CN102533726A - Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease - Google Patents
Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease Download PDFInfo
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Abstract
The invention provides a kit for amplifying a herpes simplex virus 1 (HSV-1) alkaline nuclease gene fragment UL12. The kit comprises a primer pair of which the nucleotide sequences are shown as SEQ ID NO:1-2, and other related reagents. The invention also provides a method for preparing HSV-1 alkaline nuclease by using the kit. By a genetic engineering method, a great amount of HSV-1 alkaline nuclease with high bioactivity is obtained, and the method has important significance for further searching anti-virus medicines and illustrating action mechanisms of the medicines, and lays an experimental basis for constructing medicine screening molecular models for the HSV-1 alkaline nuclease in vitro.
Description
Technical field
The present invention relates to a kind of method that obtains viral protein, be specifically related to a kind ofly, obtain the method for HSV-1 alkalescence nucleicacidase through genetic expression with round pcr amplification HSV-1 alkalescence nuclease gene.
Background technology
Herpes simplex virus I-type (HSV-1) is tunicary dna virus, is the healthy common disease substance of harm humans, and causes recurrent infection and latent infection easily.The HSV-1 infection site is extensive, can cause herpes labialis, genital herpes, encephalitis, keratitis etc., and relevant with cervical cancer and lip cancer.HSV-1 has neurotoxicity, can invade cns, destroy neurocyte.
The alkalescence nucleicacidase is by UL12 genes encoding in the HSV-1 genome, it has 5 '～3 ' exonuclease and endonuclease activity, because of its reaction optimum pH higher, so be considered to alkaline nucleicacidase.It plays an important role in the duplicating of HSV-1, and has research to confirm fracture and the breach of alkaline nucleicacidase in can correct DNA plerosis reproduction process, and can make the viral genome that is in replication status be easy to packing.The HSV-1 two mutants duplicated almost and stops after this encoding sox was knocked out, and explained that alkaline nucleicacidase is that breeding institute is essential in the virosome.Experiment showed, in addition in the non-UL12 genetic flaw virus that produces in the cell of holding, owing to gene unconventionality is difficult to infect new cell.Above-mentioned research proves that alkaline nucleicacidase is very crucial in virus replication and course of infection; For setting forth anti-HSV-1 drug mechanism and setting up a kind of new intervention means thinking is provided, also evidence is provided for alkaline nucleicacidase becomes a kind of new anti-HSV-1 drug screening target spot.Therefore, it is extremely important that purifying obtains a large amount of alkaline nucleicacidases.
Yet because of the UL12 gene fragment is long, GC content is high, is difficult to obtain easily and accurately through the PCR method of routine the UL12 gene fragment of complete sequence, and then obtains large-scale purification and activated alkaline nucleicacidase.Document Purification and Characterization of Herpes Simplex Virus Type 1 Alkaline Exonuclease Expressed in Escherichia coli.JOURNAL OF VIROLOGY.1996 (3): among the 2008-2013, reported the method that obtains alkaline nucleicacidase with gene engineering method: the HindIII-PstI fragment of from the HSV-1 genome, separating 4.3kb; This fragment cloning is made up pHP-AE in PUC19; Through three subclones the most at last target gene fragment insert among the expression plasmid pET-25b (+); Expression plasmid is converted into the intestinal bacteria abduction delivering; The sex change renaturation obtains activated protein.The technical scheme of this method is complicated, and is difficult to obtain a large amount of alkaline nucleicacidases, and cost is high, the industrial applications difficulty.Do not see the commercially available article that alkaline nucleicacidase is arranged yet.
Summary of the invention
In order to overcome above-mentioned defective, the invention provides the method that obtains HSV-1 alkalescence nucleicacidase with genetic engineering technique in a large number.
Therefore, one of the object of the invention provides a kind of test kit of the UL12 gene fragment that is used to increase, comprise the primer of nucleotide sequence shown in SEQ ID NO:1～2 to 1 with other related reagents.This test kit preferably also comprises the T carrier.
Another object of the present invention provides a kind of method that obtains activated alkaline nucleicacidase of expressing, and its step is following:
(1) amplifying target genes: extract the HSV-1 viral genome as template; With the UL12 gene fragment of the nucleotide sequence shown in SEQ ID NO:1～2 as primer amplification HSV-1; Amplification program is: 96 ℃ of preparatory sex change 3min, and 95 ℃ of sex change 40s, 2min30s is extended in 68 ℃～72 ℃ annealing; 31 circulations, 72 ℃ are extended 10min; Glue reclaims the UL12 gene fragment of amplification;
(2) construction recombination plasmid: the UL12 gene fragment that obtains is connected with expression plasmid, obtains recombinant expression plasmid;
(3) abduction delivering: with the recombinant expression plasmid transformed into escherichia coli of step (2) acquisition; The intestinal bacteria that cultivation contains recombinant expression plasmid are copied to it to be in logarithmic phase; Add sec.-propyl 2b-D2 thiogalactoside enzyme (IPTG) abduction delivering; Centrifugal collection thalline;
(4) sex change: the thalline that broken step (3) obtains, centrifugal collecting precipitation with the washings washing precipitation that comprises denaturing agent, obtains protein solution;
(5) renaturation: the protein solution with renaturation solution renaturation step (4) obtains obtains activated protein.
The method of the said amplifying target genes of above-mentioned steps (1) is preferably: extract the HSV-1 viral genome as template; With the UL12 gene fragment of the nucleotide sequence shown in SEQ ID NO:1～2 as primer amplification HSV-1; Amplification program is: 96 ℃ of preparatory sex change 3min, and 95 ℃ of sex change 40s, 2min30s is extended in 68 ℃～72 ℃ annealing; 31 circulations, 72 ℃ are extended 10min; Glue reclaims the UL12 gene fragment of amplification; UL12 gene fragment and T carrier that amplification is obtained are connected to form reorganization T carrier; Be template with this reorganization T carrier again; Further amplification obtains the UL12 target gene fragment of nucleotide sequence shown in SEQ ID NO:3 as primer with the nucleotide sequence shown in SEQ ID NO:1～2, and amplification condition is constant.Said annealing, elongating temperature all are preferably 68 ℃.
Expression plasmid is a pET series plasmid in the above-mentioned steps (2), is preferably pET32a-UL12 or pET28a-UL12 plasmid.
Intestinal bacteria are E.coli BL12 bacterial strain in the above-mentioned steps (3), perhaps are E.coli Rosetta bacterial strain.
The method that sex change is adopted in the above-mentioned steps (4) is:
Thalline with twice washing step of washings A (3) acquisition; Freezing ultrasonication thalline, centrifugal collecting precipitation; With washings B, C, D wash successively, centrifugal, collecting precipitation; With washings E washing precipitation, obtain protein solution;
Described washings A: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl;
Washings B: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 1%NP40 (emulsifying agent NP40 series);
Washings C: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 3%TritonX-100,1mM PMSF;
Washings D: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 0.5%TritonX-100,1.5M Guanidinium hydrochloride, 1mM PMSF;
Washings E: the solution that comprises 100mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 10mM beta-mercaptoethanol, 1mM PMSF and 4～6M Guanidinium hydrochloride.Wherein concentration of guanidine hydrochloride is preferably 4M.
Renaturation solution is for to comprise the solution that 50mM Tris-HCl [pH8.0], 2mM EDTA, 50mMNaCl, 5% glycerine, 1% glycocoll and concentration of guanidine hydrochloride progressively reduce in the above-mentioned steps (5), and said concentration of guanidine hydrochloride is followed successively by 4M, 3M, 2M, 1M and 0M.
Advantage of the present invention: overcome the obstacle of prior art, the fragment length that accurately increased reaches 1881bp, and GC content is higher than 71% UL12 gene fragment, obtains sequence and GeneBank and has announced that sequence homology is up to 99.2%.Obtain having the alkaline nucleicacidase of higher endonuclease and exonuclease activity through construction expression plasmid, abduction delivering, sex change and renaturation series of steps.This has crucial meaning for further illustrating mechanism of drug action, and has established experiment basis for the drug screening molecular model of external structure alkalescence nucleicacidase.
Below, foregoing of the present invention is remake further detailed description through the embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings:
The UL12 gene fragment gel electrophoresis result that amplification obtains among Fig. 1 embodiment 1.Swimming lane M:DNA Marker DL2000; Swimming lane 1:UL12 gene; Swimming lane 2:UL12 gene.
The agarose gel electrophoresis figure of the UL12 gene fragment that the further amplification of Fig. 2 obtains.Swimming lane 1 is DL2, and 000 DNA Marker serves as a mark; Swimming lane 2 is for using
EcoR I/
HinD III cutting purification of Recombinant pMD19 T carrier; Swimming lane 3 is for using
EcoR I/
HinD III cutting purifying pET28a-UL12; Swimming lane 4 is unloaded plasmid pET28a (+), uses
EcoR I/
HinD III handles the back as contrast; Swimming lane 5 is not for there to be enzyme to cut the pET28a-UL12 of processing; Swimming lane 6 be λ-
HinD III digest DNA Marker serves as a mark.
The 1st ~ 80 base pair answered the 259th ~ 180 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 a-1.
The 81st ~ 160 base pair answered the 179th ~ 100 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 a-2.
The 161st ~ 240 base pair answered the 99th ~ 20 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 a-3, and the 241st ~ 259 base pair answered the 19th ~ 1 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 a-4.
The sequence of target gene periphery when the 259th ~ 320 base is for order-checking among Fig. 3 a-4.
The sequence of target gene periphery when the 321st ~ 400 base is for order-checking among Fig. 3 a-5.
The sequence of target gene periphery when the 401st ~ 464 base is for order-checking among Fig. 3 a-6.
The 1st ~ 80 base pair answered the 835th ~ 756 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-1.
The 81st ~ 160 base pair answered the 755th ~ 676 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-2.
The 161st ~ 240 base pair answered the 675th ~ 596 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-3.
The 241st ~ 320 base pair answered the 595th ~ 516 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-4.
The 321st ~ 400 base pair answered the 515th ~ 436 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-5.
The 401st ~ 480 base pair answered the 435th ~ 356 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-6.
The 481st ~ 560 base pair answered the 355th ~ 276 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-7.
The 561st ~ 640 base pair answered the 275th ~ 196 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-8.
The 641st ~ 663 base pair answered the 195th ~ 173 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 b-9.The 1st ~ 80 base pair answered the 774th ~ 853 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-1.
The 81st ~ 160 base pair answered the 854th ~ 933 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-2.
The 161st ~ 240 base pair answered the 934th ~ 1013 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-3.The 241st ~ 320 base pair answered the 1014th ~ 1093 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-4.
The 321st ~ 400 base pair answered the 1094th ~ 1173 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-5.
The 401st ~ 480 base pair answered the 1174th ~ 1253 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-6.
The 481st ~ 560 base pair answered the 1254th ~ 1333 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-7.
The 561st ~ 640 base pair answered the 1334th ~ 1413 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-8.
The 641st ~ 703 base pair answered the 1414th ~ 1476 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 c-9.
The 1st ~ 80 base pair answered the 1446th ~ 1525 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-1.
The 81st ~ 160 base pair answered the 1526th ~ 1605 base figure in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-2.
The 161st ~ 240 base pair answered the 1606th ~ 1685 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-3.
The 241st ~ 320 base pair answered the 1686th ~ 1765 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-4.
The 321st ~ 400 base pair answered the 1766th ~ 1845 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-5, the sequence of target gene periphery when its 437 ~ 480 bases are order-checking.
The 401st ~ 436 base pair answered the 1846th ~ 1881 base in the HSV-1 17 strain gene orders shown in the SEQ ID NO:4 among Fig. 3 d-6, the sequence of target gene periphery when its 481st ~ 538 base is order-checking.
The sequence of target gene periphery when the 481st ~ 538 base is for order-checking among Fig. 3 d-7.
Fig. 4-1 is 1-400 base of UL12 gene fragment.
Fig. 4-2 is 401-800 base of UL12 gene fragment.
Fig. 4-3 is 801-1200 base of UL12 gene fragment.
Fig. 4-4 is 1201-1600 base of UL12 gene fragment.
Fig. 4-5 is 1601-1881 base of UL12 gene fragment.
The different abduction delivering times of Fig. 5 are to the figure as a result that influences of expressing quantity.Swimming lane 3 to swimming lane 8 corresponding induction times are 10h, 8h, 6h, 4h, 3h, 0h.Swimming lane 2 and swimming lane 10 conduct contrasts do not add IPTG, expression time 10h.Protein molecular weight standard (low) at swimming lane 1 and swimming lane 9, serves as a mark.
Fig. 6 pET32a-UL12's
E.coliBL21 transformant expression of results figure.Swimming lane 1 is pET32a-UL12's
E .coliTotal bacterial protein after BL21 transformant IPTG induces; Swimming lane 2 is pET32a's
E .coliTotal bacterial protein after BL21 transformant IPTG induces; Swimming lane 3 is pET32a-UL12's
E .coliInoculum supernatant after BL21 transformant IPTG induces; Swimming lane 4 is pET32a's
E .coliInoculum supernatant after BL21 transformant IPTG induces; Swimming lane 5 is protein molecular weight standard (low), serves as a mark; Swimming lane 6 is induced back pET32a-UL12's for IPTG
E .coliThe supernatant of BL21 transformant behind ultrasonic degradation; Swimming lane 7 is induced back pET32a's for IPTG
E .coliThe supernatant of BL21 transformant behind ultrasonic degradation; Swimming lane 8 is pET32a-UL12's
E .coliThe BL21 transformant is induced after the deposition behind the ultrasonic degradation through IPTG; Swimming lane 9 is pET32a's
E .coliThe BL21 transformant is induced after the deposition behind the ultrasonic degradation through IPTG.
Fig. 7 pET28a-UL12's
E.coliBL21 transformant expression of results figure.Swimming lane 1 is protein molecular weight standard (low), serves as a mark; Swimming lane 2 is pET28a's
E .coliTotal bacterial protein after BL21 transformant IPTG induces; Swimming lane 3 is pET28a-UL12's
E .coliTotal bacterial protein after BL21 transformant IPTG induces; Swimming lane 4 is pET28a's
E .coliThe BL21 transformant is induced after the deposition behind the ultrasonic degradation through IPTG; Swimming lane 5 is pET28a-UL12's
E .coliThe BL21 transformant is induced after the deposition behind the ultrasonic degradation through IPTG; Swimming lane 6 is induced back pET28a's for IPTG
E .coliThe supernatant of BL21 transformant behind ultrasonic degradation; Swimming lane 7 is induced back pET28a-UL12's for IPTG
E .coliThe supernatant of BL21 transformant behind ultrasonic degradation.
After the different washings denaturing treatment of Fig. 8, the target protein existence form is identified figure.Swimming lane 1 is the supernatant after handling with denaturing agent A and denaturing agent C; Swimming lane 2 is through the supernatant after denaturing agent A and the denaturing agent B processing; Swimming lane 3 is the depositions after handling with denaturing agent A, B, C, D and E, and the concentration of Guanidinium hydrochloride is 4M among the denaturing agent E; Swimming lane 4 is the depositions after handling with denaturing agent A, B, C, D and E, and the concentration of Guanidinium hydrochloride is 6M among the denaturing agent E; Swimming lane 5 is protein molecular weight standard (low), serves as a mark; Swimming lane 6 is through the deposition after denaturing agent A and the denaturing agent B processing; Swimming lane 7 is the depositions after handling with denaturing agent A and denaturing agent C.
The figure as a result that Fig. 9 protein vigor is measured.
E.coliBL21 with
E.coliRecombinant protein after the renaturation that Rosetta produces carries out the result of endonuclease reaction to the pcDNA3.0 plasmid: swimming lane M:
λ HindIII digest DNA Marker; Swimming lane 1:pcDNA3.0 contrast; After the renaturation
E .coliRecombinant protein that Rosetta expresses and pcDNA3.0 react on 37 ℃ and hatch 5min (swimming lane 2), 20 min (swimming lane 4), 40 min (swimming lane 6), 60 min (swimming lane 8); After the renaturation
E .coliRecombinant protein that BL21 expresses and pcDNA3.0 react on 37 ℃ and hatch 5min (swimming lane 3), 20 min (swimming lane 5), 40 min (swimming lane 7), 60 min (swimming lane 9).
The figure as a result that Figure 10 protein vigor is measured, albumen does
E.coliRecombinant protein after the renaturation that Rosetta produces.M:Marker λ-Hind III; Swimming lane 1～4: be respectively the recombinant protein liquid of pcDNA3.1 plasmid after and handle 60min, 40min, 20min, 5min with renaturation; Swimming lane 5～8: be respectively the recombinant protein liquid of linear UL12 gene after and handle 5min, 20min, 40min, 60min with renaturation.
Embodiment
Experiment material and instrument
Used experiment material and instrument in following examples remove special instruction, are commercially available purchase.
(1) material:
Standard herpes simplex virus I-type (HSV-1) SM44 strain is available from Chinese biological goods calibrating institute;
The Vero cell is purchased in the Huaxi Hospital Attached to Sichuan Univ Ministry of Health and is transplanted engineering and transplantation immunity key lab;
E.coli BL12 (DE3) plysS bacterial strain, E.coli Rosetta (DE3), prokaryotic expression carrier pET32a (+), pET28a (+) are available from Novagen company;
High-fidelity Taq enzyme, T4 dna ligase, DL2; 000 DNA Marker, λ-Hind III digest DNAMarker, protein molecular weight standard (low), restriction enzyme EcoR I and Hind III, E.coli JM109 bacterial strain, pMD19-T carrier, dNTPs, LA Taq enzyme, GC buffer, EDTA, NP-40, TritonX-100 purchase the TaKaRa company in Dalian;
Nucleic acid sequencing is accomplished by Dalian TaKaRa company;
Guanidinium hydrochloride, isopropylthio-β-D galactoside (IPTG) are U.S. AMRESCO product;
Plasmid extraction kit, glue reclaim test kit, available from U.S. OMEGA company;
Primer or the nucleotide sequence of nucleotide sequence shown in SEQ ID NO:1～2, synthetic by Shanghai SangonBiotech company;
PMSF (PMSF) is available from Sigma company;
DdH
2O, MgCl
2, ethanol, PBS damping fluid, Tris-HCl damping fluid, NaCl, glycerine, glycocoll, SDS-PAGE and agarose gel electrophoresis various conventional reagent.
(2) instrument:
The PCR appearance is available from U.S. Thermo company;
Ultraviolet-visible pectrophotometer is available from U.S. Bio-Rad company;
Electro-heating standing-temperature cultivator is the product of Qingdao Haier;
The ultrafiltration pipe is available from U.S. MILLIPORE company;
Whizzer is available from German Eppendorf company;
The ultrasonic cell disintegration appearance.
(1) extract viral genome: with the RPMI 1640 culture medium culturing vero cells that contain 10% foetal calf serum, inoculation HSV-1 treats cytopathy, and promptly CPE reaches ++ +～++ ++, harvested cell is with the full genome of viral DNA extraction agent box extracting HSV-1;
The nucleotide sequence of the UL12 gene fragment of (2) announcing according to GenBank, with PRIMER5.0 software design primer, primer sequence is shown in SEQ ID NO:1～2;
(3) amplifying target genes: the full genome of HSV-1 that obtains with extraction is a template, is primer with nucleotide sequence primer shown in SEQ ID NO:1～2 to 1, amplifying target genes UL12 gene fragment:
Reaction system:
Reaction conditions: 96 ℃ of preparatory sex change 3min, 95 ℃ of sex change 40s, 2min30s is extended in 68 ℃ of annealing, 31 circulations, 72 ℃ are extended 10min.
(4) product reclaims: 0.8% conventional agarose gel electrophoresis separates the PCR product, reclaims test kit with glue and reclaims amplified production.
The gel electrophoresis result shows that the dna fragmentation that amplification obtains is 1881bp, and is identical with known UL12 gene fragment size.(result sees Fig. 1)
(1) preparation template: the UL12 gene fragment that under the effect of T4 dna ligase, embodiment 1 is obtained is connected with the pMD-19T carrier, is built into reorganization pMD19T carrier.
(2) amplifying target genes: the reorganization pMD19T carrier that is connected with the UL12 gene fragment that obtains with step (1) is a template, with nucleotide sequence shown in SEQ ID NO:1～2 primer to being primer amplification goal gene UL12 gene fragment:
Reaction system:
Reaction conditions: 96 ℃ of preparatory sex change 3min, 95 ℃ of sex change 40s, 2min30s is extended in 68 ℃ of annealing, 31 circulations, 72 ℃ are extended 10min.
(3) product reclaims: 0.8% conventional agarose gel electrophoresis separates the PCR product, reclaims test kit with glue and reclaims amplified production.
(4) result detects: glue is reclaimed the UL12 gene fragment that obtains be connected with the pMD-19T carrier, be built into reorganization pMD19T carrier, the UL12 gene fragment that agarose gel electrophoresis and order-checking check amplification obtain.
Result such as Fig. 2, Fig. 3 and Fig. 4.Fig. 2 shows: the gel electrophoresis result shows that the dna fragmentation that amplification obtains is approximately 1.9kb, and is identical with known UL12 gene fragment size.Fig. 3 is the colored oscillogram of nucleotide sequence order-checking, proves through the inventive method amplification to have obtained the UL12 gene fragment of nucleotide sequence shown in SEQ ID NO:3.UL12 gene fragment order and the standard international strain UL12 gene fragment order comparing result of nucleotide sequence shown in SEQ ID NO:3 that Fig. 4 obtains for the present invention's amplification; Can know from Fig. 4; The UL12 actual measurement sequence of nucleotide sequence shown in SEQ ID NO:3 is 1866/1881=99.20% with the homology of the UL12 standard world strain sequence of nucleotide sequence shown in SEQ ID NO:4, proves the present invention's UL12 target gene fragment that increased exactly.
The expression of embodiment 3 alkaline nucleicacidases
(1) construction of recombinant expression plasmid
The UL12 gene fragment that embodiment 1 or embodiment 2 are obtained under EcoRI, the effect of HindIII restriction endonuclease and pET28a (+) plasmid or pET32a (+) be connected to form pET28a-UL12 recombinant expression plasmid or pET32a-UL12 recombinant expression plasmid; The recombinant plasmid that makes up contains T7 rna polymerase promoter; Control proteic expression; And include 2 His labels, can be used for purifying protein.
(2) recombinant expression plasmid abduction delivering in e. coli bl21
The pET28a-UL12 recombinant expression plasmid that step (1) is obtained passes through CaCl
2Mediated transformation is to the E.coliBL21 competent cell, and 37 ℃ are incubated on the LB culture plate that contains 50 μ g/ml kalamycins, and the 220r/min jolting is spent the night;
Picking obtains the escherichia coli cloning of recombinant expression plasmid;
The escherichia coli cloning direct inoculation is contained to 50ml in the LB substratum of 50 μ g/ml kalamycins, at 37 ℃ of shake-flask culture, is 0.4～0.6 up to the OD600 value;
Add final concentration and induce, shake bottle after 1～10 hour, centrifugal collection thalline at 37 ℃ for 0.5mM IPTG.
(3) recombinant expression plasmid abduction delivering in intestinal bacteria Rosetta
The pET32a-UL12 recombinant expression plasmid that step (1) is obtained passes through CaCl
2Mediated transformation is to the E.coliRosetta competent cell, and 37 ℃ are incubated on the paraxin LB culture plate that contains 30 μ g/ml penbritins and 34 μ g/ml 220r/min jolting incubated overnight;
Picking obtains the escherichia coli cloning of recombinant expression plasmid;
The escherichia coli cloning direct inoculation is contained to 50ml in the LB substratum of paraxin of 30 μ g/ml penbritins and 34 μ g/ml, at 37 ℃ of shake-flask culture, up to OD
600Value is 0.4～0.6;
Add final concentration and induce, shake bottle after 1～10 hour, centrifugal collection thalline at 37 ℃ for 0.5mM IPTG.
To induce the result to carry out agarose gel electrophoresis, result such as Fig. 5 show that the target protein band weakens successively in the swimming lane 3～8, and just along with the minimizing of induction time, its expressing quantity reduces gradually.Therefore, confirm that optimum induction time is 10h.
The evaluation of albumen existence form
With perhaps (3) gained thalline of step (2), it is resuspended to add 500 μ l PBS solution, and 50 μ l bacterium liquid keep sample, and all the other bacterium liquid carry out ultrasonication, and (3min, 400w), (4 ℃, 12000rmp 15min), collects respectively and goes up cleer and peaceful deposition frozen centrifugation, precipitates resuspended with PBS.Bacterium liquid keeps sample, goes up cleer and peaceful deposition and respectively adds equal-volume 2 * SDS sample loading buffer, and 100 ℃ of heating 5min sex change, sampling find that with the 12%SDS-PAGE check target protein is present in the inclusion body.Result such as Fig. 6 and shown in Figure 7.Known alkaline nucleicacidase size is 86kDa, and Fig. 6 shows the E.coli BL21 transformant expression of results of pET32a-UL12; Fig. 7 shows the E.coliBL21 transformant expression of results of pET28a-UL12.
Fig. 6 swimming lane 1 shows the total bacterial protein after the E.coli BL21 transformant IPTG of pET32a-UL12 induces; Swimming lane 6 shows that IPTG induces the supernatant of E.coli BL21 transformant behind ultrasonic degradation of back pET32a-UL12; The E.coli BL21 transformant of swimming lane 8 demonstration pET32a-UL12 is induced after the deposition behind the ultrasonic degradation (inclusion body) through IPTG.There is size to be the purpose band of 86kD in swimming lane 1 and the swimming lane 8, then do not have at other swimming lanes.The result proves; The intestinal bacteria that target protein is present in recombinant plasmid transform in daughter bacteria liquid and the deposition behind ultrasonic degradation; That is to say; Target protein exists with the form of inclusion body, with the Quantity One image analysis software that Bio-Rad company provides the target protein expression amount of Fig. 6 the 1st swimming lane is analyzed, and the target protein expression amount accounts for the 17.4%. of bacterial protein
Fig. 7 swimming lane 3 shows the total bacterial protein after the E.coli BL21 transformant IPTG of pET28a-UL12 induces; The E.coli BL21 transformant of swimming lane 5 demonstration pET28a-UL12 is induced after the deposition behind the ultrasonic degradation (inclusion body) through IPTG; The E.coli BL21 transformant that swimming lane 7 shows pET28a-UL12 through ultrasonic degradation after the supernatant of IPTG after inducing.There is size to be the purpose band of 86kD in swimming lane 3 and the swimming lane 5, then do not have at other swimming lanes.The result proves; The intestinal bacteria that target protein is present in recombinant plasmid transform in daughter bacteria liquid and the deposition behind ultrasonic degradation; That is to say; Target protein exists with the form of inclusion body, with the Quantity One image analysis software that Bio-Rad company provides the target protein expression amount of Fig. 7 the 3rd swimming lane is analyzed, and the target protein expression amount accounts for total expression amount more than 28%.
(4) sex change
1) with step (2) perhaps (3) gained thalline wash twice with 5ml washings A (50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl);
2) under 4 ℃, collect bacterium on ice bath through the 4min supersound process, smudge cells obtains bacterium liquid;
3) under 4 ℃, with the supersound washing bacterium liquid of 5ml washings B (50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 1%NP40, i.e. emulsifying agent NP40 series);
4) under 4 ℃, the centrifugal 15min of 12000rpm, collecting precipitation;
5) under 4 ℃, precipitate with 5ml washings C (50mM Tris-HCl [pH8.0], 2mM EDTA, 100mMNaCl, 3%TritonX-100,1mM PMSF, i.e. methanesulfonyl fluoride) supersound washing;
6) the centrifugal 15min of 12000rpm, collecting precipitation;
7) under 4 ℃, precipitate with 5ml washings D (50mM Tris-HCl [pH8.0], 2mM EDTA, 100mMNaCl, 0.5%TritonX-100,1.5M Guanidinium hydrochloride and 1mM PMSF) supersound washing;
8) the centrifugal 15min of 12000rpm, collecting precipitation;
9) with 5ml washings E (4M or 6M Guanidinium hydrochloride, 100mM Tris-HCl [pH8.0], 2mMEDTA, 100mM NaCl, 10mM β mercaptoethanol and 1mM PMSF) washing precipitation;
10) collect supernatant-80 ℃ of preservations, protein concn is measured with the Bio-Rad spectrophotometer.
The sex change optimal conditions is as shown in Figure 8, and when the concentration of guanidine hydrochloride among the washings E was 4M, the deposition after the processing did not almost have target protein, and target protein is dissolved in washings, is very beneficial for renaturation.
(5) renaturation
Get the solution of the target protein that 1mL step (4) sex change obtains, it is diluted to 0.1-1mg/ml with renaturation solution (Guanidinium hydrochloride that 50mMTris-HCl [pH8.0], 2mM EDTA, 50Mm NaCl, 5% glycerine, 1% glycocoll and concentration are successively decreased, the concentration of Guanidinium hydrochloride is followed successively by 4M, 3M, 2M, 1M and 0M); Renaturation in dialysis tubing; The renaturation time is 24h, at last with ultrafiltration pipe (4500rpm, 10min; 4 ℃) centrifugal collection recombinant protein solution, place-20 ℃ of refrigerators and preserve.
Two, test for protein:
1, aminoacid sequence comparison
Through calculating; (a top row is the international strain sequence of standard as follows in the aminoacid sequence comparison of the HSV-1 alkalescence nucleicacidase that the aminoacid sequence of above-mentioned target protein and GenBank announce; Below one row be the aminoacid sequence of target protein, underscore is partly represented the amino acid variation part):
1 MESTVGPACPPGRTVTKR
PWALAEDTPRGPDSPPKRPRPNSLPLTTTFRPLPPPPQTTSA
||||||||||||||||||.|||||||||||||||||||||||||||||||||||||||||
1 MESTVGPACPPGRTVTKR
SWALAEDTPRGPDSPPKRPRPNSLPLTTTFRPLPPPPQTTSA
61?VDPSSHSPDNPPRDQHATDTADEKPRAASPALSDASGPPTPDIPLSPGGTHARDPDADPD
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
61 VDPSSHSPDNPPRDQHATDTADEKPRAASPALSDASGPPTPDIPLSPGGTHARDPDADPD
121 SPDLDSMWS
ASVIPNALPSHILAETFERHLRGLLRGVRAPLAIGPLWARLDYLCSL
DVVL
|||||||||.||||||||||||||||||||||||||||||||||||||||||||||.|||
121 SPDLDSMWS
TSVIPNALPSHILAETFERHLRGLLRGVRAPLAIGPLWARLDYLCSL
AVVL
181 EEAGMVDRGLGRHLWRLTRRGPPAAADAVAPRPLMGFYEAATQNQADCQLWALLRRGLTT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
181 EEAGMVDRGLGRHLWRLTRRGPPAAADAVAPRPLMGFYEAATQNQADCQLWALLRRGLTT
241 ASTLRWGPQGPCFSPQWLKHNASLRPDVQSSAVMFGRVNEPTARSLLFRYCVGRADDGGE
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
241 ASTLRWGPQGPCFSPQWLKHNASLRPDVQSSAVMFGRVNEPTARSLLFRYCVGRADDGGE
301 AGADTRRFIFHEP
SDLAEENVHTCGVLMDGHTGMVGASL
DILVCPRD
IHGYLAPVPKTPL
|||||||||||||：||||||||||||||||||||||||：||||||.||||||||||||
301 AGADTRRFIFHEP
GDLAEENVHTCGVLMDGHTGMVGASL
GILVCPRD
THGYLAPVPKTPL
361 AFYEVKCRAKYAFDPMDPSDPTASAYEDLMAHRSPEA
FRAFIRSIPKPSVRYFAPGRVPG
|||||||||||||||||||||||||||||||||||||：|||||||||||||||||||||
361 AFYEVKCRAKYAFDPMDPSDPTASAYEDLMAHRSPEA
LRAFIRSIPKPSVRYFAPGRVPG
421 PEEALVTQDQAWSEAHASGEKRRCSAADRALVELNSGVVSEVLLFGAPDLGR
HTISPVSW
||||||||||||||||||||||||||||||||||||||||||||||||||||：||||||
421 PEEALVTQDQAWSEAHASGEKRRCSAADRALVELNSGVVSEVLLFGAPDLGR
QTISPVSW
481 SSGDLVRREPVFANPRHPNFKQILVQGYVLDSHFPDCPPHPHLVTFIGRHRTSAEEGVTF
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
481 SSGDLVRREPVFANPRHPNFKQILVQGYVLDSHFPDCPPHPHLVTFIGRHRTSAEEGVTF
541 RLEDGAGALGAAGPSKASILPNQAVPIALIITPVRIDPEIYKAIQRSSRLAFDDTLAELW
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
541 RLEDGAGALGAAGPSKASILPNQAVPIALIITPVRIDPEIYKAIQRSSRLAFDDTLAELW
601 ASRSPG
SGPAAAETTSSSPTTGRSSR
||||||.|||||||||||||||||||
601 ASRSPG
PGPAAAETTSSSPTTGRSSR
The HSV-1 alkalescence nucleicacidase aminoacid sequence similarity that the aminoacid sequence of target protein and GenBank announce is 98.56% (617/626); Degree of variation is 1.44% (9/626); Through further calculating, above-mentioned variation is not at the critical sites of protein structure, and is little to the protein-active influence.
2, vigor detects
Get the recombinant protein solution dilution for preparing to 0.12mg/ml, as substrate, carry out enzyme assay with pcDNA3.0 plasmid, pcDNA3.1 plasmid or linear UL12 gene fragment.
Reaction system is:
Reaction conditions: 37 ℃, 5/20/40/60min.
5 μ l reaction solutions were taken out in the back between reacting phase was seasonable, with 1 μ l, 6 * Loading Buffer termination reaction, were put in-20 ℃ of refrigerators, and the agarose gel electrophoresis check is done in sampling.
Result such as Fig. 9 and shown in Figure 10; The albumen of the present invention preparation can effectively cut cyclic plasmid pcDNA3.0 and pcDNA3.1 and the cleaved products oligonucleotide is degraded; Explain that it has endonuclease activity and 5 prime excision enzyme activity; The linear UL12 gene fragment of also can effectively degrading further specifies the albumen that the present invention prepares and has exonuclease activity, and the conclusion that draws with aforementioned aminoacid sequence comparison between calculation results is consistent.
Above-mentioned experiment proves; We express the target protein that obtains from the UL12 gene fragment that simplexvirus HSV-1 amplification obtains through gene engineering method, and character such as its molecular weight, enzyme work are consistent with HSV-1 alkalescence nucleicacidase; Though the gene fragment of the present invention's amplification is at nucleotide sequence; And expressed proteins on the aminoacid sequence with Genbank on the standard sequence announced have few meristic variation, but these variations do not cause and express the albumen changes on structure and activity that obtain, in the scope of genetic engineering permission; Therefore, we think that the target protein that the present invention obtains is exactly alkaline nucleicacidase.
In sum, the method for preparing HSV-1 alkalescence nucleicacidase provided by the invention, easy and simple to handle, with low cost, and the HSV-1 for preparing alkalescence nuclease is higher and output is big, for its suitability for industrialized production provides possibility.
Claims (12)
1. test kit of herpes simplex virus I-type alkalescence nuclease gene fragment UL12 that is used to increase is characterized in that: it comprise the primer of nucleotide sequence shown in SEQ ID NO:1～2 to other related reagents.
2. test kit according to claim 1 is characterized in that: also comprise the T carrier.
3. test kit according to claim 2 is characterized in that: said T carrier is the pMD19-T carrier.
4. a method for preparing herpes simplex virus I-type alkalescence nucleicacidase is characterized in that: comprise the steps:
(1) amplifying target genes: extract the herpes simplex virus I-type viral genome as template; With the UL12 gene fragment of the nucleotide sequence shown in SEQ ID NO:1～2 as primer amplification HSV-1; Amplification program is: 96 ℃ of preparatory sex change 3min, and 95 ℃ of sex change 40s, 2min30s is extended in 68 ℃～72 ℃ annealing; 31 circulations, 72 ℃ are extended 10min; Glue reclaims the UL12 gene fragment of amplification;
(2) construction recombination plasmid: the UL12 gene fragment that obtains is connected with expression plasmid, obtains recombinant expression plasmid;
(3) abduction delivering: with the recombinant expression plasmid transformed into escherichia coli of step (2) acquisition; The intestinal bacteria that cultivation contains recombinant expression plasmid are copied to it to be in logarithmic phase; Adding the enzyme induction of sec.-propyl 2b-D2 thiogalactoside expresses; Centrifugal collection thalline;
(4) sex change: the thalline that broken step (3) obtains, centrifugal collecting precipitation with the washings washing precipitation that comprises denaturing agent, obtains protein solution;
(5) renaturation: the protein solution with renaturation solution renaturation step (4) obtains obtains activated protein.
5. method according to claim 4; It is characterized in that: the method for the said amplifying target genes of step (1) is: extract the herpes simplex virus I-type viral genome as template, with the UL12 gene fragment of the nucleotide sequence shown in SEQ IDNO:1～2 as primer amplification HSV-1, amplification program is: 96 ℃ of preparatory sex change 3min; 95 ℃ of sex change 40s; 2min30s is extended in 68 ℃～72 ℃ annealing, 31 circulations, and 72 ℃ are extended 10min; Glue reclaims the UL12 gene fragment of amplification; UL12 gene fragment and T carrier that amplification is obtained are connected to form reorganization T carrier; Be template with this reorganization T carrier again; Further amplification obtains the UL12 target gene fragment of nucleotide sequence shown in SEQ ID NO:3 as primer with the nucleotide sequence shown in SEQ ID NO:1～2, and amplification condition is constant.
6. according to claim 4 or 5 described methods, it is characterized in that: the said annealing of said step (1), elongating temperature are 68 ℃.
7. method according to claim 4 is characterized in that: expression plasmid is a pET series plasmid in the said step (2).
8. method according to claim 7 is characterized in that: said expression plasmid is pET32a-UL12 or pET28a-UL12 plasmid.
9. method according to claim 4 is characterized in that: intestinal bacteria are E.coli BL12 bacterial strain in the said step (3), perhaps are E.coli Rosetta bacterial strain.
10. method according to claim 4 is characterized in that: the method that sex change is adopted in the said step (4) is:
Thalline with twice washing step of washings A (3) acquisition; Freezing ultrasonication thalline, centrifugal collecting precipitation; With washings B, C, D wash successively, centrifugal, collecting precipitation; With washings E washing precipitation, obtain protein solution;
Described washings A: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mMNaCl;
Washings B: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 1%NP40;
Washings C: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 3%TritonX-100,1mM PMSF;
Washings D: the solution that comprises 50mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 0.5%TritonX-100,1.5M Guanidinium hydrochloride, 1mM PMSF;
Washings E: the solution that comprises 100mM Tris-HCl [pH8.0], 2mM EDTA, 100mM NaCl, 10mM beta-mercaptoethanol, 1mM PMSF and 4～6M Guanidinium hydrochloride.
11. method according to claim 10 is characterized in that: concentration of guanidine hydrochloride is 4M among the said washings E.
12. method according to claim 4; It is characterized in that: renaturation solution is for to comprise the solution that 50mM Tris-HCl [pH8.0], 2mM EDTA, 50mM NaCl, 5% glycerine, 1% glycocoll and concentration of guanidine hydrochloride progressively reduce in the said step (5), and said concentration of guanidine hydrochloride is followed successively by 4M, 3M, 2M, 1M and 0M.
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| Title |
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| 《British Journal of Pharmacology》 20080616 C-Y Hsiang1 and T-Y Ho Emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type 1 yields in cell cultures 227-235 4-12 第155卷, 第2期 * |
| C-Y HSIANG1 AND T-Y HO: "Emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type 1 yields in cell cultures", 《BRITISH JOURNAL OF PHARMACOLOGY》 * |
| J C. BRONSTEIN AND P C. WEBER: "Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli", 《JOURNAL OF VIROLOGY》 * |
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