CN115386592B - Preparation method of bovine leukemia virus full-length infectious clone - Google Patents
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Abstract
The invention discloses a preparation method of bovine leukemia virus full-length infectious clone, which is characterized by comprising the following steps: the method comprises the following steps: primer design, gene amplification, plasmid construction, virus rescue identification and identification results. The invention can construct virus infectious clone taking bacterial plasmid as skeleton and can successfully rescue virus.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a preparation method of bovine leukemia virus full-length infectious clone.
Background
Bovine leukemia virus (Bovine leukemia virus, BLV) belongs to the family of retroviruses (Retroviridae), the genus T-retrovirus (Deltaretroviruses), and has a genome length of about 8.7kb. The viral genome comprises long terminal repeats LTRs at both ends, structural genes gag, pro, pol, env and non-structural genes tax, rex, G4, R3, AS. The structural proteins gp51 (env) and p24 (gag) are indicators of active replication of the virus and are commonly used for serological diagnosis as they are the major antigenic proteins. Infection with this virus causes persistent Proliferation (PL) of bovine lymphocytes, resulting in bovine endemic leukemia (enzooticbovine leukosis, EBL). To date, researchers at home and abroad have made some work on BLV in terms of viral biological properties, pathogenesis, diagnostic methods, vaccine studies, and the like. However, there is still no effective means of control and no report on the construction of full-length infectious clones of bovine leukemia virus at home. Therefore, the construction of the full-length infectious clone of the bovine leukemia virus is beneficial to the deep development of researches on molecular biological characteristics, replication mechanism, pathogenesis and the like of the virus, and is beneficial to the thorough understanding of BLV, thereby providing theoretical basis for developing efficient BLV diagnostic reagents and vaccines.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a preparation method of bovine leukemia virus full-length infectious clone.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of bovine leukemia virus full-length infectious clone comprises the following steps:
primer design: designing a primer according to the genome sequence of the target strain;
gene amplification and construction of plasmids: performing PCR amplification on the target gene by using the primer;
virus rescue: culturing cells, transfecting, then carrying out passage, making IF for each generation after melting, and observing the proliferation condition;
virus rescue identification: performing syncytial observation, genetic stability analysis, antigen detection and reverse transcriptase activity measurement;
identification result: and (5) observing the virus rescue identification result.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in the design of the primer, the primer aims at the whole genome sequence of the BLV strain.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in the primer design, the obtained primers comprise SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in the gene amplification and construction plasmid, the SEQ ID No.1 and the SEQ ID No.2 are matched with an amplification gene A, the SEQ ID No.3 and the SEQ ID No.4 are matched with an amplification gene B, the SEQ ID No.5 and the SEQ ID No.6 are matched with an amplification gene C, and the SEQ ID No.7 and the SEQ ID No.8 are matched with an amplification gene D.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in the gene amplification and construction plasmid, PCR is used for gene amplification, and fusion PCR is carried out on the target genes amplified by the SEQ ID No.1 and the SEQ ID No.2 and the target genes amplified by the SEQ ID No.3 and the SEQ ID No. 4.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in the gene amplification and construction plasmid, PCR is used for gene amplification, and the target genes amplified by SEQ ID No.5 and SEQ ID No.6 and the target genes amplified by SEQ ID No.7 and SEQ ID No.8 are subjected to fusion PCR.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: in virus rescue, the medium used was DMEM medium with 2mL of 10% fetal bovine serum.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: when the virus is rescued, subculturing is carried out, half cells are selected for 3 times of freeze thawing in each generation, and IF detection is carried out once in every interval generation.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: virus rescue identification included genetic stability analysis, and after 13 passages, genetic stability was detected using IF and RT-PCR.
As a preferred embodiment of the method for producing a bovine leukemia virus full-length infectious clone of the present invention, there is provided a method comprising: virus rescue identification includes reverse transcriptase activity assay, which includes transfection, completion of transfection culture, 3 times of cell freeze thawing of cell culture in mobile phone, centrifugation, supernatant filtration, ultracentrifugation and resuspension for use.
The invention provides application of the primer or the preparation method in preparation of BLV diagnosis practical and/or vaccine and bovine leukemia virus full-length infectious clone prepared by the preparation method. The invention can construct virus infectious clone taking bacterial plasmid as skeleton and can successfully rescue virus. The invention provides an effective platform for the deep development of the basic and application research of BLV, and has important scientific application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic diagram showing the genomic structure of bovine leukemia virus of the present invention;
FIG. 2 is a schematic diagram of the results of syncytial observations;
FIG. 3 is a schematic diagram showing the effect of the identification step, wherein FIG. 3a is a schematic diagram showing the results of indirect immunofluorescence detection of antigen and genetic stability analysis, and FIG. 3b is a schematic diagram showing the results of RT-PCR of Tb 1Lu cells after transfection;
FIG. 4 is a schematic diagram showing the results of a reverse transcriptase activity assay;
FIG. 5 is a diagram showing comparison of the sequencing results of the obtained gene fragment and pCAGGS-BLV 17985.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The materials used in the embodiment of the invention comprise the following:
a BLV strain (GenBank accession numbers: OL 451224) was prepared, and Tb 1Lu cells, vero cells, pCAGGS vector, DH5a competent cells were stored by the present laboratory, and the sequence of the BLV strain (GenBank accession numbers: OL 451224) was represented by Seq ID No.9, the storage number was C202766, and the storage institution was China center for type culture Collection (university of Wuhan).
Restriction enzyme PrimeSTAR GXL DNA Polymerase was purchased from baori doctor materials technology limited; clone Express Ultra One Step Cloning Kit, DL5000DNA Marker and DL15000DNA Marker were purchased from Nanjinouzan Biotech Co., ltd; giemsa stain was purchased from Soy technologies Co., ltd; BLV-p24 polyclonal antibody was synthesized by american biological company; FITC-Goat Anti-Mouse IgG from KPL; DAPI staining solution was purchased from bi yun tian biotechnology limited; high Pure PCR Template Preparation Kit from Roche; axyPrep Plasmid Miniprep Kit from Axygen; reverse Transcriptase Assay Kit from Thermo Fisher Scientific; the enzyme-labeled instrument is purchased from TECAN; gel imaging systems were purchased from Tanon corporation; PCR instrument was purchased from Applied Biosystems company; inverted phase contrast microscope was purchased from Olympus corporation; fluorescence microscopy was purchased from Leica, germany.
Example 1
The primers were designed by selecting (GenBank accession numbers: AB987702, MH170030, LC 080656) the whole genome sequence according to the currently published BLV strain, and synthesized by Nanjing Pentagon Corp. The prepared primer sequence table is shown in table 1.
TABLE 1 primers required for full Length infectious clone
Note that: f: forward primer, R: and (3) a reverse primer.
Genomic DNA of BLV17985 was extracted. The genome DNA is used as a template, and primers A (SEQ ID No. 1-2), B (SEQ ID No. 3-4), C (SEQ ID No. 5-6) and D (SEQ ID No. 7-8) are respectively used for PCR amplification to obtain A, B, C, D fragments; recovering A, B, C, D fragment for standby after gel purification; then, two fragments A, B and C, D are respectively subjected to fusion PCR to obtain fragments A+ B, C +D, wherein the specific conditions of the PCR are A, B and C, D, and the two fragments are respectively subjected to fusion PCR by using primer pairs A-F/B-R, C-F/D-R, and the amplification conditions are as follows: 98 ℃ for 10s; 15s at 56 ℃;68 ℃ for 4min20s. Fragment a+ B, C +d was obtained. Then, the pCAGGS carrier is subjected to double-enzyme tangential digestion by using SacI and Xho I endonucleases, and gel is purified and recovered for later use. Finally, clone Express Ultra One Step CloningKit recombinant A+B fragment, C+D fragment and linearization vector are used to obtain pCAGGS-BLV17985, which is sent to Nanjing qing department of biological Co-Ltd for full length sequencing.
The full-length genome sequence of the recombinant virus in the pCAGGS-BLV17985 infectious clone plasmid is shown in OL451224; the genome structure is shown in FIG. 1. The pCAGGS-BLV17985 infectious clone plasmid is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2022291.
According to Lipofectamine TM 3000 kit description operation, when a monolayer of Tb 1Lu cells grows on a 6-well cell culture plate and spreads to 70% -80%, firstly discarding the cell culture supernatant, then adding 2mL of fresh DMEM culture medium containing 10% fetal bovine serum, taking 2.5ug of sample/well for transfection, and placing at 37 ℃ and 5% CO 2 And (3) starting to carry out passage by culturing in an incubator until 68 hours, collecting half cells for 3 times of freeze thawing for each generation, inoculating a freeze thawing product of cells of the previous generation (except the first generation) for each passage, carrying out IF (intermediate frequency) for each generation, monitoring virus proliferation, and after the passage reaches 11 generations, stabilizing the virus proliferation, collecting the cells for freeze thawing at the moment, and designating the rescued virus as pBLV17985.
Example 2
Syncytia observation:
env proteins of BLV can cause some cells to form syncytia. The 4 cells of Cos 7, MDBK, tb 1Lu and Vero stored in this laboratory were transfected with pCAGGS-BLV17985 according to the 1.5 transfection procedure, cytopathic effect was observed at 24h and 48h, and part of the cells were found to form syncytia at 24h, and a large number of syncytia were found to be formed at 48 h. Discarding the cell culture solution, fixing the cells with methanol for 2-3min, staining with Giemsa staining solution at room temperature for 15-30min, washing with tap water, drying, and performing microscopic examination.
Genetic stability analysis of pBLV17985 on Tb 1Lu cells:
the cells were grown to 70% -80% Tb 1Lu cells using pCAGGS-BLV17985 transfection, serially passaged 13 times on Tb 1Lu cells, half cells were harvested for each passage to freeze-thaw, the freeze-thaw products of the previous generation cells (except the first generation) were inoculated at each passage, and then the genetic stability of pBLV17985 on Tb 1Lu cells was examined using IF and RT-PCR.
Indirect immunofluorescence detection of antigen:
as described above, after transfection of Tb 1Lu cells with pCAGGS-BLV17985, the cells were serially passaged 13 times, and 96-well cell culture plates were plated as IF every other cell, while a blank control group was set. Washing with PBS for 2 times, adding methanol: acetone (1:1), -fixing at 20deg.C for 30min, and washing with PBS for 2 times; sealing with 5% skimmed milk for 2 hr, and washing with PBS for 3 times; the primary antibody is BLV-p24 polyclonal antibody, and is incubated for 3 hours at 37 ℃ and washed with PBS for 5 times; the secondary antibody is FITC-Goat Anti-Mouse IgG, incubated for 2h at 37 ℃ in the dark, and washed with PBS for 5 times; DAPI staining solution is stained for 5min at room temperature in a dark place; fluorescence was then observed.
Reverse transcriptase activity assay:
transfection procedure was as described in previous 1.5, with empty pCAGGS plasmid wells transfected and negative wells not transfected being used as controls. Placing at 37deg.C and 5% CO 2 The cells of the test group were collected from the culture supernatant after culturing in the incubator until 68 hours. The collected cell suspension was freeze-thawed 3 times, centrifuged at 2000g at 4℃for 10min and the centrifuged supernatant was then taken and passed through a 0.45 μm filter. The filtered liquid was ultracentrifuged at 30000rpm/min and centrifuged at 4℃for 2 hours, and finally the obtained pure virus was resuspended in 100. Mu.LPBS and placed at-80℃for further use. The transfection-empty pCAGGS plasmid group and the negative control group were treated in the same manner as the test group.
Reverse transcriptase activity was measured using a microplate reader after treatment of the samples as per Reverse Transcriptase Assay Kit instructions.
Example 3
Syncytia observation:
a large number of syncytia were observed in Tb 1Lu cells and Vero cells, and no syncytia were observed in the remaining 2 cells (FIG. 2).
Analysis of genetic stability of pBLV 17985:
the IF and qPCR results showed that pBLV could stably inherit 13 passages on Tb 1Lu cells and virus titers slightly increased with increasing passage numbers (fig. 3a. B.).
Indirect immunofluorescence detection of antigen:
specific green fluorescence is continuously observed in the passage process of Tb 1Lu transfected by pCAGGS-BLV, which indicates that p24 protein is expressed in cells; whereas normal Tb 1Lu cells as a blank had no specific fluorescence (fig. 3 a).
Reverse transcriptase activity assay:
the results showed that the test group showed stronger reverse transcriptase activity than the negative group tested positive (fig. 4).
The amplified 4 BLV fragments A, B, C, D were ligated to T vector, sent to the nanjing qing biosciences, inc, for sequencing, and the sequencing results were spliced. The resulting splice results were compared with the sequencing results of pCAGGS-BLV 17985. As shown in FIG. 5, the results of the comparison are shown in FIG. 5, and the present invention has good and accurate cloning results for pCAGGS-BLV 17985.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (9)
1. A preparation method of bovine leukemia virus full-length infectious clone is characterized by comprising the following steps: the method comprises the following steps:
primer design: designing a primer according to the genome sequence of the target strain;
gene amplification and construction of plasmids: carrying out PCR amplification on a target gene by using a primer, and constructing the target gene into a plasmid pCAGGS by using a homologous recombination method;
virus rescue: culturing cells, transfecting, then carrying out passage, carrying out indirect immunofluorescence on each generation after melting, and observing proliferation conditions;
virus rescue identification: performing syncytial observation, genetic stability analysis, antigen detection and reverse transcriptase activity measurement;
identification result: observing the virus rescue identification result;
in the design of the primers, the nucleotide sequences of the obtained primers are SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, and the sequence of the strain is SEQ ID No.9.
2. The method for preparing bovine leukemia virus full-length infectious clone according to claim 1, wherein: in the primer design, the primer is aimed at the whole genome sequence of the BLV strain.
3. The method for preparing bovine leukemia virus full-length infectious clone according to claim 2, wherein: in the gene amplification and construction plasmid, SEQ ID No.1 and SEQ ID No.2 are matched with an amplification gene A, SEQ ID No.3 and SEQ ID No.4 are matched with an amplification gene B, SEQ ID No.5 and SEQ ID No.6 are matched with an amplification gene C, and SEQ ID No.7 and SEQ ID No.8 are matched with an amplification gene D.
4. The method for preparing bovine leukemia virus full-length infectious clone according to claim 3, wherein: in the gene amplification and construction plasmid, PCR is used for gene amplification, and fusion PCR is carried out on target genes amplified by SEQ ID No.1 and SEQ ID No.2 and target genes amplified by SEQ ID No.3 and SEQ ID No. 4.
5. The method for preparing bovine leukemia virus full-length infectious clone according to claim 3, wherein: in the gene amplification and construction plasmid, PCR is used for gene amplification, and the target genes amplified by SEQ ID No.5 and SEQ ID No.6 and the target genes amplified by SEQ ID No.7 and SEQ ID No.8 are subjected to fusion PCR.
6. The method for preparing bovine leukemia virus full-length infectious clone according to claim 1, wherein: in virus rescue, the medium used was DMEM medium with 2mL of 10% fetal bovine serum.
7. The method for preparing bovine leukemia virus full-length infectious clone according to claim 1, wherein: when the viruses are rescued, subculturing is carried out, half cells are selected for 3 times of freeze thawing in each generation, and IF detection is carried out once in each generation.
8. The method for preparing bovine leukemia virus full-length infectious clone according to claim 1, wherein: the virus rescue identification comprises genetic stability analysis, and after 13 passages, the genetic stability is detected by adopting IF and RT-PCR.
9. The method for preparing bovine leukemia virus full-length infectious clone according to claim 1, wherein: the virus rescue identification comprises reverse transcriptase activity measurement, wherein the reverse transcriptase activity measurement comprises transfection, the transfection is completed, cultured cells are collected and thawed for 3 times, centrifugation is carried out, supernatant is taken and filtered, and then ultracentrifugation and resuspension are carried out for later use.
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| CA2952724A1 (en) * | 2014-06-20 | 2015-12-23 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for rapid generation of an infectious rna virus |
| CN114214338A (en) * | 2021-08-26 | 2022-03-22 | 扬州大学 | Porcine PIV5 full-length infectious clone and preparation method and application thereof |
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| CA2952724A1 (en) * | 2014-06-20 | 2015-12-23 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Method for rapid generation of an infectious rna virus |
| CN114214338A (en) * | 2021-08-26 | 2022-03-22 | 扬州大学 | Porcine PIV5 full-length infectious clone and preparation method and application thereof |
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| 高旭 ; 沈荣显 ; 相文华 ; 周建华 ; .逆转录病毒基因组RNA的二聚作用.病毒学报.2008,(第06期),487-491. * |
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