CN102703502A - Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent - Google Patents
Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent Download PDFInfo
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Abstract
The invention discloses a preparation method for a replication and transcription activator (Rta) protein and the application of the Rta protein to a nasopharynx cancer detection reagent and relates to a medical diagnosis reagent. The preparation method disclosed by the invention comprises the following steps of: 1, constructing a recombinant expression vector by taking a BRLF1 full-length gene as an exogenous gene; 2, transfecting: transfecting the recombinant expression vector into an eukaryotic expression system to obtain a positive transfected cell; and 3, expressing and purifying: culturing the positive transfected cell so as to enable the positive transfected cell to express an interest protein, and separating and purifying the interest protein, wherein the eukaryotic expression system refers to a Chinese hamster ovary (CHO) cell. The Rta protein prepared by the method disclosed by the invention is used for detecting nasopharynx cancer; the sensitivity of the Rta protein is 96 percent (288/300), and the specificity of the Rta protein is 96.7 percent (290/300). The sensitivity and the specificity are superior to those of antigens respectively prepared by a prokaryotic expression system and a pichia expression system, and the sensitivity and the specificity on clinical early diagnosis on the nasopharynx cancer are greatly improved.
Description
Technical field
The present invention relates to medical diagnosis reagent, particularly, the present invention relates to a kind of eukaryotic expression system that utilizes and express the method that the BRLF1 full-length gene obtains the expression product Rta of purifying, and be applied in the serology detection of nasopharyngeal carcinoma with the Rta albumen that obtains.
Background technology
Epstein-Barr virus is a kind of gamma herpes viruses, and there is this virus in the whole world near adult's infection of 95%.Ebv infection can be divided into latent infection phase and cracking replicative phase two states; After primary infection; This virus can be set up lifelong latent infection in host; The Epstein-Barr virus cracking replication status of persistence infects can cause a series of human malignancies (Rickinson AB, Lee SP, Steven NM.Cytotoxic T lymphocyte responses to Epstein-Barr virus.Curr Opin Immunol.1996Aug; 8 (4): 492-7).The BRLF1 gene is positioned at Epstein-Barr virus genome ORF 50, is the immediate early gene that Epstein-Barr virus gets into cracking replication status early expression.BRLF1 genes encoding transcription activating protein Rta (claiming EB1 again), length is 1818bp.There are some researches show that the BRLF1 gene can effectively activate Epstein-Barr virus and get into cracking replication status (Zalani S in bone-marrow-derived lymphocyte and epithelial cell; Holley-Guthrie E, Kenney S.Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.Proc Natl Acad Sci U S is Aug 20 A.1996; 93 (17): 9194-9; Ragoczy T, Heston L, Miller G.The Epstein-Barr virus Rta protein activates lytic cycle genes and can disrupt latency in B lymphocytes.J Virol.1998Oct; 72 (10): 7978-84).Rta albumen can activate the promotor of several diseases virus gene; Like BMLF1; BaRF1; (Ragoczy T, Heston L, Miller G.The Epstein-Barr virus Rta protein activates lytic cycle genes and can disrupt latency in B lymphocytes.J Virol.1998 Oct such as BALF5; 72 (10): 7978-84).Therefore the Rta albumen by BRLF1 genetic expression is that Epstein-Barr virus gets into the essential active element of cracking replication status.
Epstein-Barr virus produces and the oral infection at pars oralis pharyngis throughout one's life, and primary infection can cause infectious monocytosis, is mainly in children and Young Adults.Through discovering that Epstein-Barr virus all has relevant with nasopharyngeal carcinoma, cancer of the stomach, lung cancer and a series of common lymphoma for many years.Wherein nasopharyngeal carcinoma and Epstein-Barr virus relation are the closest; In recent years through the viral genome in the tumour epithelial cell and detection of antigens having been confirmed the two closely related property (zur Hausen H, Schulte-Holthausen H, Klein G; Henle W; Henle G, Clifford P, Santesson L.EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx.Nature.1970 Dec 12; 228 (5276): 1056-8; Yeung WM, Zong YS, Chiu CT, Chan KH, Sham JS, Choy DT, Ng MH.Epstein-Barr virus carriage by nasopharyngeal carcinoma in situ.Int J Cancer.1993 Mar 12; 53 (5): 746-50).Nasopharyngeal carcinoma is the south east asia kinds of tumor, with esophagus cancer, liver cancer and title China three big tumours, and occurred frequently in China south number province, in the various malignant tumours in south, hold pride of place.Main policies for Epstein-Barr virus related neoplasms control is: 1, treat 2, the generation ideal of disease is postponed to mean lifetime, 3, prevention after the early detection diagnosis.
Because Epstein-Barr virus can't be separately in vitro culture, and the difficulty that a large amount of cell cultures and purifying bring makes with unsafe factor and directly is difficult in realization clinically from the morphologic detection Epstein-Barr virus, more can't be applied to extensive screening.Therefore the Epstein-Barr virus related neoplasms is carried out early diagnosis is to control Epstein-Barr virus related neoplasms efficient ways at present more for early diagnosis antigen, antibody or the genetic material of using virus-specific, and the leading indicator of diagnosing the Epstein-Barr virus relative disease now is Epstein-Barr virus shell antigen (VCA), EA (EA), NA (EBNA) and antibody thereof.Wherein EBNA1 all has expression in Epstein-Barr virus latent period and burst times, though and VCA and EA nasopharyngeal carcinoma (Nasopharngeal carcinoma) (NPC) among the patient recall rate high, its specificity is bad, in healthy population, still has higher recall rate.Therefore selecting sensitivity and the good detection index of specificity is one of present this research field urgent problem.
At Chinese patent ZL200610113403, the author is through proteic two gene fragments of the recombinant expressed Rta of protokaryon, and with mixed form as detecting antigen, be used for the auxiliary detection of nasopharyngeal carcinoma, obtained good effect.Though this scheme shows the diagnosis effect that is higher than international standard; Also there is a big difference but with the tolerance range of nasopharyngeal carcinoma biopsy pathologic finding diagnosis; Cause this test kit not giving play to Rta molecular target inherent, the highly sensitive that should reach and high specific index aspect clinical definite and the anticancer therapy monitoring, be used for antibody test or when using, have omission or tire lower problem as vaccine; Be prone to cross reaction takes place, Interference Detection causes the result false positive to occur.Only selected for use the part reading frame of BRLFl gene to carry out antibody test simultaneously among the Chinese patent ZL200610113403, but the sensitivity aspect is unsatisfactory as antigen.
Summary of the invention
In order to overcome the above defective that above-mentioned field still exists, the invention provides the proteic method of Rta of eukaryotic expression, and the Rta albumen of being expressed by this method is as the antigenic application of nasopharyngeal carcinoma immunodetection.
The proteic preparation method of Rta, its step is following:
(1) make up recombinant expression vector: with the BRLF1 full-length gene is foreign gene;
(2) transfection: change said recombinant expression vector in the eukaryotic expression system positive cell that changes of acquisition,
(3) expression and purifying: cultivate said positive transformant and make it express target protein, separation and purification target protein; It is characterized in that: said eukaryotic expression system refers to Chinese hamster ovary celI.
Said recombinant expression vector refers to pcDNA4.1A-BRLF1; Be to be skeleton carrier with pcDNA4.1A; PcDNA4.1A and BRLF1 full-length gene cDNA are through the EcoR/XbaI double digestion; Connect the double digestion title product of pcDNA4.1A nuclear BRLF1 full-length gene cDNA, product transformed into escherichia coli competent cell gets after screen in succession.
Said transfection refers to that pcDNA4.1A-BRLF1 mixes back transfection CHO/DG44 cell with liposome Lipofectamine2000.
Said expression referred in containing the DMEM substratum of 20% calf serum, 1% Regular Insulin and 1%EGF cultured continuously 48 hours.
Said DMEM substratum is available from HyClone, Code:SH3002201B.
Said purifying refers to: culturing cell is hatched with Ni-NTA superflow beads after the cracking of RIPA lysis buffer, and the 20mM imidazoles is washed 4 times, carries out wash-out with the 400mM imidazoles then; With HPLC binding molecule sieve technology recombinant fusion protein is carried out purifying again.
A kind of Elisa enzyme mark that detects nasopharyngeal carcinoma detects carrier, it is characterized in that, said enzyme mark detects Rta albumen that carrier obtains with above-mentioned preparation method as envelope antigen.
Said Rta albumen is 0.05-0.5ng/ml as the concentration that encapsulates of envelope antigen.
The application of the Rta albumen that above-mentioned preparation method obtains in preparation nasopharyngeal carcinoma detection reagent.
Its sensitivity of detection and specificity that the Rta albumen of method preparation of the present invention is used for nasopharyngeal carcinoma are respectively 96% (288/300); 96.7% (290/300); The result is superior to prokaryotic expression system and the prepared antigenic result of pichia yeast expression system, improves the sensitivity and the specificity of the clinical early diagnosis of nasopharyngeal carcinoma greatly.Study carefully its major cause: adopt prokaryotic expression system to express eukaryotic protein; Though it is identical with eukaryotic protein on primary structure; Modify but lack behind the protein translation further processing, so recombinant protein there is bigger difference two, on the tertiary structure with the albumen of eukaryotic expression, only can simulates the antigenic determinant of forming by the adjacent amino acid residue in the parent protein; Be linear epitope, cause a little less than the antigen-antibody binding ability; Prokaryotic expression adopts the intestinal bacteria system more; In purified product, often have micro-tropina, and persons quite a lot infected intestinal bacteria among the crowd, had Chinese People's Anti-Japanese Military and Political College's enterobacteria antibody in the body; Therefore be prone to cross reaction takes place, Interference Detection causes the result false positive to occur.And the albumen of method preparation of the present invention also is superior to the pichia yeast expression system expressed proteins; Possible cause is: pichia yeast expression system is as the low eukaryotic cell that waits; When expressing the albumen of Mammals or parasitic Mammals life entity; Still there are some defectives; Glairy glycosylation modified still incomplete, the processing of the heterogeneity of product albumen matter, signal peptide not exclusively, inner degraded, polymer formation etc., these defectives all might influence expressed specificity and sensitivity when being used for nasopharyngeal carcinoma diagnosis.
Rta protein preparation method of the present invention has also overcome some problems of existence in the employing CHO eukaryotic expression albumen: as: the output efficiency of CHO eukaryotic expression system is low excessively, can't obtain the Rta full-length proteins by ordinary method.The present invention has increased the output efficiency of CHO eukaryotic expression system through the adjustment to the developing medium composition, has successfully realized the expression of Rta full-length proteins.
Among the proteic preparation method of Rta of the present invention, make up the BRLF1 gene eukaryotic expression vector, contained the full-length gene of BRLF1 gene, had the HIS label simultaneously, be convenient to expression and purification.
Description of drawings
Fig. 1. use EcoRI, XbaI carries out double digestion to the pEASY carrier that contains BRLF1 gene gene order.
Fig. 2. use the carrier universal primer to carry out bacterium liquid PCR and identify.
Fig. 3. with EcoR I/XbaI double digestion pcDNA4.1A-BRLF1.
SDS-PAGE detected result behind Fig. 4 .pcDNA4.1A-BRLF1 transfecting eukaryotic cells CHO expression product purifying.
Embodiment
The all commercially available acquisition of biomaterial (mikrobe, Chinese hamster ovary celI, expression vector) that the present invention adopts, also there is preservation in our unit, can guarantee to be used for proof test from the applying date to public's granting.
1. primer design is with synthetic
PCR primer sequence according to known BRLF1 sequence (GenBank gi:94734074) design does; Rta-full-lowprimer:ccTCTAGAaaataagctggtgtc (sequence 2 in the sequence table) Rta-full-upprimer:gcGAATTCatgaggcctaaaaaggatg (sequence 3 in the sequence table); Have EcoRI respectively; The XbaI enzyme cutting site, it is synthetic to give birth to the worker by Shanghai.
The BRLF1 sequence is corresponding, and expressed to go out proteic sequence be GenBank gi:94734074.
2. obtain the cDNA of BRLF1 gene and be cloned on the carrier:
Use 12-o-tetradecanoylphorbol-13-acetate, and 12-o-myristoyl Buddhist ripple acetic ester-13 (Cell Signaling Technology, Inc., Danvers, MA USA) induces B95-8 cell (ATCC Number:CRL-10624
TM) in EBV get into burst times; Adopt the RT-PCR method to obtain the cDNA of BRLF1 gene; And be cloned on pEASY-blunt-simple (Beijing Quanshijin Biotechnology Co., Ltd) carrier, be transfected into competent escherichia coli cell, after the picking mono-clonal is cultivated; Extract plasmid, use universal primer to carry out two-way mensuration.The sequence of universal primer is: M13R:5 '-CAGGAAACAGCTATGAC-3 ' (sequence 4 in the sequence table)/M13F:5 '-GTAAAACGACGGCCAGT-3 ' (sequence 5 in the sequence table); Through the order-checking proof, it is in full accord that institute's cloned sequence and GenBank gi:94734074 are announced.
3. subclone is to the carrier for expression of eukaryon pcDNA4.1A that cuts through same enzyme
Use EcoRI, XbaI carries out double digestion (Fig. 1) to the pEASY carrier that contains BRLF1 gene gene order.
Agarose gel electrophoresis reclaims the purpose fragment, uses EcoRI simultaneously, XbaI double digestion carrier for expression of eukaryon pcDNA4.1A (Invitrogen); Target fragment is connected with carrier; 16 ° of C connections are spent the night, 42 ° of C thermal shock Transformed E .coli TOP10, coating LB+Amp
+Flat board is inverted for 37 ℃ and is cultivated 16h.The picking mono-clonal shakes bacterium after cultivating, and uses carrier universal primer M13R/M13F to carry out bacterium liquid PCR (Fig. 2), and the product size is consistent with expection.Use EcoR I/XbaI double digestion simultaneously, sepharose evaluation enzyme is cut product, qualification result such as Fig. 3, and size is consistent called after pcDNA4.1A-BRLF1 with expection.The pcDNA4.1A carrier itself has his and myc label, therefore will have the his sequence label at expressed proteins C end, and the recombinant protein aminoacid sequence that has the his label is shown in sequence in the sequence table 1.
4. recombinant expression plasmid pcDNA4.1A-BRLF1 transfection CHO cell
PcDNA4.1A-BRLF1 transformed into escherichia coli competent cell DH5a cultivates in the LB substratum in a large number, extracts and purification of Recombinant expression plasmid pcDNA4.1A-BRLF1 with plasmid purification system (QIAGEN).
PcDNA4.1A-BRLF1 mixes back transfection CHO/DG44 (Invitrogen) cell with liposome Lipofectamine2000 (Invitrogen company); Containing the DMEM substratum of 20% calf serum, 1% Regular Insulin and 1%EGF (HyClone, SH3002201B) middle cultured continuously 48 hours (experimental group).Control group: (HyClone does not add 20% calf serum, 1% Regular Insulin, 1%EGF cultured continuously 48 hours in SH3002201B) to above-mentioned DMEM substratum.
5. the evaluation of expression product:
Above-mentioned cultured cells is carried out conventional SDS-PAGE electrophoretic separation after the cracking of RIPA damping fluid.In changeing the film damping fluid, above-mentioned separated albumen is transferred on the nitrocellulose filter after the electrophoretic separation.Hatch with mouse anti His monoclonal antibody, fully wash behind the film again with anti-mouse IgG two anti-the hatching of HRP labelled goat.Wash and add luminous substrate behind the film (ImmoBilon Western MILLIPORE) and at dark indoor film (KODAK) makes public.Antibody is available from mountain gold bridge company in Beijing.
6. the purifying of expression product:
Culturing cell is hatched with Ni-NTA superflow beads (Qiagen) after the cracking of RIPA lysis buffer, and the 20mM imidazoles is washed 4 times, carries out wash-out with the 400mM imidazoles then.With HPLC binding molecule sieve technology recombinant fusion protein is carried out purifying again.Being specially and selecting chromatography column for use is that Sephorose-100 has adorned post (Amersham Biosciences company).With 2 times of column volumes degerming PBS balance chromatography column, will pass through on the albumen that affinitive layer purification obtains in the appearance adding post, be the protein content that the A260nm wavelength is observed flowing liquid in the post.Only collect peak nose part protein.Identify its purity with SDS-PAGE.With spectrophotometer protein concentration is measured.The SDS-PAGE qualification result is illustrated in fig. 4 shown below, and experimental group only has single band, and protein purification >=95% is described; And control group does not have band, explains in the expression product of control group not obtain target protein.
Embodiment 2, set up human serum Rta-IgG detection method with the Rta antigen of eukaryotic expression
The Rta albumen for preparing with embodiment 1 is antigen; Set up the method that indirect method detects Rta-IgG antibody in the human serum; Its principle is: on capillary strip, encapsulate high purity eukaryotic expression Rta albumen in advance, with Rta antibodies in serum or the plasma sample, the goat anti-human igg who adds the HRP mark again combines with it; Add the tmb substrate colour developing then, use the stop buffer termination reaction again.Detect absorbancy (A value) through ELIASA.Abs value size is directly proportional with the Rta-IgG AC.
Step is following:
1. coated elisa plate preparation:
With encapsulate damping fluid (pH9.6, the carbonate buffer solution of 0.05mol/L) with the eukaryotic expression Rta antigen diluent behind the purifying to 0.1ng/ml (0.05-0.5ng/ml), join in the enzyme plate; Every hole 100ul, 37 ° of C reacted 2 hours, got rid of coating buffer; Clean 2 times each 30 seconds with washings.Every hole adds the 200ul confining liquid, and 37 ° of C reacted 2 hours, got rid of confining liquid, claps and does, in 30 ° of C loft drier oven dry 3-5 hour, with the vacuum-packed preservation of aluminium foil bag.
2. other liquid dosage:
Enzyme joins thing, selects the goat anti-human igg of the HRP mark of KPL company production, arrives working concentration for 8000 times with two anti-diluted; Sample diluting liquid contains the phosphoric acid buffer of BSA, pH7.4; Substrate solution is divided into A, B two liquid, and A is a urea peroxide solution, and B is TMB (TMB) solution; Stop buffer is the 2N sulphuric acid soln; Concentrated washing lotion is the phosphate buffered saline buffer that contains 10% polysorbas20.
3. detection method is set up
3.1. prepare: take out test kit, room temperature (18-25 ° of C) balance 30 minutes.20 times of concentrated cleaning solutions are done 20 times of dilutions with zero(ppm) water, sample to be checked is pressed dilution (desirable 10ul serum is with 100ul sample diluted and mixing) in 10: 100 with the sample diluent.
3.2. application of sample: standard substance 6 holes are all established in each test, and the recommended standard article repeat 2 holes for every part and detect, to improve the test accuracy.Other each holes add 100ul and have diluted sample (for improving the accuracy of test, advising sample diplopore reinspection to be checked).
3.3. incubation: behind non-setting adhesive bar shrouding, 37 ° of C incubators or water-bath 30 minutes.
3.4. wash plate: behind the incubation, remove the non-setting adhesive bar, inhale and remove liquid in the hole, every hole adds the 300ul washing lotion, leaves standstill 30 seconds, discards liquid, claps dry plate.Repeat above step 5 time again.
Join thing 3.5. add enzyme: every hole adds enzyme and joins thing 100ul.
3.6. incubation: behind non-setting adhesive bar shrouding, 37 ° of C incubators or water-bath 30 minutes.
3.7. wash plate: repeating step 4
3.8. colour developing: every hole adds substrate solution A, each 50ul of B respectively, 37 ° of C lucifuge incubations 10 minutes.
3.9. stop: every hole adds stop buffer 50ul, termination reaction.Shake mixing gently.
3.10. value of reading: ELIASA is set in dual wavelength 450nm/630nm, measures each hole A value.Should be after termination reaction the value of reading in 30 minutes.
4. result
Detect patients serum's sample 300 examples that case is diagnosed as nasopharyngeal carcinoma in order to last method, control serum samples 300 examples comprise hepatitis class patients serum 30 examples; Liver cancer patient blood serum 20 examples; Autoimmune disorder patients serum's 20 examples, pharynx nasalis benign lesion patient 20 examples, 210 parts of health examination human serums; Detected result is a judgment value with OD value 0.3, the Rta-IgG antibody test result such as the table 1 of each group:
Table 1. detected result
Compare test (its characteristics are that Rta albumen is prokaryotic expression, the multi-disc section), result such as table 2 with the Rta-IgG test kit that has gone on the market simultaneously.
Table 2. results of comparison
See from the result; Test kit with the Rta antigen prepd of eukaryotic expression; Its sensitivity in nasopharyngeal carcinoma diagnosis is significantly higher than the test kit with prokaryotic expression; And specificity is also very high in normal people's group and other several kinds of disease group, and this result has proved that with eucaryon Rta albumen be the human serum Rta-IgG detection kit that antigen is set up, the huge meaning in nasopharyngeal carcinoma diagnosis.
Claims (8)
1.Rta proteic preparation method comprises that step is following:
(1) make up recombinant expression vector: with the BRLF1 full-length gene is foreign gene;
(2) transfection: change said recombinant expression vector in the eukaryotic expression system positive cell that changes of acquisition;
(3) expression and purifying: cultivate said positive transformant and make it express target protein, separation and purification target protein; It is characterized in that: said eukaryotic expression system refers to Chinese hamster ovary celI.
2. preparation method according to claim 1; It is characterized in that: said recombinant expression vector refers to pcDNA4.1A-BRLF1; Be to be skeleton carrier with pcDNA4.1A, pcDNA4.1A and BRLF1 full-length gene cDNA connect the double digestion title product of pcDNA4.1A nuclear BRLF1 full-length gene cDNA through the EcoR/XbaI double digestion; Product transformed into escherichia coli competent cell gets after screen in succession.
3. preparation method according to claim 1 is characterized in that: said transfection refers to that pcDNA4.1A-BRLF1 mixes back transfection CHO/DG44 cell with liposome Lipofectamine2000.
4. preparation method according to claim 1 is characterized in that: said expression referred in containing the DMEM substratum of 20% calf serum, 1% Regular Insulin and 1%EGF cultured continuously 48 hours.
5. preparation method according to claim 1 is characterized in that: said purifying refers to: culturing cell is hatched with Ni-NTA superflow beads after the cracking of RIPA lysis buffer, and the 20mM imidazoles is washed 4 times, carries out wash-out with the 400mM imidazoles then; With HPLC binding molecule sieve technology recombinant fusion protein is carried out purifying again.
6. an Elisa enzyme mark that detects nasopharyngeal carcinoma detects carrier, it is characterized in that, said enzyme mark detects Rta albumen that carrier obtains with the arbitrary said preparation method of claim 1～5 as envelope antigen.
7. the Elisa enzyme mark of detection nasopharyngeal carcinoma according to claim 6 detects carrier, and it is characterized in that: said Rta albumen is 0.05-0.5ng/ml as the concentration that encapsulates of envelope antigen.
8. the application of the Rta albumen of the arbitrary said preparing method's acquisition of claim 1～5 in preparation nasopharyngeal carcinoma detection reagent.
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| WO2018169983A1 (en) * | 2017-03-13 | 2018-09-20 | President And Fellows Of Harvard College | Methods of modulating expression of target nucleic acid sequences in a cell |
| CN110251657A (en) * | 2019-06-14 | 2019-09-20 | 中山大学 | Application of EBV BRLF1 and its functional small peptides in inhibiting inflammasome activity |
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| CN116948978A (en) * | 2023-07-13 | 2023-10-27 | 兰州大学 | A human B lymphoma cell line stably expressing EBV cleavage protein ZEBRA and its construction method and application |
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| CN103131674A (en) * | 2012-12-11 | 2013-06-05 | 同昕生物技术（北京）有限公司 | Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line |
| CN103134936A (en) * | 2012-12-11 | 2013-06-05 | 同昕生物技术（北京）有限公司 | Reaction carrier and kit for detecting Epstein Barr (EB) virus replication and transcription activator (Rta)-immunoglobulin G (IgG) antibody |
| CN103131674B (en) * | 2012-12-11 | 2015-01-28 | 同昕生物技术（北京）有限公司 | Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line |
| WO2018169983A1 (en) * | 2017-03-13 | 2018-09-20 | President And Fellows Of Harvard College | Methods of modulating expression of target nucleic acid sequences in a cell |
| US11674138B2 (en) | 2017-03-13 | 2023-06-13 | President And Fellows Of Harvard College | Methods of modulating expression of target nucleic acid sequences in a cell |
| CN110251657A (en) * | 2019-06-14 | 2019-09-20 | 中山大学 | Application of EBV BRLF1 and its functional small peptides in inhibiting inflammasome activity |
| CN110790825A (en) * | 2020-01-03 | 2020-02-14 | 同昕生物技术（北京）有限公司 | NPCT8 polypeptide for nasopharyngeal carcinoma screening, kit and application thereof |
| CN116948978A (en) * | 2023-07-13 | 2023-10-27 | 兰州大学 | A human B lymphoma cell line stably expressing EBV cleavage protein ZEBRA and its construction method and application |
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