CN1120238C - Recombined pure herpes virus built by using adhesive particles as basis and use thereof - Google Patents
Recombined pure herpes virus built by using adhesive particles as basis and use thereof Download PDFInfo
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- CN1120238C CN1120238C CN98101753A CN98101753A CN1120238C CN 1120238 C CN1120238 C CN 1120238C CN 98101753 A CN98101753 A CN 98101753A CN 98101753 A CN98101753 A CN 98101753A CN 1120238 C CN1120238 C CN 1120238C
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Abstract
The present invention relates to a method for generating a recombined herpes simplex virus conveniently with high efficiency by using 5 clay particles (cos 6, cos 28, cos 14, cos 56 and cos 48) containing an HSV1 complete genome as a base. Equimolar mix is carried out on a clay particle containing an exogenous gene and the other 4 clay particles, cells sensitive to HSV-1 infection are cotransfected after PacI endonuclease digestion, and the recombined herpes simplex virus containing the exogenous gene is generated by the homologous recombination of 5 HSV segments. According to the method, the herpes simplex virus HSV1-1 acZ100 capable of expressing a gene of colibacillus lacz is generated. The HSV1-1 acZ100 can be used as a tracer agent for researching nerve conduction, a target virus of an anti-herpes simplex virus medicine and other antiviral medicines, and a helper virus in HSV1 amplicon carrier research for sensitively monitoring the titer change of the helper virus.
Description
Technical field the invention belongs to the biotechnology invention field, and being specifically related to a kind of is the easy of fundamental construction recombinant herpes simplex virus and high-efficiency method with the clay, and the recombinant herpes simplex virus HSV1-lacZ100 that makes up in this way and uses thereof.
The strategy that background technology is built with infective herpes simplex virus vector has two: the recombinant type virus strategy and micro-(" mini ") the virus strategy that depends on helper virus that need not helper virus.The former is based on whole virus genomic operation, and goal gene is inserted in the viral genome, is built into the recombinant virus herpes simplex virus type; The latter is built into recombinant plasmid herpes simplex virus type carrier based on to duplicating and be packaged into the necessary virus of the infectious viral particle understanding and the application of basic cis element.The latter can be regarded as the former a kind of extreme example.
Because hsv genome very huge (about 152kb), thereby be difficult to insert foreign gene by genome is carried out direct control.So far the common methods that makes up reorganization HSV virus has 2 kinds: 1) exogenous gene expression unit is inserted in the HSV thymidine kinase gene (HSV tk) of cloning in plasmid, with this plasmid and HSV-1 genomic dna cotransfection sensitive cells (as vero or bhk cell), utilize the tk gene order of exogenous gene expression unit both sides and the corresponding sequence generation homologous recombination among the HSV-1DNA and produce reorganization HSV-1 virus.Utilize negative screening effect (as the using nucleoside analog ganciclovir) recombinant celo virus of tk gene.In the reorganization HSV virus that this method obtains, exogenous gene expression unit is inserted in the tk gene.2) other gene fragment in the clone HSV-1 genome is inserted reporter gene such as lacZ genetic expression unit therein, carries out homologous recombination with the HSV-1 genomic dna in cell and produces reorganization HSV virus.Utilize the color reaction of reporter gene product to pick out recombinant virus.
More than two kinds of methods all have the shortcoming that recombination fraction is low, background is high (it is many promptly to produce wild-type virus), cause the screening efficiency of recombinant virus low and process is loaded down with trivial details.
Summary of the invention the objective of the invention is to set up a kind of novel method that obtains reorganization HSV virus efficiently and fast.Design a kind of utilization for this reason and contained the strategy that the complete genomic clay of HSV-117 strain makes up reorganization HSV virus.The invention has the advantages that the genome that HSV-1 is huge " is changed greatly little ", realized the genomic operability of HSV-1; Do not need and wild-type HSV-1 genomic dna cotransfection, thereby reduced background, the probability that obtains to contain the recombinant virus of foreign gene in theory should reach or near 100%.We have successfully obtained to express the recombinant herpes simplex virus HSV1-lacZ100 of lacZ gene with this method, and actual recombination probability is more than 50%.
The feature of HSV1-lacZ100 recombinant virus of the present invention is to have inserted the CMV-lacZ-polyA of lacZ genetic expression unit that is about 4.3kb in the XbaI site of the genomic UL44 gene of HSV-117 strain virus.
Being used for starting materials of the present invention has following several:
1.Set C clay:
Comprise cos6, cos28, cos14, cos56, cos48 be totally 5 clays.This cover clay for Davison AJ. provide (Conningham C, Davision AJ.A cosmid-based system for constructing mutants ofherpes simplex virus type 1.Virology, 1993,197:116-124).
2.tgCMV-HyTK：(Lupton?SD.，Brunton，LL.，Kalberg?VA.and?Overell?RW.Dominant?positive?and?negative?selection?using?a?hygromycinphosphotransferase-thymidine?kinase?fusion?gene.Mol.Cell?Biol.1991，11：3374-3378)
3.pSV-the β-#E1081 of Gallactosidase:Promega company wherein contains the lacZ-SV40polyA fragment.
The building process of HSV-lacZ100 recombinant virus of the present invention is as follows: the 1) structure of pCMV-lacZ-polyA plasmid
The lacZ-SV40polyA fragment (derive from pSV-β-Gallactosidase), be inserted into constitute in the corresponding site of pCMV-HyTK plasmid (cutting the polyA of 154bp with HindIII and BamHI) in interstitial granules pCMV-HyTK-lacZ-polyA; Interstitial granules is mended flat end back and is constituted pCMV-lacZ1 from connecting to remove the HyTK gene of 2.1kb in being somebody's turn to do with Nhe I and Hind III double digestion again; Cut this plasmid with Xho I and Bam HI, add Xba I linker (8mer) behind the end-filling, behind XbaI enzyme cutting, link to each other, be built into pCMV-lacZ-polyA.Cut and the CMV-lacZ-polyA expression cassette of 4.5kb can be cut out with Xba I enzyme.The bacterial classification called after E.coli DH5 α/pCMV-lacZ-polyA that contains this plasmid.2) structure of cos56-lacZ
The CMV-lacZ-polyA fragment that two ends is added with Xba I site is inserted in the Xba I site of cos56, is built into cos56-lacZ.Utilize HSV-1 fragment among the clay cos56 in the UL44 gene, to contain the characteristics of single Xba I restriction enzyme site, with the CMV-lacZ-polyA fragment of above-mentioned 4.5kb with link to each other with the linearizing cos56 of Xba I, transform MAX Efficiency DH5 α (GIBCO BRL company product) competence bacteria, coat on the amp+ flat board that contains X-gal and IPTG, the blue look bacterium colony of picking extracts recombinant plasmid, identifies with XbaI enzyme cutting.Xba I enzyme is cut qualification result and is shown that the CMV-lacZ-polyA fragment of 4.5kb successfully is inserted in the Xba I site of cos56 (direction of insertion is not identified).This site is arranged in the HSV-1UL44 gene, and this genes encoding HSV-1 gC (gC) duplicates optional for the infection and the toxigenicity of HSV-1 in culturing cell.3) 5 clay cotransfection bhk cells
With cos6, cos28, cos14, cos56-lacZ, the cos48 balanced mix, cut with PacI (Biolab) enzyme and to spend the night, use phenol, phenol/chloroform and chloroform extracting successively, ethanol sedimentation DNA, centrifugal 10 minutes of 12000rpm, abandon most supernatant, the DNA precipitation is dissolved in an amount of aseptic TE liquid, make total DNA concentration reach about 1 μ g/ μ l.Get the above-mentioned dna solution of 10 μ l and 20 μ l lipofectamine (Gibco BRL) press product description transfection bhk cell (2 * 10
6), change the 37 ℃ of cultivations of 1640 liquid that contain 2% foetal calf serum behind the 24h into.After 2-3 days, under light microscopic, can be observed plaque and occur also increasing in time.After 5 days, the cytopathy more than 80% (becoming the big circle that becomes).The cell of this moment is arised from-20 ℃ and 37 ℃ of multigelations 3 times together with nutrient solution one, and centrifugal 10 minutes of 1500rpm gets 0.1ml supernatant (all the other packing be stored in-20 ℃ standby) and is used to infect 80% bhk cell that is paved with (about 2 * 10
6), with X-gal staining fluid dyeing 30-60 minute, observe a large amount of blue plaques under the light microscopic after 24 hours.Show the recombinant herpes simplex virus that has successfully obtained to carry and express the lacZ gene.4) purifying of HSV1-lacZ virus
Through suitable dilution postoperative infection bhk cell, 12 good plaques separated from one another of picking are inoculated in the bhk cell of 12 orifice plates and carry out amplification cultivation behind the 36h, keep each hole supernatant behind the 3d respectively with above-mentioned viral supernatant.With X-gal liquid staining cell.In 12 plaques, what indigo plant was dyed has 7, do not dyed by indigo plant 5.Get the corresponding supernatant in the 12nd hole that indigo plant is dyed, carry out again increasing in a large number behind a plaque purifying.
HSV1-lacZ100 virus of the present invention can be used for 1) as the tracer agent of studying nerve conduction; 2) as the target virus of anti-herpes simplex virus medicament and other antiviral; 3) change to monitor the helper virus titre more delicately as the helper virus in the research of HSV1 amplicon vector.
Cos56-lacZ clay of the present invention can be stored in MAX Efficiency DH5 α (the GIBCO BRL company product) strain.The bacterial strain called after MAX Efficiency DH5 α/cos56-lacZ that contains the cos56-lacZ clay is in the LB substratum that contains 50----100 μ g/ml penbritin (1%tryptone, 0.5%yeast extract, 1%NaCl) cultivation of going down to posterity.
The HSV1-lacZ100 virus of the present invention cultivation of can in bhk cell or other sensitive cells, going down to posterity.The nutrient solution or the lysate supernatant behind the cell multigelation that contain HSV1-lacZ100 virus all can be stored in-20 ℃ to-70 ℃.The culture supernatant that contains HSV1-lacZ100 virus has been stored in China Committee for Culture Collection of Microorganisms microorganism center on April 30th, 1998, that registers on the books is numbered CGMCC No.0348.
The two ends that will be different from the foreign gene X of CMV-lacZ-polyA add XbaI contact (wherein on behalf of any length, X be no more than the 15kb exogenous dna fragment), insert in the XbaI site of cos56, cos56-X that obtains and cos6, cos48, cos28, cos14 cotransfection sensitive cells (as Vero, BHK), can obtain to have inserted the segmental reorganization of X HSV1 virus in the XbaI site of HSV1 UL44 gene.
Foreign gene X also can be inserted in the XbaI site of cos6, and with cos56, cos48, cos28, cos14 cotransfection sensitive cells can obtain to have inserted the segmental reorganization of X HSV-1 virus in the XbaI site of HSV1UL2 gene.
Embodiment following examples have been done detailed description to preparation and the purposes of HSV1-lacZ100 of the present invention, but do not mean that restriction content of the present invention.
The titer determination of embodiment 1HSV1-lacZ100 virus inoculates 1 * 10 in 6 orifice plates
6/ hole bhk cell is cultivated 24h for 37 ℃.Change liquid, every hole adds the 1ml nutrient solution.In the 1st hole, add the above-mentioned supernatant liquor of 100 μ l, get 100 μ l behind the mixing and add in the 2nd hole, take out 100 μ l again and add in the 3rd hole, carry out dilution in 1: 10 successively.Cultivate after 3 hours for 37 ℃, discard nutrient solution in each hole, add covering liquid (Eagle ' the s nutrient solution that contains 1.5% methylcellulose gum and 2% foetal calf serum) 5ml/ hole, cultivated 3 days for 37 ℃.Discard nutrient solution, wash cell 3 times with the PBS damping fluid, wash 1 time with the PBS damping fluid after 5 minutes in 4 ℃ of fixed cells with the PBS damping fluid that contains 2% formaldehyde and 0.2% glutaraldehyde, add the X-gal staining fluid and (contain 1mg/ml X-gal, the 5mmol/L Tripotassium iron hexacyanide, the 5mmol/L yellow prussiate of potash, 2mmol/L MgCl
2The PBS damping fluid) cover cell.Observe after 30-60 minute and counting locus coeruleus number.Count the 5th hole and the 6th hole locus coeruleus number and be respectively 20 and 4 (locus coeruleus is all overstocked in 1 to 4 hole, is difficult to counting).The titre of calculating HSV1-lacZ thus is 2-4 * 10
6Pfu/ml.
Going down to posterity of embodiment 2HSV1-lacZ100 virus
To upload for 5 generations at bhk cell continuously through the HSV1-lacZ100 of 2 plaque purifying, the locus coeruleus rate that detects this recombinant virus infection is 100% still, and the HSV-lacZ100 virus that shows acquisition can be stablized and goes down to posterity.
Other cell of embodiment 3HSV1-lacZ100 virus infection
Infect the Vero cell of cultivating with HSV1-lacZ100 virus (moi=0.1 ~ 1), the HeLa cell, the Wish cell, the A549 cell detects with X-gal behind 37 ℃ of cultivation 24h, all can be observed the cell that a large amount of indigo plants are dyed.Illustrate that this virus has infectivity external to various kinds of cell, and express the lacZ gene.
Embodiment 4 usefulness HSV1-lacZ100 Preliminary detection V-H
+The antivirus action of atpase inhibitor ConA
V-H
+Atpase inhibitor ConA is developed by medical biotechnology institute of Chinese Academy of Medical Sciences Viral Laboratory and provides.Said preparation concentration is 625umol/L (among the 50%DMSO).This experiment is a model with HSV1-lacZ100 virus infection bhk cell, preliminary observation the anti-herpes simplex virus effect of ConA.
At 6 orifice plate moderates inoculation bhk cell, 27 ℃ of overnight incubation, cell about 80% is paved with before using.With 1640 complete culture solutions (containing 10%FBS) ConA being diluted to final concentration respectively is 5umol/L, 1umol/L, 200nmol/L, 40nmol/L, 8nmol/L.Discard nutrient solution, 1,2,3,4,5 holes add the ConA liquid 2ml of above-mentioned concentration successively, and the 6th hole adds the nutrient solution that does not contain ConA.Cultivate 2h for 37 ℃.Every hole adds 100ul HSV1-lacZ100 virus liquid (about 10
6Pfu/ml), cultivate 24h for 37 ℃.Discard nutrient solution, wash cell 3 times, add stationary liquid (2% formaldehyde, 0.2% glutaraldehyde is in the PBS liquid), discard stationary liquid, wash once with PBS liquid in 4 ℃ of fixing 5min with PBS liquid.Add X-gal solution (the 5mmol/L Tripotassium iron hexacyanide, 5mmol/L yellow prussiate of potash, 2mmol/L MgCl2, X-gal 1mg/ml) and cover 1h for 37 ℃.Visual inspection sees that intensive locus coeruleus appears in the 5th hole and the 6th hole, and 1,2,3,4 holes are all not obvious.Under inverted microscope, observe, see that cell indigo plant in blocks is dyed in the 5th hole and the 6th hole, and cell mostly is individual cells indigo plant and dyes in 1,2,3,4 holes.The ConA of this results suggest concentration 40nmmol/L to 5umol/L can obviously suppress duplicating of HSV1 virus, and concentration is that the ConA of 8nmol/L does not have restraining effect substantially.The result shows that ConA has duplicated the obvious suppression effect to the toxigenicity of virus.
Replace the characteristics of the antivirus action of wild-type HSV-1 research medicine to be with HSV1-lacZ100, can the result be observed and judge more responsive and objective.
Claims (10)
1. method that is used to produce recombinant herpes simplex virus is characterized in that it is made up of following steps:
(1) two ends with foreign gene X expression cassette dna fragmentation add the XbaI joint, constitute cos56-X in the single XbaI site of insertion reorganization clay cos56, with the combination called after Set X of cos6, cos28, cos14, cos56-X and five clays of cos48;
(2) with Set X cotransfection HSV sensitive cells after the PacI enzyme is cut, homologous recombination takes place and generates recombinant virus HSV1-X seed culture of viruses in five fragments that are imported in cell;
(3) seed culture of viruses that obtains is carried out plaque select and purifying, obtain recombinant virus HSV1-X.
2. the method for generation recombinant herpes simplex virus according to claim 1 is characterized in that, wherein foreign gene X expression cassette inserts in the XbaI site of cos56.
3. the method for generation recombinant herpes simplex virus according to claim 1, it is characterized in that, foreign gene X expression cassette is that the length that two ends have an XbaI site is the CMV-lacZ-polyA of 4.3kb, wherein CMV is upright morning of the promotor of human cytomegalic inclusion disease virus, lacZ is an e.coli, and polyA is SV40 little t antigen intron of virus and tailing signal.
4. the method for generation recombinant herpes simplex virus according to claim 1 is characterized in that, the recombinant virus HSV1-X that is wherein obtained, and X can be e.coli.
5. the method for generation recombinant herpes simplex virus according to claim 4 is characterized in that, wherein produces a kind of recombinant herpes simplex virus HSV1-lacZ100 that expresses the intestinal bacteria beta-galactosidase enzymes, is made up of following steps:
(1) makes up the recombinant plasmid pCMV-lacZ-polyA that contains the CMV-lacZ-polyA expression cassette.Wherein CMV is upright morning of the promotor of human cytomegalic inclusion disease virus, and lacZ is an e.coli, and polyA is SV40 little t antigen intron of virus and tailing signal;
(2) the XbaI site is built into cos56-CMV-lacZ-polyA among the intestinal bacteria lacZ expression casette insertion cos56;
(3) the clay cos6 that will recombinate, cos28, cos14, mole such as cos48 and five clays of cos56-CMV-lacZ-polyA mix and form Set-lacZ;
(4), obtain to express the recombinant herpes simplex virus HSV1-lacZ100 seed culture of viruses of intestinal bacteria beta-galactosidase enzymes with Set-lacZ cotransfection bhk cell;
(5) the HSV1-lacZ100 seed culture of viruses behind the plaque purifying, in bhk cell, can stablize go down to posterity and in a large number the amplification.
6. the method for generation recombinant herpes simplex virus according to claim 1 is characterized in that, the recombinant virus HSV1-X of acquisition is a reorganization HSV1 virus of having inserted foreign gene X expression cassette in the XbaI site of the genomic UL44 gene of people HSV1.
7. the method for generation recombinant herpes simplex virus according to claim 5, it is characterized in that, wherein cos56-CMV-lacZ-polyA can with cos6, cos28, cos14, the modifier of cos48 forms the new recombinant virus of combination results, reaches the purpose of monitoring newly-generated recombinant virus with the activity of intestinal bacteria beta-galactosidase enzymes.
8. a recombinant virus is characterized in that having inserted foreign gene X expression cassette in the XbaI site of the genomic UL44 gene of people HSV1.
9. recombinant virus according to claim 8 is characterized in that, it is HSV1-LacZ100 (CGMCCNo.0348).
10. claim 8 or 9 described recombinant viruses are as the target virus of anti-herpes simplex virus medicament and other antiviral.
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| EP3113801B1 (en) * | 2014-03-03 | 2020-10-07 | Albert Einstein College of Medicine | Recombinant herpes simplex virus 2 (hsv-2) vaccine vectors |
| CN104946678B (en) * | 2015-06-04 | 2017-12-12 | 中国农业科学院哈尔滨兽医研究所 | Marek's disease virus infectivity recombinant clone system and its construction method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1052896A (en) * | 1989-12-04 | 1991-07-10 | 阿克佐公司 | Recombinant herpesvirus of turkeys and deutero-live vector vaccine thereof |
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| CN1052896A (en) * | 1989-12-04 | 1991-07-10 | 阿克佐公司 | Recombinant herpesvirus of turkeys and deutero-live vector vaccine thereof |
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