CN103993032A - Method for preparing recombinant poliovirus-like particles - Google Patents
Method for preparing recombinant poliovirus-like particles Download PDFInfo
- Publication number
- CN103993032A CN103993032A CN201410185984.5A CN201410185984A CN103993032A CN 103993032 A CN103993032 A CN 103993032A CN 201410185984 A CN201410185984 A CN 201410185984A CN 103993032 A CN103993032 A CN 103993032A
- Authority
- CN
- China
- Prior art keywords
- spinal cord
- disease virus
- protein expression
- grey matter
- recombinant expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biology, especially relates to a method for preparing recombinant poliovirus-like particles, and further provides related recombinant expression vectors. The invention provides the recombinant expression vectors of the recombination poliovirus-like particles. The recombinant expression vectors contain a P1 protein expression box and a 3C protein expression box, wherein the P1 protein expression box and the 3C protein expression box are located in the same recombinant expression vector or are respectively located in two recombinant expression vectors. The virus-like particles obtained by the method are similar to a poliovirus in the structure and can be used as a vaccine antigen; and the antigen is composed of a plurality of monomers, has huge molecular weight, enables a granular repetitive structure to be observed under an electron microscope, and has stronger immunogenicity.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method of preparing recombining spinal cord grey matter disease virus sample particle, and relevant recombinant expression vector is further provided.
Background technology
Poliomyelitis (Poliomyelitis) is to infect the caused acute infectious intestinal disease of limb paralysis as master of take by poliovirus (Poliovirus), mainly pass through fecal-oral transmission, affect five years old following children, be commonly called as poliomyelitis, children's majority that paralysis disease occurs leaves limping, disables all the life.Poliomyelitis, without specific treatment method, can only be usingd vaccine as prevention at present.
Poliovirus belongs to Picornaviridae (Picornaviridae) enterovirus genus (Enteroviruse), includes sub-thread positive chain RNA gene, and protein coat is the three-dimensional symmetrical structure of icosahedron, without coating.Virogene of poliomyelitis group RNA is about 7.5kb.Gene element is 5' end non-coding region, polyprotein coding region, 3' end non-coding region and 3' end Poly (A) tail four parts.Wherein polyprotein coding region coding produces a polyprotein precursor, is divided into P1, P2He P3 district; P1 district can produce capsid protein VP1, VP2, VP3 and VP4 through protease hydrolysis, and P2He P3 district hydrolyzable produces proteolytic enzyme, rna replicon enzyme and for identifying other albumen of cell, regulatory gene.The VP1 of 5 copies, VP2, VP3 and VP4 have formed pentamer, and 12 pentamers form icosahedron nucleocapsid.Poliovirus has three serotypes, i.e. I, II, III type, various between without cross-immune reaction.
Poliomyelitis live vaccine (oral) (oral polio vaccine, OPV) was succeeded in developing the listing in the U.S. in 1958, extended to afterwards the whole world.The advantage of OPV comprises that inoculation simple (oral), expense are low, can produce stable Intestinal Mucosal Immunization, effectively blocks Polio virus propagation etc.But along with the propelling of whole world elimination ridge ash target, the effect of Biosafety in containment Polio virus is propagated is more and more important.Take the relevant paralysis ridge ash of the vaccine producing after OPV (Vaccine associated paralytic poliomyelitis, VAPP) case, Vaccine-derived poliovirus (Vaccine-derived poliovirus, VDPV) and the circulation of caused Vaccine-derived poliovirus (Circulating vaccine-derived polioviruses, cVDPVs) and immune deficiency Vaccine-derived poliovirus (Immunodeficient related vaccine-derived poliovirus, iVDPV) case more and more receive publicity.
The poliomyelitis vaccine,Salk that the people such as Salk succeed in developing (Inactivated poliovirus vaccine, IPV), for the production of street strain comprise I type Mahoney strain, II type MEF strain, III type Saukett strain.The immunogenicity of IPV and security are all very desirable, can effectively prevent the outburst of ridge ash.And IPV does not exist and causes VAPP, cVDPVs equivalent risk, and its herd immunity is better than OPV.Although the cost ratio OPV of every IPV is expensive, consider conventional ridge ash immunity and booster immunization, if now still using 148 countries of OPV to use IPV immunization instead, also can not increase the weight of financial burden.
The IPV that reality is used in the world is at present traditional ridge ash inactivated vaccine (Conventional inactivated poliovirus vaccine, cIPV) of producing by the Wild poliovirus strain with formalin-inactivated.After the Poliomyelitis Eradication virus of the whole world, stringent regulations Ji Hui street strain more, so the exploitation of Poliomyelitis Vaccine of new generation is extremely urgent.
Can spontaneous assembling assembly virus-like particle after the virus structural proteins of a lot of viruses are recombinant expressed, antigenicity and the euvirus of this virus-like particle are as broad as long, but lack viral nucleic acid thereby do not have infectivity.Virus-like particle has become an open available strategy of virus disease vaccine safely and effectively, and therefore, for the virus-like particle of poliovirus, effectively preparing is the effective way of producing OPV.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the present invention is to provide a kind of method of preparing recombining spinal cord grey matter disease virus sample particle, and the recombinant expression vector of relevant recombining spinal cord grey matter disease virus sample particle is further provided, for solving the problems of the prior art.
For achieving the above object and other relevant objects, first aspect present invention provides a kind of recombinant expression vector of recombining spinal cord grey matter disease virus sample particle, described recombinant expression vector includes P1 protein expression box and 3C protein expression box, and described P1 protein expression box and 3C protein expression box are arranged in same recombinant expression vector or lay respectively at two recombinant expression vectors.
Preferably, the aminoacid sequence of described P1 albumen is as shown in SEQ ID NO:1; Described 3C Argine Monohydrochloride sequence is as shown in SEQ ID NO:2.
Preferably, in described P1 protein expression box, the expressed sequence that contains promotor and be subject to the P1 albumen of promoter regulation.P1 protein expression box can be expressed P1 albumen.
Preferred, in described P1 protein expression box, the expressed sequence of P1 albumen is as shown in SEQ ID NO:3.5 ' and 3 ' end of the expressed sequence of described P1 albumen is respectively equipped with the restriction enzyme site sequence of BstBI enzyme and KpnI enzyme.
Preferred, described promotor is AOX1 promotor (expressed sequence of regulation and control P1 albumen).AOX1 promoter sequence specifically can be referring to pPICZA, B, & C Pichia Vectors specification sheets and carrier information (Lifetechnologies, article No. V190-20).
Preferred, in P1 protein expression box, the AOX1 (TT) of take is transcription termination sequence.AOX1 (TT) Transcription Termination region sequence specifically can be referring to pPICZA, B, & C Pichia Vectors specification sheets and carrier information (Lifetechnologies, article No. V190-20).
Further preferably, in described P1 protein expression box, with 5 ' to 3 ' direction, comprise expressed sequence, AOX1 (TT) transcription termination sequence of AOX1 promotor, P1 albumen.
Preferably, in described 3C protein expression box, the expressed sequence that contains promotor and be subject to the 3C albumen of promoter regulation.3C protein expression box can be expressed 3C albumen.
Preferred, in described 3C protein expression box, the expressed sequence of 3C albumen is as shown in SEQ ID NO:4.5 ' and 3 ' end of the expressed sequence of described 3C albumen is respectively equipped with the restriction enzyme site sequence of BstBI enzyme and KpnI enzyme.
Preferred, described promotor is truncation type AOX1 promotor (expressed sequence of regulation and control 3C albumen), and the sequence of described truncation type AOX1 promotor is as shown in SEQ ID NO:5.
Preferred, in 3C protein expression box, the AOX1 (TT) of take is transcription termination sequence.
Further preferably, in described 3C protein expression box, with 5 ' to 3 ' direction, comprise expressed sequence, AOX1 (TT) transcription termination sequence of truncation type AOX1 promotor, 3C albumen.
Preferably, described P1 protein expression box and 3C protein expression box are arranged in same recombinant expression vector, are preferably Yeast expression carrier.
When P1 protein expression box and 3C protein expression box are arranged in same recombinant expression vector, can successively arrange P1 protein expression box of the present invention and 3C protein expression box with 5 ' to 3 ' direction, also can arrange conversely.
Preferred, described recombinant expression vector obtains after inserting pPICZB by P1 protein expression box and 3C protein expression box.
Preferred, P1 protein expression box and 3C protein expression box are replaced the 7-1411 base of pPICZB.
Further preferred, the concrete complete sequence of the 7-1411 base of described P1 protein expression box and 3C protein expression box replacement pPICZB is as shown in SEQ ID NO:8.In described SEQ ID NO:8, include P1 protein expression box and 3C protein expression box.
Second aspect present invention provides the preparation method of the recombinant expression vector of described recombining spinal cord grey matter disease virus sample particle, by building the carrier for expression of eukaryon of regulating and expressing P1 albumen and 3C albumen simultaneously, prepares.
Preferably, the recombinant expression vector of described recombining spinal cord grey matter disease virus sample particle is prepared by the carrier for expression of eukaryon that AOX1 promoter regulation is expressed and 3C albumen is expressed by truncation type AOX1 promoter regulation by building P1 albumen.
Preferred, the recombinant expression vector of described recombining spinal cord grey matter disease virus sample particle is to be obtained by the transformation of pPICZB plasmid, and its concrete preparation method comprises the steps:
1) structure of pPICZdA plasmid: build truncation type AOX1 promotor Insert Fragment SEQ ID NO:5, truncation type AOX1 promotor Insert Fragment is replaced to AOX1 promoter fragment in pPICZB, obtain plasmid pPICZdA;
2) structure of 3C-pPICZdA plasmid: the expressed sequence SEQ ID NO:4 of 3C albumen is inserted to the multiple clone site of pPICZdA, obtain 3C-pPICZdA; In 3C-pPICZdA, contain 3C expression cassette;
3) structure of P1-pPICZ plasmid: the expressed sequence SEQ ID NO:3 of P1 albumen is inserted to the multiple clone site of pPICZ, obtain P1-pPICZ;
4) structure of P13C-pPICZ plasmid: enzyme combination treatment P1-pPICZ obtains P1 expression cassette, and P1 expression cassette is inserted to 3C-pPICZdA, obtain P13C-pPICZ, before or after on position is positioned at 3C expression cassette.
Preferred, in described step 1, the construction process of pPICZdA plasmid is specially: build two ends, left and right and be respectively BglII restriction enzyme site and BstBI restriction enzyme site, the truncation type AOX1 promotor Insert Fragment that truncation type AOX1 promotor SEQ ID NO:5 is contained in centre; Then BglII is combined to enzyme with BstBI and cut processing truncation type AOX1 promotor Insert Fragment and pPICZB, both connect rear acquisition plasmid pPICZdA.
Preferred, in described step 2, the construction process of 3C-pPICZdA plasmid is specially: expressed sequence SEQ ID NO:4 and the pPICZdA of BstBI enzyme and KpnI enzyme combination treatment 3C albumen, both connect rear acquisition 3C-pPICZdA.
Preferred, in described step 3, the construction process of P1-pPICZ plasmid is specially: expressed sequence SEQ ID NO:3 and the pPICZ of BstBI enzyme and KpnI enzyme combination treatment P1 albumen, both connect rear acquisition P1-pPICZ.
Preferred, in described step 4, the construction process of P13C-pPICZ plasmid is specially: BamHI enzyme BglII enzyme combination treatment P1-pPICZ obtains P1 expression cassette, re-uses BamHI enzyme and processes 3C-pPICZdA, after the 3C-pPICZdA after enzyme is processed is connected with P1 expression cassette, obtains P13C-pPICZ.
Further preferred, in described step 4, before the 3C-pPICZdA after enzyme is processed is connected with P1 expression cassette, also use alkaline phosphatase treatment to use BamHI enzyme to process the product of 3C-pPICZdA gained.
Third aspect present invention discloses a kind of engineering bacteria of recombining spinal cord grey matter disease virus sample particle, for including the recombinant bacterial strain of the recombinant expression vector of described recombining spinal cord grey matter disease virus sample particle.
Preferably, described recombinant bacterial strain is yeast.
Preferred, described yeast is selected from yeast saccharomyces cerevisiae (Saccharomycescerevisiae), multiple-shaped nuohan inferior yeast (Hansenula polymorpha), pichia pastoris phaff (Pichiapastoris), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces Lactis Vickers yeast (Kluyveromyces lactis), and one or more the combination in chestnut wine fission yeast (Schizosaccharomyces pombe).
Fourth aspect present invention provides a kind of method of preparing recombining spinal cord grey matter disease virus sample particle, comprises the steps:
1), by the recombinant expression vector transformed yeast cell of described recombining spinal cord grey matter disease virus sample particle, obtain expressing the recombinant yeast cell of P1 gene and 3C gene;
2) use the recombinant yeast cell of step 1 gained, by yeast expression system, express P1 and 3C, after purifying, obtain poliovirus sample particle.
Those skilled in the art can rule of thumb, screen the recombinant yeast cell of step 1 gained, and after screening, bacterial classification be activated.
Preferably, the concrete steps of described screening are: by bacterium liquid coating YPD dull and stereotyped (Zeocin), after 30 ℃ of incubated overnight, picking is partly cloned line YPD dull and stereotyped (Zeocin), is inverted for 30 ℃ and cultivates 2-3 days, and the good strain of picking growing state is as fermented bacterium.
Preferably, the concrete steps of described activation are: 30 ℃ of shaking culture in constant-temperature shaking culture device, substratum is YPD substratum, records the OD of activation solution
600when 1～2 scope, stop cultivating, microscopy is transferred activation solution into YPD seed culture medium after without miscellaneous bacteria, and in constant-temperature shaking culture device, 30 ℃ of overnight incubation obtain the seed culture fluid of activation.
Preferably, described yeast is selected from yeast saccharomyces cerevisiae (Saccharomycescerevisiae), multiple-shaped nuohan inferior yeast (Hansenula polymorpha), pichia pastoris phaff (Pichiapastoris), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces Lactis Vickers yeast (Kluyveromyces lactis), and one or more the selection in chestnut wine fission yeast (Schizosaccharomyces pombe).
Those skilled in the art can rule of thumb use described recombinant yeast cell, adopt suitable yeast expression system to express P1 and 3C.
Adopt BSM substratum, initial temperature is set as 30 ℃, initial pH5～6, DO value 100%, add PTM1 trace salt, the initial multiplicative stage cultivates 20～24 hours, oxygen dissolving value 20～40%, when thalline weight in wet base reaches approximately 60～90g/L, add glycerine, maintain oxygen dissolving value 20～40%, add 4～8 hours, when detection thalline weight in wet base is increased to 100～200g/L, stop feed supplement; Maintain oxygen dissolving value higher than 20～40%, by Temperature Setting, at 30 ℃, pH value is controlled and is adjusted to 6 ± 0.05, starts to add methanol induction, and the speed of adding of methyl alcohol is 50～100mL/min, induces 40 hours fermentation ends.
Preferably, the concrete grammar of described purifying is: fermented liquid is carried out abandoning supernatant after solid-liquid separation, collect bacterium mud; Bacterium mud is added brokenly to bacterium buffered soln, after fully stirring, obtain thalline suspension, the broken thalline suspension of high pressure obtains broken bacterium liquid; To break the centrifugation of bacterium liquid, collecting supernatant liquor and in supernatant liquor, adding solid ammonium sulfate (final concentration is 16.4g-22.6g/100ml) to saturation ratio is 30～40%, and after placement is spent the night, recentrifuge is separated, and supernatant discarded retains throw out; After adding buffered soln, fully stir, centrifugation is also collected centrifugal supernatant sample again; Density gradient centrifugation, collects required gradient sample and carries out membrane filtration, obtains the virus-like particle protein sample of purifying.
When those skilled in the art can rule of thumb judge density gradient centrifugation, the gradient at target sample place.
Preferred, the medium that described density gradient centrifugation is used is sucrose.
Further preferred, the concrete grammar of described density gradient centrifugation is: in centrifuge tube, slowly add successively from the bottom to top 60% sucrose solution, 50% sucrose solution, 40% sucrose solution, 30% sucrose solution, described centrifugal supernatant sample, whiteruss, the sample in centrifugal rear collection 50-60% saccharose gradient.
Fifth aspect present invention provides recombinant expression vector, the engineering bacteria of described recombining spinal cord grey matter disease virus sample particle, and the purposes of the method for preparing recombining spinal cord grey matter disease virus sample particle in Poliomyelitis Vaccine preparation field.
The present invention is according to the DNA sequence dna of pichia spp codon-bias composite coding P1 and 3C albumen, two genes of synthetic gained are connected on yeast expression vector, obtain expressing the double expression plasmid (non-secretion expression plasmid) of P1 precursor protein and HRV 3CP simultaneously.Recombinant plasmid is incorporated in pichia spp genome by gene engineering method, through Large-scale Screening, obtains high expression level bacterial strain, take this recombinant strains as seed, and fermentation expression obtains P1 and 3C albumen.3C enzyme is cracked into VP1, VP3 and VP0 in yeast cell inside by P1 precursor protein, and three kinds of capsid protein subunits are self-assembled into as virus-like particle (VLPs).By the purification process such as column chromatography, obtain high purity, form homogeneous, VLPs that proterties is stable, the VLPs after purifying adsorbs suitable adjuvant (as aluminium adjuvant) becomes recombiant vaccine preparation.The recombiant vaccine preparation that contains poliovirus sample particle prepared by the inventive method through experimentation on animals checking has good immunogenicity.
Beneficial effect of the present invention: the present invention, by using different promotors, regulates the expression amount of viral capsid proteins P1 and protease 3 C, the shearing of regulation and control virus-like particle precursor protein P1, thereby the virus-like particle of acquisition form homogeneous.In the virus-like particle structure that the inventive method obtains, be similar to poliovirus, can be used as vaccine antigen, this antigen consists of a plurality of monomers, molecular weight is huge, under Electronic Speculum, can be observed particulate state repetitive structure, there is stronger immunogenicity (the immune group of 0.01 μ g dosage has certain positive rate of rotation, and the immune group mouse of 0.1 μ g dosage sun rate of rotation can reach 83.3%); And vaccine antigen prepared by the present invention, containing virulent genetic material, does not have potential infection potential, it is vaccine candidate antigen more suitably.
Accompanying drawing explanation
Fig. 1: P13C-pPICZdual plasmid map.
Fig. 2: P13C-pPICZdual enzyme is cut evaluation figure:
Swimming lane 1:P13C-pPICZdual plasmid (enzyme is not cut);
Swimming lane 2:P13C-pPICZdual plasmid (BglII+SacI);
Swimming lane 3:250bp DNA ladder (TAKARA).
Fig. 3: recombining spinal cord grey matter disease virus sample particle Western-Blot detected result:
The empty Host Strains abduction delivering of swimming lane 1:X-33 result;
Swimming lane 2: standard protein molecular weight Marker;
Swimming lane 3:P13C-X33 abduction delivering result.
Fig. 4: recombining spinal cord grey matter disease virus sample particle electron microscopic observation (30000 times).
Embodiment
Below, by specific specific examples explanation embodiments of the present invention, those skilled in the art can understand other advantages of the present invention and effect easily by the disclosed content of this specification sheets.The present invention can also be implemented or be applied by other different embodiment, and the every details in this specification sheets also can be based on different viewpoints and application, carries out various modifications or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term using in the embodiment of the present invention is in order to describe specific specific embodiments, rather than in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, unless should be understood that the present invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology of using in the present invention and scientific terminology and those skilled in the art of the present technique understand conventionally.The concrete grammar using in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, can also with to the method described in the embodiment of the present invention, equipment, material is similar or any method, equipment and the material of the prior art that is equal to are realized the present invention.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt the routine techniques of molecular biology, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and the association area of the art routine.These technology are existing perfect explanation in existing document, specifically can be referring to MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
The preparation of embodiment 1 virion
1. the selection of expressing gene and the optimization design of codon
The DNA sequence dna of coding poliomyelitis I type Mahoney strain P1 albumen and 3C albumen is with reference to GeneBank:V01149.1, and the aminoacid sequence of P1 albumen is as shown in SEQ ID NO:1, and the aminoacid sequence of 3C albumen is as shown in SEQ ID NO:2.
Wild-type DNA sequence dna to the 3C albumen shown in P1 albumen shown in SEQ ID NO:1 and SEQ ID NO:2 of encoding in I type Mahoney strain is transformed, codon adopts the higher codon of frequency of utilization in pichia spp as far as possible, has avoided affecting transcription factor land, tumor-necrosis factor glycoproteins and the RNA higher structure of expression simultaneously.After codon optimized, obtain encoding the recombination sequence of P1 albumen as shown in SEQ ID NO:3, the recombination sequence of coding 3C albumen is as shown in SEQ ID NO:4.
2.P13C-pPICZdual the structure of recombinant expression vector
Design 5 ' end and 3 ' end contain respectively the P1 protein expression sequence of BstBI restriction enzyme site and KpnI restriction enzyme site and carry out full gene and synthesize, and obtain the nucleotide fragments of sequence shown in SEQ ID NO:3; Design 5 ' end and 3 ' end do not contain the 3C protein expression sequence of BstBI restriction enzyme site and KpnI restriction enzyme site and carry out full gene and synthesize, and obtain the nucleotide fragments of sequence shown in SEQ ID NO:4.The transformation of P13C-pPICZdual plasmid, from pPICZ plasmid (Life Technologies), is summarized as follows:
Adopt the primer of sequence shown in SEQ ID NO:6-7 to increase from pPICZB plasmid and obtain truncation type AOX1 promotor Insert Fragment.BglII+BstBI associating enzyme is cut and is processed truncation type AOX1 promotor (SEQ ID NO:5) Insert Fragment and pPICZB, and two fragments obtain pPICZdA after connecting.Adopt the mixed enzyme enzyme of BstBI enzyme and KpnI enzyme to cut processing P1 protein expression sequence (SEQ ID NO:3) and pPICZB plasmid, two fragments obtain P1-pPICZ after connecting.Adopt the mixed enzyme enzyme of BstBI enzyme and KpnI enzyme to cut 3C protein expression sequence and pPICZdA plasmid, two fragments obtain 3C-pPICZdA after connecting.Adopt the mixed enzyme enzyme of BglII enzyme and BamHI enzyme to cut P1-pPICZ plasmid, BamHI enzyme enzyme is cut 3C-pPICZdA, (the BamHI+BglII enzyme buffer3 that cuts use is alkaline phosphatase optimal reaction buffer to use alkaline phosphatase again, without changing or adding other damping fluids) process 3C-pPICZdA, two fragments obtain P13C-pPICZdual (Fig. 1) after connecting.P13C-pPICZdual enzyme is cut evaluation figure as shown in Figure 2.
Primer 1:5 '-CCCC
aGATCTcATTCCAATTCCTTCT-3 ' (SEQ ID NO:6)
Primer 2: 5 '-GGCCCCGT
tTCGAAtAATTAGTTG-3 ' (SEQ ID NO:7)
3.P13C-pPICZdual recombinant strains builds
Pichi strain X33 is purchased from Life Technologies.By the P13C-pPICZdual plasmid of recombination to construct, by the linearizing of SacI enzyme, electricity turns pichia spp, and it is 1500V that electricity turns condition, 120 Ω, 50 μ F.Electricity turns rear bacterium liquid coating YPD dull and stereotyped (200 μ g/mlZeocin), and 30 ℃ of inversions of spending the night are cultivated.Picking is partly cloned line YPD dull and stereotyped (1500 μ g/ml Zeocin), is inverted for 30 ℃ and cultivates 2-3 days, and the good strain of picking growing state is as fermented bacterium, called after P13C-X33.
4. the fermentor cultivation of recombinant protein
Adopt YPD substratum to activate recombinant strains P13C-X33,200rpm in constant-temperature shaking culture device, 30 ℃ of overnight incubation, shaking culture records the OD of activation solution about 20 hours
600scope 1～2, stops cultivating.
Microscopy is got 1ml activation solution after without miscellaneous bacteria and is transferred into 500ml seed culture medium (YPD), in constant-temperature shaking culture device with 200rpm, 30 ℃ of overnight incubation.Microscopy is pressed 1:15 inoculation fermentation tank after without miscellaneous bacteria.
Use BIOENGINEERING RALF Basic fermentor tank, fermentation adopts basic salt culture medium BSM.Fermentation initial temperature is set as 30 ℃, initial pH5～6, rotating speed 300rpm, air flow 0.5vvm, DO value 100%, adds PTM1 trace salt (PTM1 mother liquor formula: copper sulfate 0.6%, sodium iodide 0.008%, manganous sulfate 0.3%, Sodium orthomolybdate 0.02%, boric acid 0.002%, cobalt chloride 0.12%, ferric sulfate 6.5%, zinc chloride 2.0%, sulfuric acid 0.5%, vitamin H 0.02% (mass volume ratio), in fermentation, PTM1 mother liquid concentration is 12ml/L).The initial multiplicative stage cultivates about 20～24 hours, by regulating mixing speed, air flow quantity, tank pressure and adding pure oxygen, maintains oxygen dissolving value 20～40%.When thalline weight in wet base reaches approximately 60～90g/L, add 50% glycerine solution (mass percent concentration) 200-300 gram.Maintain oxygen dissolving value 20～40%, add 4～8 hours.When detection thalline weight in wet base is increased to 100～200g/L, stop feed supplement.By Temperature Setting, at 30 ℃, pH value is controlled and is adjusted to 6 ± 0.05, starts to add methanol induction, and the speed of adding of methyl alcohol is 50～100mL/min.Maintain oxygen dissolving value higher than 20～40%, Temperature Setting is at 30 ℃, and it is 6 ± 0.05 that pH value is controlled, and induces 40 hours fermentation ends.
Adopt refrigerated centrifuge fermented liquid to be carried out to solid-liquid separation, centrifugal rotational speed 8000rpm, centrifugal 10 minutes.After centrifugal end, abandon supernatant, collect bacterium mud, the bacterium mud of results is placed in-20 ℃ of refrigerators frozen.
5. virus-like particle purifying
Get 200g fermentation thalline, broken buffered soln (50mM PB, 0.2M NaCl, 0.5%Tween-80, pH7.0) 800ml bacterium for that adds 4 ℃ of precoolings is stirred to completely and mixes, acquisition thalline suspension on magnetic stirring apparatus.Adopt the thalline suspension of the broken preparation of high pressure homogenizer (ATS AH-100B) high pressure, regulate homogenizing valve, controlling broken bacterium pressure is 1200bar.Repeat above high pressure shattering process 3～4 times, collect broken bacterium liquid.
Adopt the broken bacterium liquid of high speed freezing centrifuge (ThermoFisher lynx4000) centrifugal clarification, the 10000rpm that imposes a condition, 30min, 8 ℃, centrifugation, collects the supernatant liquor after centrifugal.In supernatant liquor, adding solid ammonium sulfate (final concentration is 16.4g-22.6g/100ml) to saturation ratio is 30～40%, after 4 ℃ of placements are spent the night, press 10000rpm, 30min, 8 ℃ of condition centrifugations for the second time, supernatant discarded retains throw out, add buffered soln (50mM PB pH7.0) 100ml, stir 2～4h.Finally again press 10000rpm, 30min, 8 ℃ of condition centrifugations, retain supernatant for next step separation.
In centrifuge tube, slowly add successively from the bottom to top 60% sucrose solution 1ml, 50% sucrose solution 1ml, 40% sucrose solution 1ml, 30% sucrose solution 1ml, above-mentioned centrifugal supernatant sample 0.5ml, whiteruss 0.5ml.Use Beckman Coulter Optima Max tabletop ultracentrifuge, MLS-50 rotor, at 4 ℃, centrifugal 4 hours of 40000rpm.Collect sample in 50%-60% saccharose gradient, with 100KD super filter tube (Millipore company), remove sucrose and concentrate 20 times, use 0.45um membrane filtration, obtain the virus-like particle protein sample of purifying.
6. virus-like particle Property Identification
The virus-like particle protein sample of purifying detects through Western-blot, can with polyclonal antibody (the how anti-employing of the present embodiment CalBioreagents company product of poliovirus VP1, article No. P046, the two prosperous Bioisystech Co., Ltd of anti-employing Beijing ancient cooking vessel states, the anti-goat IgG of article No. SH-0071 horseradish enzyme labelling rabbit) be specific color reaction (Fig. 3).The virus-like particle protein sample of purifying, adds 20g/L phosphoric acid tungsten dye liquor dyeing 0.5-1 minute, naturally dries rear electron microscopic observation, and Electronic Speculum (Philips) is observed and presented virus-like particle (Fig. 4).
The antigen Performance Detection of embodiment 2 virions
1. virus sample particle vaccines preparation
The poliovirus sample granule protein Al adsorption adjuvant (50 μ g albumen: 500 μ g, aluminium adjuvant) that purifying is obtained, prepares and has immunogenic poliovirus vaccine.
2. the immunogenic mensuration of poliovirus sample particle
The SPF level BALB/c mouse of choosing 6～8 week age, is divided into 4 groups, every group of 6 mouse.Wherein 1 group of mouse carries out immunity (as negative control group) with the damping fluid (50mM PB pH7.0) that contains aluminium adjuvant, other 3 groups of each immunizing doses be 1 μ g/ only, 0.1 μ g/ only, 0.01 μ g/ only (immunizing dose refers to the quality of virus-like particle protein amount herein), respectively at subcutaneous injection immunity in the 0th, 14 days, immunity twice altogether, blood sampling in two weeks after immunity for the second time.By the blood collecting, after 4 ℃ of placements are spent the night, the centrifugal 10min of 5000g, draws supernatant, obtains mouse polyvalent antibody, deposits in-20 ℃, and detects the positive rate of rotation of this mouse polyvalent antibody, and concrete grammar is as follows:
With coating buffer (0.1M pH9.8NaHCO
3) poliovirus sample granule protein to the 1 μ g/ml of dilution purifying, in each hole of enzyme plate, respectively add 0.1ml, 4 ℃ of coated spending the night.Remove coating buffer, wash plate.Every hole adds 0.3ml confining liquid (5% skim-milk+PBST) in 37 ℃ of insulations 2 hours.Remove coating buffer, every hole adds each 0.1ml of tested serum with 1:1000 dilution with dilution buffer liquid (2% skim-milk+PBST), in 37 ℃ of insulations 1 hour, removes serum solution, washes plate.Then to every hole, add goat anti-mouse igg (the ancient cooking vessel state with the HRP mark of 1:5000 dilution with dilution buffer liquid (2% skim-milk+PBST), article No. SH-0011 horseradish enzyme labelling goat anti-mouse igg) each 0.1ml, 37 ℃ of insulations were removed enzyme mark liquid after 0.5 hour, washed plate; Then in every hole, add 0.1ml DAB nitrite ion, the effect of room temperature lucifuge adds 2M H after 10 minutes
2sO
40.05ml stop buffer termination reaction, and measure OD with enzyme mark photoelectric color comparator
450value, the positive rate of rotation result of three test set is as shown in table 1.The OD of the tested serum antibody of the negative contrast of Cutoff value
450the mean value of value adds 3 times of standard deviations, OD
450the mouse that value is greater than Cutoff value is judged to be the positive, OD
450the mouse that value is less than Cutoff value is judged to be feminine gender.
Table 1 poliovirus sample particle sun rate of rotation result
| Group | 1 μ g group | 0.1 μ g group | 0.01 μ g group |
| Sun rate of rotation | 100％ | 83.3％ | 16.7％ |
Experimental result explanation, the immune group mouse sun rate of rotation of 0.1 μ g dosage can reach 83.3%, illustrates that the poliovirus vaccine that aforesaid method prepares has high immunogenicity.
In sum, the present invention has effectively overcome various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all can, under spirit of the present invention and category, modify or change above-described embodiment.Therefore, such as in affiliated technical field, have and conventionally know that the knowledgeable, not departing from all equivalence modifications that complete under disclosed spirit and technological thought or changing, must be contained by claim of the present invention.
Claims (20)
1. the recombinant expression vector of a recombining spinal cord grey matter disease virus sample particle, described recombinant expression vector includes P1 protein expression box and 3C protein expression box, and described P1 protein expression box and 3C protein expression box are arranged in same recombinant expression vector or lay respectively at two recombinant expression vectors.
2. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 1, is characterized in that, the aminoacid sequence of described P1 albumen is as shown in SEQ ID NO:1; Described 3C Argine Monohydrochloride sequence is as shown in SEQ ID NO:2.
3. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 1, is characterized in that, in described P1 protein expression box, and the expressed sequence that contains promotor and be subject to the P1 albumen of promoter regulation.
4. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 3, it is characterized in that, in described P1 protein expression box, the expressed sequence of P1 albumen is as shown in SEQ ID NO:3, described promotor is AOX1 promotor, and the AOX1 (TT) of take is transcription termination sequence.
5. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 4, it is characterized in that, in described P1 protein expression box, with 5 ' to 3 ' direction, comprise expressed sequence, AOX1 (TT) transcription termination sequence of AOX1 promotor, P1 albumen.
6. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 1, is characterized in that, in described 3C protein expression box, and the expressed sequence that contains promotor and be subject to the 3C albumen of promoter regulation.
7. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 6, it is characterized in that, in described 3C protein expression box, the expressed sequence of 3C albumen is as shown in SEQ ID NO:4, described promotor is truncation type AOX1 promotor, the sequence of described truncation type AOX1 promotor is as shown in SEQ ID NO:5, and the AOX1 (TT) of take is transcription termination sequence.
8. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 7, it is characterized in that, in described 3C protein expression box, with 5 ' to 3 ' direction, comprise expressed sequence, AOX1 (TT) transcription termination sequence of truncation type AOX1 promotor, 3C albumen.
9. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 1, it is characterized in that, described P1 protein expression box and 3C protein expression box are arranged in same Yeast expression carrier, and described recombinant expression vector obtains after inserting pPICZB by P1 protein expression box and 3C protein expression box.
10. the recombinant expression vector of a kind of recombining spinal cord grey matter disease virus sample particle as claimed in claim 9, is characterized in that, P1 protein expression box and 3C protein expression box are replaced the 7-1411 base of pPICZB.
The recombinant expression vector of 11. a kind of recombining spinal cord grey matter disease virus sample particles as claimed in claim 10, it is characterized in that, the concrete complete sequence of the 7-1411 base of described P1 protein expression box and 3C protein expression box replacement pPICZB is as shown in SEQID NO:8.
The preparation method of the recombinant expression vector of the recombining spinal cord grey matter disease virus sample particle as described in 12. claims as arbitrary in claim 1-11, the recombinant expression vector of described recombining spinal cord grey matter disease virus sample particle is to be obtained by the transformation of pPICZB plasmid, and its concrete preparation method comprises the steps:
1) structure of pPICZdA plasmid: build truncation type AOX1 promotor Insert Fragment SEQ ID NO:5, truncation type AOX1 promotor Insert Fragment is replaced to AOX1 promoter fragment in pPICZB, obtain plasmid pPICZdA;
2) structure of 3C-pPICZdA plasmid: the expressed sequence SEQ ID NO:4 of 3C albumen is inserted to the multiple clone site of pPICZdA, obtain 3C-pPICZdA; In 3C-pPICZdA, contain 3C expression cassette;
3) structure of P1-pPICZ plasmid: the expressed sequence SEQ ID NO:3 of P1 albumen is inserted to the multiple clone site of pPICZ, obtain P1-pPICZ;
4) structure of P13C-pPICZ plasmid: enzyme combination treatment P1-pPICZ obtains P1 expression cassette, and P1 expression cassette is inserted to 3C-pPICZdA, obtain P13C-pPICZ.
13. preparation methods as claimed in claim 12, it is characterized in that, in described step 1, the construction process of pPICZdA plasmid is specially: build two ends, left and right and be respectively BglII restriction enzyme site and BstBI restriction enzyme site, the truncation type AOX1 promotor Insert Fragment that truncation type AOX1 promotor SEQ ID NO:5 is contained in centre; Then BglII is combined to enzyme with BstBI and cut processing truncation type AOX1 promotor Insert Fragment and pPICZB, both connect rear acquisition plasmid pPICZdA.
14. preparation methods as claimed in claim 12, it is characterized in that, in described step 2, the construction process of 3C-pPICZdA plasmid is specially: expressed sequence SEQ ID NO:4 and the pPICZdA of BstBI enzyme and KpnI enzyme combination treatment 3C albumen, both connect rear acquisition 3C-pPICZdA.
15. preparation methods as claimed in claim 12, it is characterized in that, in described step 3, the construction process of P1-pPICZ plasmid is specially: expressed sequence SEQ ID NO:3 and the pPICZ of BstBI enzyme and KpnI enzyme combination treatment P1 albumen, both connect rear acquisition P1-pPICZ.
16. preparation methods as claimed in claim 12, it is characterized in that, in described step 4, the construction process of P13C-pPICZ plasmid is specially: BamHI enzyme BglII enzyme combination treatment P1-pPICZ obtains P1 expression cassette, re-use BamHI enzyme and process 3C-pPICZdA, after the 3C-pPICZdA after enzyme is processed is connected with P1 expression cassette, obtain P13C-pPICZ.
The engineering bacteria of 17. 1 kinds of recombining spinal cord grey matter disease virus sample particles, for including the recombinant bacterial strain of the recombinant expression vector of the recombining spinal cord grey matter disease virus sample particle as described in claim as arbitrary in claim 1-11.
18. 1 kinds of methods of preparing recombining spinal cord grey matter disease virus sample particle, comprise the steps:
1), by the recombinant expression vector transformed yeast cell of the recombining spinal cord grey matter disease virus sample particle as described in claim as arbitrary in claim 1-11, obtain expressing the recombinant yeast cell of P1 gene and 3C gene;
2) by yeast expression system, express P1 and 3C, obtain poliovirus sample particle.
19. a kind of methods of preparing recombining spinal cord grey matter disease virus sample particle as claimed in claim 18, it is characterized in that, described yeast is selected from yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast, pichia pastoris phaff, Kluyveromyces fragilis, Kluyveromyces Lactis Vickers yeast, and one or more the selection in chestnut wine fission yeast.
Recombinant expression vector, the engineering bacteria as claimed in claim 17 of the recombining spinal cord grey matter disease virus sample particle as described in 20. claims as arbitrary in claim 1-11, and the purposes of the method for preparing recombining spinal cord grey matter disease virus sample particle as described in claim as arbitrary in claim 18-19 in Poliomyelitis Vaccine preparation field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410185984.5A CN103993032A (en) | 2014-05-05 | 2014-05-05 | Method for preparing recombinant poliovirus-like particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410185984.5A CN103993032A (en) | 2014-05-05 | 2014-05-05 | Method for preparing recombinant poliovirus-like particles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103993032A true CN103993032A (en) | 2014-08-20 |
Family
ID=51307363
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410185984.5A Pending CN103993032A (en) | 2014-05-05 | 2014-05-05 | Method for preparing recombinant poliovirus-like particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103993032A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802923A (en) * | 2016-04-14 | 2016-07-27 | 中国科学院生物物理研究所 | Recombinant poliomyelitis virus-like particles and preparation method and application thereof |
| CN108130333A (en) * | 2016-12-01 | 2018-06-08 | 上海泽润生物科技有限公司 | I virus-like particle of recombinant poliovirus |
| CN117089576A (en) * | 2023-10-20 | 2023-11-21 | 浙江迪福润丝生物科技有限公司 | Method for screening plasmid and PV protease inhibitor and evaluating drug effect |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876688A (en) * | 2012-10-15 | 2013-01-16 | 北京工业大学 | Method for preparing recombinant polio I virus-like particles |
-
2014
- 2014-05-05 CN CN201410185984.5A patent/CN103993032A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876688A (en) * | 2012-10-15 | 2013-01-16 | 北京工业大学 | Method for preparing recombinant polio I virus-like particles |
Non-Patent Citations (1)
| Title |
|---|
| 胡世界等: "巴斯德毕赤酵母表达系统及其高水平表达策略", 《生物技术》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802923A (en) * | 2016-04-14 | 2016-07-27 | 中国科学院生物物理研究所 | Recombinant poliomyelitis virus-like particles and preparation method and application thereof |
| CN108130333A (en) * | 2016-12-01 | 2018-06-08 | 上海泽润生物科技有限公司 | I virus-like particle of recombinant poliovirus |
| CN117089576A (en) * | 2023-10-20 | 2023-11-21 | 浙江迪福润丝生物科技有限公司 | Method for screening plasmid and PV protease inhibitor and evaluating drug effect |
| CN117089576B (en) * | 2023-10-20 | 2024-02-06 | 浙江迪福润丝生物科技有限公司 | Method for screening plasmid and PV protease inhibitor and evaluating drug effect |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103255163B (en) | A kind of EV71 virus-like particle and preparation method thereof and application | |
| CN103436553B (en) | A kind of method preparing restructuring coxsackie virus A 16-type virus-like particle | |
| CN103525855B (en) | A kind of method preparing restructuring enterovirus EV 71 virus-like particle | |
| CN104404074B (en) | The preparation method of hoof-and-mouth disease virus capsid protein series connection coexpression and virus-like particle | |
| CN108546302B (en) | Composite multi-epitope expression cassette, recombinant virus composed of same and application thereof | |
| CN102559615B (en) | EV71 vaccine preparation method and the vaccine prepared by the method | |
| CN113388587B (en) | Recombinant bovine nodavirus expressing bovine viral diarrhea E2 gene and application thereof | |
| CN104611358A (en) | Method for preparing Norovirus virus-like particles in escherichia coli | |
| CN105002146A (en) | RHVT (recombinant Herpesvirus of Turkey)-H9HA (H9 hemagglutinin) and construction method thereof | |
| CN104745606A (en) | Coxsackie A16 type virus-like particles | |
| CN106367400A (en) | Variation strain for porcine epidemic diarrhea virus and isolated culturing method and application thereof | |
| WO2023246621A1 (en) | Coxsackievirus a10 strain and use thereof | |
| CN106692964A (en) | Serum type 4 fowl adenovirus inactivated vaccine and preparation method thereof | |
| CN109689862A (en) | Recombinate purifying and its vaccine preparation method of EV71 virus-like particle | |
| CN103993032A (en) | Method for preparing recombinant poliovirus-like particles | |
| CN105349562A (en) | Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain | |
| CN106318955A (en) | Recombinant adenovirus expressing human enterovirus 71 capsid protein and vaccine prepared from same and application thereof | |
| CN102680699B (en) | ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection | |
| CN106754765A (en) | A kind of NDV sample particle, preparation method and applications | |
| CN110124025A (en) | A kind of bird flu and 4 type bigeminy genetic engineering subunit vaccine of aviadenovirus and preparation method thereof | |
| CN103333849A (en) | Staphylococcus aureus mutant strain, and preparation method and applications thereof | |
| CN104725503A (en) | Fugu rubripes nervous necrosis virus capsid protein egg yolk antibody and application thereof | |
| CN105396129A (en) | Inactivated vaccine produced through poliomyelitis attenuated strains | |
| CN108315306A (en) | One plant height fertility swine fever virus and its construction method | |
| CN101979502A (en) | Recombinant brucella expressing VP1 gene of O-type foot-and-mouth disease virus and method for producing vaccines thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140820 |