BCBL1 cell line with p300 gene knockout function as well as construction method and application thereof
阅读说明：本技术 一种敲除p300基因的BCBL1细胞系及其构建方法与应用 (BCBL1 cell line with p300 gene knockout function as well as construction method and application thereof ) 是由 卢杰 孙传凯 郭祎珍 于 2020-05-28 设计创作，主要内容包括：本发明公开了一种敲除p300基因的BCBL1细胞系及其构建方法与应用,属于生物技术领域。本发明提供的特异性靶向p300基因的sgRNA,可通过CRISPR/Cas9技术构建p300基因敲除的细胞系。通过本发明方法可以得到稳定敲除p300的BCBL1细胞系。本发明所提供的p300基因敲除细胞系能够为卡波西肉瘤相关疱疹病毒在宿主细胞中复制的研究提供细胞模型,也能够为p300基因与肿瘤的发生机制和药物治疗研究提供细胞模型。本发明构建方法还可以为其他悬浮细胞系得到稳定敲除的细胞株提供经验。(The invention discloses a BCBL1 cell line with a p300 gene knocked out and a construction method and application thereof, belonging to the technical field of biology. The sgRNA specifically targeting the p300 gene can construct a p300 gene knockout cell line by using a CRISPR/Cas9 technology. The method can obtain the BCBL1 cell line with stably knocked-out p 300. The p300 gene knockout cell line provided by the invention can provide a cell model for research of replication of Kaposi sarcoma-associated herpesvirus in host cells, and can also provide a cell model for research of a generation mechanism and drug therapy of p300 gene and tumor. The construction method can also provide experience for obtaining stably knocked-out cell strains of other suspension cell lines.)
1. A sgRNA sequence targeted for knock-out of a p300 gene, comprising: the nucleotide sequence is as follows: 5'-GCGG CCTAAACTCTCATCTC-3' are provided.
2. Use of the sgRNA sequence for targeted knock-out of the p300 gene according to claim 1 for preparing a cell line for knock-out of the p300 gene.
3. A construction method of a BCBL1 cell line with a p300 gene knocked out is characterized in that: the method comprises the following steps:
(1) according to the sgRNA sequence of the targeted knockout p300 gene, adding CACCG at the 5 ' end of the sgRNA sequence to obtain a forward oligonucleotide chain, simultaneously obtaining a corresponding DNA complementary chain according to the sgRNA sequence of the targeted knockout p300 gene, adding AAAC at the 5 ' end of the sgRNA sequence, adding C at the 3 ' end of the sgRNA sequence to obtain a reverse oligonucleotide chain, and annealing the synthesized forward oligonucleotide chain and the reverse oligonucleotide chain to form a double chain;
(2) connecting the double strands prepared in the step (1) with a Cas9 vector to obtain a recombinant knockout expression vector;
(3) co-transfecting the recombinant knockout expression vector prepared in the step (2) and a slow virus packaging system plasmid into a cell package to prepare slow virus, and then harvesting the virus, purifying and concentrating to obtain a virus particle concentrated solution;
(4) infecting BCBL1 cells with the virus particle concentrated solution prepared in the step (3), culturing, and screening stably transformed cells to obtain a BCBL1 cell line with the p300 gene knocked out.
4. The method for constructing a p300 gene-knocked-out BCBL1 cell line according to claim 3, wherein:
the Cas9 vector in the step (2) is lentiCRISPR v2 vector;
the cell in the step (3) is a HEK 293T cell;
the slow virus packaging system plasmids in the step (3) are pMD2.G and psPAX 2;
the co-transfection method in the step (3) is a lipofection method;
the liposome used in the lipofection method is Lipofectamin 3000;
the purification in the step (3) is realized by a polyvinylidene fluoride pin filter with the aperture of 0.45 mu m;
the concentration in the step (3) is realized by an ultra-high speed refrigerated centrifuge;
the setting conditions of the ultra-high speed refrigerated centrifuge are that the rotating speed is 15000-25000 rpm, and the time is 2-4 h;
the culture medium used for the culture in the step (4) is an RPMI1640 complete culture medium containing polybrene with the final concentration of 7-9 mug/mL;
the culture conditions in step (4) were 37 ℃ and 5% CO2Culturing in an incubator;
the screening in the step (4) is puromycin screening.
5. A p300 gene knock-out BCBL1 cell line, characterized in that: is constructed by the construction method described in claim 3 or 4.
6. Use of the p300 gene knock-out BCBL1 cell line according to claim 5 for studying KSHV replication mechanism and/or tumorigenic mechanism.
7. Use of the p300 gene knock-out BCBL1 cell line of claim 5 in the preparation of a medicament for treating and/or preventing primary effusion lymphoma.
8. Use of the p300 gene knock-out BCBL1 cell line of claim 5 for modulating KSHV lytic replication and/or virion production.
Use of the p300 gene for the regulation of KSHV lytic replication and/or virion production.
The application of the p300 gene as a target molecule in preparing a diagnostic reagent and/or a therapeutic drug for primary exudative lymphoma.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a p300 gene knockout BCBL1 cell line, and a construction method and application thereof.
Background
The transcription coactivator p300 is the nuclear binding target of adenovirus oncoprotein E1A, has a molecular weight of 300kDa, and is named as p 300. The p300 functional domain comprises in order: n-terminal Nuclear Receptor Interaction Domain (NRID), transcription adaptor zinc finger 1(TAZ1), and kinase-induced CREB interaction region (KIX) together; secondly, the Bromodomain (BD), which is the key to functioning as a transcriptional helper, and a combination of RING and Plant Homeodomain (PHD); following the HAT domain, it is important for the formation of the transcription complex; finally, the ZZ-type zinc finger domain, TAZ2 domain, and the IBiD domain.
The mechanism of action of p300 is mainly 3. The first is that p300 can be connected with gene-specific transcription factors, transcription complexes and the like to play a role of a bridge; secondly, p300 can be combined with different proteins to form a complex due to the fact that the p300 comprises a plurality of structural domains, so that the interaction between the proteins and DNA is facilitated; the last is p300, one of the members of the Histone Acetyltransferase (HAT) family, has histone acetyltransferase activity, can acetylate histone or non-histone (mainly some transcription factors) to enhance the activity of gene, and plays a role in assisting transcription.
In view of the mechanism of action of p300, p300 is a transcription adapter with multiple functions. It not only participates in chromosome translocation and gene mutation, but also can be used as a transcription auxiliary factor of protein participating in oncogenic pathway; the replication and repair of DNA, cell cycle arrest and apoptosis also require the involvement of p 300; p300 may also be recruited by some proteins and transcription factors that do not have acetylation, either to exert a promoting or repressing effect. In addition, p300 plays an important role in regulating cell growth and division and inhibiting tumor proliferation. Often, abnormal expression of p300 causes a range of phenomena such as tumor cell proliferation and changes in viral gene expression, and also affects the activity of genes upstream or downstream thereof.
Primary Effusion Lymphoma (PEL) is a rare non-hodgkin's lymphoma associated with human immunodeficiency virus, HIV, and occurs in a fluid cavity of a tumor of clonal B cell origin, often found in HIV-infected individuals, with pleural effusion and peritoneal effusion occurring as the main clinical manifestations of malignant effusion. PEL pathogenesis is associated with Kaposi's Sarcoma (KS) associated herpes virus (KSHV) infection, and the specific molecular mechanism is not well defined. The CHOP scheme chemotherapy and the antiretroviral therapy are combined in treatment, the prognosis is poor, and a more specific therapeutic target is to be further discovered.
The CRISPR/Cas9 is a new technology, can realize the specificity and accurate knockout of genome, has the advantages of simple principle, high editing efficiency, simple and easy operation of design process, capability of being combined with various technologies and the like compared with the traditional ZFN and TALEN technologies, and becomes a popular technology in the fields of medicine, pesticide, environmental protection and the like.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the primary object of the present invention is to provide a sgRNA sequence for targeted knockout of a p300 gene.
The invention also aims to provide a construction method of the BCBL1 cell line with the p300 gene knocked out.
It is still another object of the present invention to provide a p300 gene knock-out BCBL1 cell line.
The object of the invention is prepared by the following method:
a sgRNA sequence for targeted knockout of a p300 gene has the nucleotide sequence: 5'-GCGGCCTAAACTCTC ATCTC-3' are provided.
The sgRNA sequence of the p300 gene targeted knockout is applied to preparation of a cell line of the p300 gene knockout.
The cell line is preferably BCBL1 cell line.
A method for constructing a BCBL1 cell line with a p300 gene knocked out comprises the following steps:
(1) according to the sgRNA sequence of the targeted knockout p300 gene, adding CACCG at the 5 ' end of the sgRNA sequence to obtain a forward oligonucleotide chain, simultaneously obtaining a corresponding DNA complementary chain according to the sgRNA sequence of the targeted knockout p300 gene, adding AAAC at the 5 ' end of the sgRNA sequence, adding C at the 3 ' end of the sgRNA sequence to obtain a reverse oligonucleotide chain, and annealing the synthesized forward oligonucleotide chain and the reverse oligonucleotide chain to form a double chain;
(2) connecting the double strands prepared in the step (1) with a Cas9 vector to obtain a recombinant knockout expression vector;
(3) co-transfecting the recombinant knockout expression vector prepared in the step (2) and a slow virus packaging system plasmid into a cell package to prepare slow virus, and then harvesting the virus, purifying and concentrating to obtain a virus particle concentrated solution;
(4) infecting BCBL1 cells with the virus particle concentrated solution prepared in the step (3), culturing, and screening stably transformed cells to obtain a BCBL1 cell line with the p300 gene knocked out.
The Cas9 vector described in step (2) is preferably lentiCRISPR v2 vector.
The cells in step (3) are preferably HEK 293T cells.
The lentiviral packaging system plasmids described in step (3) are preferably pMD2.G and psPAX 2.
The co-transfection method described in step (3) is preferably a lipofection method.
The liposome used in the lipofection method is preferably Lipofectamin 3000.
The purification in the step (3) is preferably realized by a polyvinylidene fluoride (PVDF) pin filter; more preferably by a polyvinylidene fluoride pin filter having a pore size of 0.45 μm.
The concentration in step (3) is preferably carried out by means of an ultra-high speed refrigerated centrifuge.
The preferable setting conditions of the ultra-high speed refrigerated centrifuge are that the rotating speed is 15000-25000 rpm, and the time is 2-4 h; more preferably at 20000rpm for 3 h.
The BCBL1 cells in the step (4) are preferably BCBL1 cells in a logarithmic growth phase; most preferably BCBL1 cells cultured as follows: the frozen BCBL1 cells are taken and cultured in RPMI1640 complete medium containing 10% v/vFBS and 1% w/v double antibody of a green chain, and passage is carried out after the cells grow to obtain BCB L1 cells with good growth state.
The culture medium used for the culture in the step (4) is preferably RPMI1640 complete culture medium containing polybrene (polybrene) with the final concentration of 7-9 mug/mL; more preferably, it is RPMI1640 complete medium containing polybrene at a final concentration of 8. mu.g/mL.
The culture conditions in step (4) are preferably 37 ℃ and 5% CO2Culturing in an incubator.
The screening in step (4) is preferably puromycin screening.
The concentration of the puromycin is preferably 2-4 ug/mL.
A BCBL1 cell line with a p300 gene knocked out is constructed by the construction method.
The application of the p300 gene knock-out BCBL1 cell line in KSHV replication mechanism and/or tumorigenic mechanism research.
The application of the p300 gene-knocked-out BCBL1 cell line in preparing a medicament for treating and/or preventing primary exudative lymphoma.
The application of the p300 gene knocked-out BCBL1 cell line in regulation of KSHV lytic replication and/or virus particle generation.
Use of the p300 gene for regulating KSHV lytic replication and/or virion production.
The p300 gene is used as a target molecule in the preparation of a diagnostic reagent and/or a therapeutic drug for primary exudative lymphoma.
Compared with the prior art, the invention has the following advantages and effects:
(1) the sgRNA specifically targeting the p300 gene can construct a p300 gene knockout cell line, in particular to a p300 knockout BCBL1 cell line, by using a CRISPR/Cas9 technology. And (3) obtaining a cell line for stably knocking out the p300 through resistance screening, and verifying and knocking out the p300 gene through a western blot experiment method.
(2) The p300 gene knockout BCBL1 cell line provided by the invention can provide a cell model for the research of KSHV replication in host cells, and also can provide a cell model for the research of the generation of B cell lymphoma caused by p300 gene and tumors, especially KSHV and other related viruses and the drug treatment. As KSHV mainly infects endothelial cells and B cells in vivo and the pathogenic mechanism of the KSHV to the endothelial cells is widely researched, but the pathogenic mechanism of the KSHV infecting the B cells is not yet clarified, no effective treatment method exists for PEL primary exudative lymphoma at present, and the construction of the BCBL1 cell line stably knocking out the p300 gene provides materials and research directions for researching the pathogenic mechanism of the PEL related to the KSHV infection, and contributes to the deep understanding of the tumor generating mechanism.
(3) The construction method can provide experience for obtaining stably knocked-out cell strains of other suspension cell lines.
(4) After knock-out of BCBL1 cell line p300, KSHV cleaved DNA replication and virus particles were significantly reduced after 120h TPA/SB treatment, with a 50% reduction in KSHV cleaved DNA replication and an approximately 80% reduction in virus production. p300 plays an important role in the activation of KSHV lytic replication and the production of virions.
Drawings
Fig. 1 is a structural diagram of the lentiCRISPR v2 plasmid.
FIG. 2 is a map of the restriction enzyme digestion of the plasmid vector; wherein, Lane M is marker, Lane 1 is control group, and Lanes 2 and 3 are digested plasmids.
FIG. 3 is an electrophoretic detection scheme of recombinant plasmids; wherein, lane M is marker, lane 1 is control group, lane 2 is recombinant plasmid lentiCRISPRV2-sgp300-1, lane 3 is recombinant plasmid lentiCRISPRV2-sgp300-2, and lane 4 is recombinant plasmid lentiCRISPRV2-sgp 300-3.
FIG. 4 is a diagram showing the sequencing results of the recombinant plasmid.
FIG. 5 is a graph showing the result of Western Blot detection; wherein, lane 1 is wild type BCBL1 cell, lane 2 is sgctrl-BCBL1 cell, lane 3 is sgp300-1-BCBL1 cell, lane 4 is sgp300-2-BCBL1 cell, lane 5 is sgp300-3-BCBL1 cell.
FIG. 6 is a graph demonstrating the effect of p300 in KSHV DNA replication and virus production; where A is the result of the determination of the mRNA expression levels of RTA and K8, B is the result of the evaluation of the intracellular KSHV genomic copy, C is the standard curve, and D is the result of the evaluation of the KSHV genomic copy in the supernatant.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
Example 1 construction of p300 Gene knockout BCBL1 cell line
(1) Design of sgRNA:
based on the full sequence of the p300 gene to be knocked out (GeneID:2033), 3 sgRNAs targeting p300 are designed in a CDS region by utilizing an sgRNA design website (http:// cr ispr-era. stanford. edu and http:// cr ispr. mit. edu), and the sequences of the sgRNAs are as follows: sgp 300-1: 5'-GCGGCCTAAACTCTCATCTC-3' (SEQ ID NO.1), sgp 300-2: 5'-GACTGCGTAGGACCCTGATT-3' (SEQ ID NO.2), sgp 300-3: 5'-GTTCAATT GGAGCAGGCCGA-3' (SEQ ID NO.3), adding CACCG at the 5 ' end to obtain a forward oligonucleotide chain, simultaneously obtaining a corresponding DNA complementary chain according to the sgRNA sequence, adding AAAC at the 5 ' end and C at the 3 ' end to obtain a reverse oligonucleotide chain, and entrusting Shanghai Biochemical company to synthesize the reverse oligonucleotide chain, wherein the sequence information of the oligonucleotide chains is as follows:
sgp300-1F：5’-CACCGGCGGCCTAAACTCTCATCTC-3’(SEQ ID NO.4)
sgp300-1R：5’-AAACGAGATGAGAGTTTAGGCCGCC-3’(SEQ ID NO.5)
sgp300-2F：5’-CACCGGACTGCGTAGGACCCTGATT-3’(SEQ ID NO.6)
sgp300-2R：5’-AAACAATCAGGGTCCTACGCAGTCC-3’(SEQ ID NO.7)
sgp300-3F：5’-CACCGGTTCAATTGGAGCAGGCCGA-3’(SEQ ID NO.8)
sgp300-3R：5’-AAACTCGGCCTGCTCCAATTGAACC-3’(SEQ ID NO.9)；
(2) annealing of the primer: the synthesized forward oligonucleotide chain and the reverse oligonucleotide chain are annealed to form a double chain, and the reaction system and the experimental program are as follows:
reaction system:
experimental procedure: (carried out in a PCR apparatus)
(3) Digestion of lentiCRISPR v2 plasmid: the lentiCRISPR v2 plasmid was digested with BsmaI, and the reaction system and experimental procedure were as follows:
reaction system:
adding all the components in sequence, mixing gently, and placing in 55 deg.C water bath for enzyme digestion for 15 min.
(4) Enzyme digestion identification and glue recovery
After uniformly mixing the plasmid DNA enzyme digestion product with 10 × loading buffer, carrying out electrophoresis for 1h and 200mA on 1% agarose gel, carrying out enzyme digestion identification by using an ultraviolet shooting function of a gel imaging system, and taking an uncut lentiCRISPR v2 plasmid as a control, wherein the result is shown in FIG. 2; gel recovery was then performed using a gel recovery kit.
(5) Recombinant plasmid ligation and transformation
The annealing product is diluted by 200 times by using sterile water and then mixed with 50ng of vector, a proper amount of T4 DNA ligase is added, the temperature is controlled at 16 ℃ in a PCR instrument, and the mixture is connected overnight, so that three recombinant plasmids, namely lentiCRISPR v2-sgp300-1, lentiCRISPR v2-sgp300-2 and lentiCRISPR v2-sgp300-3, are obtained.
② transformation of recombinant plasmid
Taking out the frozen Stable3 competent cells, and thawing on ice; adding appropriate amount of overnight ligation product into competent cells, flicking, mixing, placing on ice, and standing for at least 30 min; thermally shocking at 42 deg.C for 90s, rapidly placing in ice, and standing for 3 min; adding 800 μ L LB liquid culture medium into the centrifuge tube, and recovering the strain at 220rpm and 30 deg.C for 1 h; taking a proper amount of shaken bacterial liquid, uniformly coating the bacterial liquid on an LB solid agar culture medium containing 100 mu g/ml of ampicillin, after the bacterial liquid is fully absorbed, putting the flat plate upside down in an incubator at 37 ℃, and culturing overnight for about 16 hours; the next day, a single colony is picked up in an LB liquid culture medium (containing 100 mu g/ml of ampicillin), the colony is shaken overnight by a constant-temperature shaking table at 37 ℃ and 220rpm, endotoxin-free plasmids are extracted, and electrophoresis detection and sequencing identification of recombinant plasmids are respectively carried out by taking lentiCRISPR v2 plasmid as a control. The results are shown in FIGS. 3 and 4.
(6) Preparation of lentiviruses and selection of stable cell lines
Preparation and concentration of lentiviruses
Recovering the frozen HEK 293T cells, culturing the cells by DMEM (containing 10% v/vFBS and 1% w/v cyan chain double antibody), subculturing the cells until the cell state is good, and then carrying out subsequent experiments; the 293T was digested with 0.25% w/v pancreatin and counted at 5X 106The individual cells were seeded at 10cm2Culture dish, placing at 37 deg.C and 5% CO2Culturing in an incubator to make the cell confluence rate reach about 70%; starvation was performed 30min before transfection by replacing the old medium with 4mL of Opti-MEM medium; preparation of plasmid DNA: the recombinant plasmid and the packaging plasmid were added to a 1.5mL centrifuge tube in the following proportions: 10 μ g of recombinant plasmid, 7.5ug of psPAX2, 5ug of pMD2.G and 45uL of P3000, and diluted to 500 μ L with Opti-MEM medium; and (3) diluting the liposome: 30 mu L of Lipofectamin 3000 reagent is taken to be placed in a 1.5mL centrifuge tube and diluted to be 500 mu L by using Opti-MEM culture medium; dropwise adding the prepared plasmid DNA into the diluted liposome mixed solution, slightly mixing, and incubating at room temperature for 20 min; gently adding the plasmid DNA-liposome compound into a culture dish, and supplementing 5mL of complete culture medium after 4-6 h; viral supernatants were collected 48h and 72h after transfection. The collected lentivirus supernatant was filtered to remove cell debris using a PVDF pin filter (used in combination with a 20mL syringe) having a pore size of 0.45 μm, then concentrated using an ultra-high speed refrigerated centrifuge, centrifuged at 20000rpm for 3 hours to obtain high titer virus, which was then suspended and used for transfection of target cells.
② screening of Stable cell lines
Recovering the frozen BCBL1 cells, transferring the cells to an RPMI1640 complete culture medium containing 10% v/v FBS and 1% w/v green chain double antibody for culture, carrying out passage after the cells are overgrown, and carrying out subsequent experiments when the passage culture is carried out until the cell state is good; the well-grown BCBL1 cells were counted using a cell counting plate at 5X 10 per well5Respectively inoculating the cell concentration in two 6-well plates, and marking an experimental group and a control group; the experimental group was inoculated with lentivirus, and RPMI1640 complete medium containing polybrene (polybrene) at a final concentration of 8. mu.g/mL was added in a volume of 1 mL; the control group was not inoculated with lentivirus, and the other conditions were the same; 4-6 h after lentivirus infection, 1mL of RPMI1640 complete medium (without polybrene) is respectively supplemented, and the mixture is placed at 37 ℃ and 5% CO2Continuously culturing in an incubator; the culture medium is replaced by new culture medium 24 hours after infection; 48h after infection, RPMI1640 complete medium containing puromycin (puromycin) with the final concentration of 4 mug/mL is used for replacing the old medium, primary screening is carried out, then the concentration is reduced to 2 mug/mL until all the cells in the control group die, the cells which survive in the experimental group are successful in infection, and subsequent verification is carried out, and the cells are named sgp300-1-BCBL1, sgp300-2-BCBL1 and sgp300-3-BCBL1 respectively.
(7) Western Blot experimental verification
2mL of BCBL1 wild type and mutant cell suspensions are respectively taken, and the supernatant is discarded by centrifugation; adding 1mL of precooled PBS and repeatedly washing for 2 times; add 100. mu.L of lysis buffer (containing 1 Xprotease inhibitor), lyse and collect protein, after SDS-PAGE gel electrophoresis, convert to PVDF membrane. After blocking, the corresponding primary antibodies (Rabbit polyclonal to KAT3B/p300 and Rabbit polyclonal to beta actin, abcam) were incubated overnight at 4 ℃ and secondary antibodies (Donkey Anti-Rabbit IgG H)&；L(Alexa 488) Abcam) room temperature for 1 h. Two-color infrared system gel permeation imaging was performed with lentiCRISPR v2 plasmid-infected BCBL1 cells as a control (sgctrl-BCBL1), and the results are shown in fig. 5. The results show that the sgp300-1-BCBL1 cells have no p300 protein expression, and the targeting of sgp300-1 can be seenThe efficiency is significantly different from other sites, and high-efficiency targeting can be realized. And selecting successfully constructed sgp300-1-BCBL1 cells for subsequent experiments.
(8) Determination of intracellular and extracellular viral DNA
Activation of KSHV lytic replication was achieved by induction of sgctrl-BCBL1 cells and sgp300-1-BCBL1 cells with phorbol ester/sodium butyrate (12-O-tetrahydrophorbol-13-acetate/sodium butyrate, TPA/SB) (working concentration of TPA 20. mu.g/mL, working concentration of SB 0.3M), respectively. RTA and K8 mRNA levels were determined using RT-qPCR at 48h and 72h post-induction, respectively; at 0h, 48h, 72h and 120h, the cells and the supernatant were collected, the KSHV genomic DNA in the cells and in the supernatant was extracted, and RT-qPCR relative quantification and absolute quantification analysis were performed using specific primers for the latent gene LANA, respectively, and the results are shown in FIG. 6.
② relative fluorescent quantitative PCR
Extracting RNA, then carrying out reverse transcription or qRT-PCR identification on a corresponding DNA product, preparing a PCR reaction solution on ice according to the following components, subpackaging the reaction solution into reaction tubes, quickly centrifuging the reaction solution to the bottom of the tubes, and carrying out qPCR on a LightCycler 96 system, wherein the cycle parameters are as follows: 95 ℃ for 30s, followed in this order by 95 ℃ for 5s, 60 ℃ for 30s, 95 ℃ for 5s, 65 ℃ for 60s, and 50 ℃ for 30 s. At the end of the run, the specificity of the primers and the purity of the amplification product were analyzed using the melting curve and according to 2-ΔctThe method calculates relative quantitative values, takes GAPDH gene as an internal reference, and carries out normalization treatment on the sample. Each sample was treated three times and the experiment was repeated at least three times independently. The sequences of the primers used were as follows:
K8-F：5’-CCTGGACGCTCTCTCACACA-3’(SEQ ID NO.10)
K8-R：5’-GGATCTGCGAGTTGGAAGCT-3’(SEQ ID NO.11)
RTA-F：5’-CAGTAATCACGGCCCCTTGA-3’(SEQ ID NO.12)
RTA-R：5’-AGACCCGGCGTTTATTAGTACGT-3’(SEQ ID NO.13)
LANA-F：5’-TTACCTCCACCGGCACTCTT-3’(SEQ ID NO.14)
LANA-R：5’-GGATGGGATGGAGGGATTG-3’(SEQ ID NO.15)
GAPDH-F：5’-ACTTCAACAGCGACACCCACTC-3’(SEQ ID NO.16)
GAPDH-R：5’-TCTCTTCCTCTTGTGCTCTTGCT-3’(SEQ ID NO.17)；
reaction system:
third, the absolute quantification of qPCR and the establishment of standard curve
A plasmid pEGFP-LANA (amplified using cDNA of BCBL-1 as a template to obtain a LANA gene fragment ligated between the enzyme cleavage sites ECOR I and Kpn I of pEGFP-C2) containing the LANA gene (GeneID: 4961527) was extracted as a template, and the concentration thereof was examined. The plasmid pEGFP-LANA was diluted in a gradient to give a concentration of 8X 107，8×106，8×105，8×104，8×103Copy/. mu.L of template was subjected to qPCR absolute quantification. And drawing a standard curve by taking the Cq value as an ordinate and taking the logarithm of the initial concentration as an abscissa. The copy number is calculated as follows: plasmid copy number per μ L ═ plasmid concentration (ng/μ L) × 1 μ L]×10-9/[ (plasmid molecular weight + insert molecular weight) × 660]×(6.02×1023copies/mol)。
In addition, extracellular viral progeny from the culture medium were also determined by quantifying the encapsidated viral DNA (fig. 6D). The results show that after p300 knock-out, KSHV cleaved DNA replication and virus were significantly reduced after 120h TPA/SB treatment (fig. 6B and 6D), with a 50% reduction in KSHV cleaved DNA replication and an approximately 80% reduction in virus production. The above results indicate that p300 plays an important role in KSHV activation and virion production.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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cagtaatcac ggccccttga 20
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> RTA-R
<400> 13
agacccggcg tttattagta cgt 23
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> LANA-F
<400> 14
ttacctccac cggcactctt 20
<210> 15
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> LANA-R
<400> 15
ggatgggatg gagggattg 19
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GAPDH-F
<400> 16
acttcaacag cgacacccac tc 22
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> GAPDH-R
<400> 17
tctcttcctc ttgtgctctt gct 23
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