST buffer. Adding FITC fluorescein isothiocyanate labeled donkey anti-goat IgG into each hole at a ratio of 1:500, incubating for 1h at 37 ℃ in a wet box, sucking away fluorescent secondary antibody, washing cells for 3 times by PBST, sucking out the washing liquid, then dripping 2 drops of 50% glycerol solution, placing under a fluorescence microscope to observe a dyeing result, and taking pictures.
9. Results
Immunofluorescence detection shows that the slam/V gene is expressed under the mediation of pseudotyped virus, the expressed protein can be recognized by slam fluorescence labeling antibody, and the expression amount of slam receptor in normal vero cells is very low. Further verifies from the protein level that the constructed sub-clone of vero/slam/V positive cells can improve the expression of slam/V receptor. As shown in fig. 2.
Example 3
1-7 the procedure is as in example 1.
8. Activity analysis after subcloning expression of slam/V by Vero/slam/V positive cells
Digesting with pancreatin, collecting vero/slam/V positive cell subclones by PBS, transferring cell lysate after cracking through 12% SDS-PAGE electrophoresis to PVDF transfer membrane, washing the membrane for 3 times by PBS, 5min each time, and sealing overnight by dripping 5% skimmed milk powder in PBS. PBST membrane washing 3 times, each time for 5min, adding 1:200 dilution of goat anti-slam primary antibody, 37 degrees C binding for 1.5 h. Washing the membrane with PBS for 3 times, 10min each time, adding donkey anti-goat enzyme-labeled secondary antibody diluted by 1:500, incubating for 1h with gentle shaking at room temperature, washing the membrane with PBST for 3 times, 5min each time, and analyzing the result by tetrachloro-1-naphthol substrate color development and amino black staining to check whether the expression product has specific immunocompetence.
9. Results
After SDS-PAGE electrophoresis, positive bands appear on the expressed exogenous protein at about 14kDa, but no specific band appears on a blank control group, which indicates that the expressed exogenous protein has an active structure, and after being transferred to a PVDF transfer membrane, the expressed exogenous protein acts with goat anti-slam primary antibody and donkey anti-goat IgG/HRP respectively, the result indicates that a specific chromogenic band (shown in figure 3) appears on the position of a target band, no band appears on a normal vero control cell, which indicates that a target protein slam/V functional zone can be recognized by slam antibody and has immunological activity.
Example 4
1-7 the procedure is as in example 1.
8. Inoculating PPRV virus, verifying PPRV susceptibility to vero/slam/V positive cell subclone
Simultaneously taking two bottles of vero/slam/V and vero normal cells, setting blank controls at the same time, abandoning the culture solution, inoculating 3ml of PPRV virus solution, incubating for 2h at 37 ℃, supplementing 7ml of maintenance solution, and placing at 37 ℃ to observe the cytopathic condition.
9. Results
After PPRV inoculation, the lesion time of vero/slam/V positive cell subclones (such as B in figure 2) caused by PPRV is obviously shortened compared with that of normal vero cells (such as A in figure 2), and as shown in figure 4, slam/V expressed by the vero/slam/V positive cell subclones has the effect of improving PPRV susceptibility. This result provides a better tool for propagating PPRV.
Example 5
1-7 the procedure is as in example 1.
8. Inoculating PPRV virus, and verifying the proliferation effect of goat slam/V expressed by subcloned transgenic positive cells on PPRV from transcription level by real-time RT-PCR
PPRV is respectively inoculated with vero/slam/V sensitive cell subclones and normal vero cells, viruses are respectively collected in 48h, 72h and 96h, after repeated freezing and thawing in a 70-degree refrigerator, 400uL of PPRV is respectively taken, RNA of the PPRV is respectively extracted by a Trizol method, the concentration of the RNA is measured by ND2000, all the RNA is kept at One concentration (100ng/uL) by dilution, 3uL of each sample is obtained, PPRV primers, probe primers and β -actin housekeeping gene primers which are verified in a laboratory are respectively diluted into 10umol/L, and the virus is cultured according to One StepPrimer ScriptTMThe PPRV and β -actin genes were amplified simultaneously in the RT-PCR Kit (Perfect Real time) protocol, and each sample was repeated 3 times.
9. Results
As can be seen from FIG. 5, the relative copy number of PPRV propagated on the subclones of the transgenic Vero/slam/V sensitive cells was higher than that of the normal Vero cells no matter at any time point of 48h, 72h or 96h, which indicates that slam stably expressed by the subclones of the Vero/slam/V sensitive cells has the function of obviously enhancing PPRV propagation.
Organization Applicant
----------------------
Street, No.1 Schmidt of salt court castle in Lanzhou city, Gansu province
City of Lanzhou
State of Gansu
Country of China
PostalCode : 730046
PhoneNumber : 0931-8343385
FaxNumber : 0931-8340977
EmailAddress : jingningcaixiong@163.com
<110> OrganizationName veterinary research institute of Lanzhou university, Chinese academy of agricultural sciences
Application Project
-------------------
<120> Title, a preparation method of sensitive cell subclone Vero-Slam-V for enhancing PPRV replication
<130>AppFileReference : V Domain of Human SLAM(CDw150) Is Essentialfor Its Function as a Measles Virus Receptor
<140>CurrentAppNumber :
<141>CurrentFilingDate : ____-__-__
Sequence
--------
<213>OrganismName : Ovis aries
<400>PreSequenceString :
ggggacaagt ttgtacaaaa aagcaggctt aatgctagat ctgcggaaag gtgact 56
<212>Type : DNA
<211>Length : 56
SequenceName : SEQ ID NO.1
SequenceDescription :
Sequence
--------
<213>OrganismName : Ovis aries
<400>PreSequenceString :
ggggacaact tttgtataca aagttgtggc atgctgaggg ccaagagtga g 51
<212>Type : DNA
<211>Length: 51
SequenceName : SEQ ID NO.2
SequenceDescription :
Claims (3)
1. A preparation method of sensitive cell subclone Vero/Slam/V for enhancing PPRV replication is characterized by comprising the steps of synthesizing a goat signal lymphocyte activating molecule Slam/V primer sequence, obtaining Slam/V replication-defective lentivirus-like particles by constructing a goat Slam/V lentivirus expression vector, infecting Vero target cells with the obtained Slam/V replication-defective lentivirus-like particles, screening Vero positive cell subclones stably expressing Slam/V, and verifying activity, genetic stability and the propagation effect of the Vero/Slam/V expressed by the positive cell subclones on PPRV replication;
the slam/V primer has a B site homology arm matched with a carrier, and the sequence of the slam/V primer is as follows:
SEQ ID NO.1: GGGGACAAGTTTGTACAAAAAAGCAGGC
TTAATGCTAGATCTGCGGAAAGGTGACT,
SEQ ID NO.2: GGGGACAACTTTTGTATACAAAGTTGTGG
CATGCTGAGGGCCAAGAGTGAG；
constructing the Slam/V expression Vector, including carrying out B, P site recombination on Slam/V and a pDONR (TM) 221 donor Vector to obtain an entry Vector pDONR/Slam/V, carrying out L, R site recombination on the pDONR/Slam/V and a pLenti6.2/V5-DEST Gateway expression Vector to obtain an expression clone pDOST/Slam/V;
screening Vero positive cell subclones for stably expressing Slam/V, namely performing resistance screening by adopting blasticidin to obtain Vero positive cell subclones for stably expressing Slam/V; the tolerance concentration of the Vero cell to blasticidin is 3.5 ug/ml.
2. The method for preparing the sensitive cell subclone Vero/Slam/V for enhancing PPRV replication of claim 1, wherein the obtaining of Slam/V replication-defective lentivirus-like particles is carried out by co-transfecting pDEST/Slam/V plasmid and packaging mixtures pLP1, pLP2 and VSV-G into human embryonic kidney cell line 293-FT, harvesting cell supernatant, and obtaining infectious Slam/V replication-defective lentivirus-like particles.
3. The method for preparing the sensitive cell subclone Vero/Slam/V for enhancing PPRV replication of claim 1, wherein the verification of the sensitive cell subclone Vero/Slam/V adopts a target gene amplification method, an indirect immunofluorescence method, a western-blot immunoblotting detection method and a cytopathic effect to respectively verify integration and transcription of a Slam/V genome, expression of Slam/V protein, reactivity and a proliferation effect of PPRV on the Slam/V-expressing positive cell subclone.
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| CN107805628A (en) * | 2017-10-26 | 2018-03-16 | 安徽农业大学 | A kind of stable expression PPR virus acceptor Nectin 4 cell line and its construction method |
| CN109266615A (en) * | 2018-08-17 | 2019-01-25 | 中国农业科学院兰州兽医研究所 | A kind of cell BHK/slam/v based on PPR virus receptor |
| CN109266614A (en) * | 2018-08-17 | 2019-01-25 | 中国农业科学院兰州兽医研究所 | A kind of cell BHK/slam based on PPR virus receptor |
| CN113416713A (en) * | 2021-05-11 | 2021-09-21 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Construction and application of recombinant adenovirus |
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