CN102586295B - Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof - Google Patents
Influenza virus H1N1 subtype neuraminidase as well as gene, inhibitor screening model and application thereof Download PDFInfo
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Abstract
The invention provides influenza A-type influenza virus neuraminidase (NA), a gene and a preparation method thereof as well as a construction method of a screening model of an influenza virus neuraminidase inhibitor. Particularly, influenza A-type influenza virus H1N1 subtype neuraminidase is secretly expressed by pichia pastoris to obtain the neuraminidase with biological activity, and particularly, the truncated neuraminidases of 82 amino acids at N end of an NA full-length sequence is removed, so that an operation process of the NA prepared by totivirus, which is complicated, consumes time and has a safety problem, is avoided. According to the invention, the A-type influenza virus H1N1 subtype neuraminidase inhibitor screening model is also constructed by utilizing the neuraminidase; and the influenza virus H1N1 subtype neuraminidase has the advantages of definite mechanism of drug action, less material consumption, quick screening speed, high sensitivity, easy realization of high throughput screening and the like.
Description
Technical field
The invention belongs to anti-influenza virus medicament screening field, be specifically related to a kind of neuraminidase and gene thereof of influenza virus H1N1 hypotype and utilize this enzyme to build its inhibitor screening model and application.
Background technology
Influenza virus for a long time serious threat always the mankind's health, in recent years its H5N1 and H1N1 hypotype in succession break out the demand of the whole world to anti-influenza virus medicament that strengthened.The medicine of the treatment influenza of FDA license at present mainly contains four kinds: amantadine (Amantadine), Rimantadine (Rimantadine), Oseltamivir (Oseltamivir) and zanamivir (Zanamivir).
Along with the continuous increase of resistance case, the mankind are being faced with the baptism of influenza virus large-scale outbreak, the research and development of novel anti-influenza virus medicament are imperative, and this makes design synthesize or from compound library, screen new type nerve propylhomoserin enzyme inhibitors becomes focus and the difficult point of current research.In the research of Development of New Drugs, it is one of vital link that searching and discovery have bioactive lead compound, and it is Main Means and the approach of finding lead compound that a large amount of compounds are carried out to screening active ingredients.In the multiple screening model of current influenza neuraminidase (Neuraminidase, NA) inhibitor, neuraminidase is mainly prepared by totivirus, and preparation process is more loaded down with trivial details consuming time, and has safety problem.The medicaments sifting model of molecular level has that material usage is few, mechanism of drug action is relatively clearer and more definite, can realize the features such as large-scale screening, has become the main method of current drug screening.
Up to the present the model Large-scale Screening anti-influenza virus medicament that, there is no the neuraminidase structure of the Influenza virus H1N1 hypotype of utilizing Pichia anomala expression both at home and abroad must be reported.Heterogenous expression is to obtain an effective way of target point protein, and with respect to Mammals and insect cell expression system, pichia yeast expression system operation is simple and easy, be easy to cultivation, fast growth, expression amount is high, cost is low; Have aox strong promoter, foreign protein is subject to the strict induction regulating controlling of methyl alcohol and expresses; There is rear Modifying Capability, can carry out to expressing protein a series of rear modifications such as glycosylation, protein phosphorylation, folding, signal sequence processing, lipid acidylate, thereby make the albumen of its expression there is biological activity.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly to prepare loaded down with trivial details consuming time for the neuraminidase using in existing influenza virus H1N1 hypotype neuraminidase inhibitor screening model, and there is the deficiency of safety problem, and a kind of influenza A virus neuraminidase and gene and preparation method are provided, and the construction process of the screening model of influenza virus neuraminidase inhibitor.
A first aspect of the present invention is to provide a kind of Neuraminidase Gene of Influenza virus H1N1 hypotype, and its nucleotide sequence is following any one:
1) nucleotide sequence shown in SEQ ID No.1 in sequence table;
2) nucleotide sequence of encoding amino acid sequence albumen as shown in SEQ ID No.2 in sequence table.
In the present invention, the nucleotide sequence total length 1407bp of the Neuraminidase Gene of nucleotide sequence shown in SEQ ID No.1,469 amino acid of encoding, codon used is conducive to NA gene expresses in pichia spp.
In the present invention, the albumen of aminoacid sequence as shown in SEQ ID No.2 in sequence table is 82 amino acid having removed total length NA albumen n end, is brachymemma NA.
A second aspect of the present invention is to provide the recombinant vectors of the Neuraminidase Gene that contains Influenza virus H1N1 hypotype as above.The carrier used of described recombinant vectors is the conventional carrier of this area, preferred vector pPICZ α A.
A third aspect of the present invention is to provide the transformant that contains recombinant vectors as above.The host cell of described transformant is the host cell of this area routine, preferably pichia spp, more preferably pichia spp KM71 or SMD1168 expression strain.
A fourth aspect of the present invention is to provide a kind of neuraminidase of Influenza virus H1N1 hypotype of restructuring, and its aminoacid sequence is in sequence table shown in SEQ ID No.2.It is 82 amino acid having removed NA full-length gene N end, is brachymemma NA, has neuraminic acid enzyme bioactivity.
A fifth aspect of the present invention is to provide a kind of method of genetic engineering bacterium of the neuraminidase of preparing secreting, expressing Influenza virus H1N1 hypotype, comprises the following steps:
1) synthesize through codon optimized nucleotide sequence the Neuraminidase Gene as shown in SEQ ID No.1 in sequence table;
2) Neuraminidase Gene is cloned into construction recombination plasmid in Yeast expression carrier, then recombinant plasmid is imported to Pichiapastoris expression strain and obtain recombinant yeast pichia pastoris, must express the genetic engineering bacterium of neuraminidase.
In the present invention, step 1) synthetic method of described Neuraminidase Gene can ordinary method, and can synthetic full-length gene.Step 2) described Neuraminidase Gene can be step 1) the synthetic Neuraminidase Gene as shown in SEQ ID No.1 in sequence table through codon optimized nucleotide sequence, also can be take the Neuraminidase Gene shown in SEQ ID No.1 in sequence table as template, that utilizes that primer amplification amplification obtains brachymemma only contains functional area, removed may with the Neuraminidase Gene of catalytic activity extraneous areas, preferably utilize primer amplification obtain encoding amino acid sequence be the Neuraminidase Gene of the brachymemma of albumen shown in SEQ ID No.2 in sequence table; The described method of utilizing primer amplification to obtain the Neuraminidase Gene of brachymemma is the ordinary method of this area, take step 1) Neuraminidase Gene of synthesized obtains the Neuraminidase Gene of encoding amino acid sequence brachymemma of albumen as shown in SEQ ID No.2 in sequence table as template amplification.Described primer is preferably: the sequence of upstream primer NA_F is 5 '-GC
cTCGAGaAAAGAGTTAAGTTGGCTGG-3 ' (as shown in SEQ ID No.3 in sequence table), the sequence of downstream primer NA_R is 5 '-GC
tCTAGAcCCTTGTCAATGGTGAATGG-3 ' (as shown in SEQ ID No.4 in sequence table), has respectively XhoI and XbaI enzyme cutting site.Described Yeast expression carrier is plasmid pPICZ α A preferably.Described Pichiapastoris expression strain is pichia spp KM71 or SMD1168 preferably.When secreting to born of the same parents, the brachymemma NA that two kinds of recons of KM71 or SMD1168 are expressed all there is natural N terminal sequence through Kex2 cutting, without the extra amino acid increasing.The cloning process that the described Neuraminidase Gene by brachymemma is cloned into Yeast expression carrier is this area ordinary method.Preferably, as above utilize primer NA_F and NA_R amplification to obtain brachymemma NA gene, build by XhoI and XbaI enzyme cutting site the recombinant vectors pPICZ α A-NAS that contains brachymemma NA gene, then with electrotransformation, the pPICZ α A-NAS of SacI linearization for enzyme restriction is imported in pichia spp, utilize Zeocin
tMresistance screening recombinant bacterial strain, finally induces recombinant yeast pichia pastoris secreting, expressing brachymemma NA.
A sixth aspect of the present invention is to provide the genetic engineering bacterium of the neuraminidase of the secreting, expressing Influenza virus H1N1 hypotype that method as above prepares.
The application of the neuraminidase of Influenza virus H1N1 hypotype that a seventh aspect of the present invention is to provide restructuring as above in neuraminidase inhibitor screening model.
A eighth aspect of the present invention is to provide a kind of construction process of neuraminidase inhibitor screening model, it is characterized in that, comprise the following steps: the Neuraminidase Gene of described Influenza virus H1N1 hypotype is cloned into construction recombination plasmid in Yeast expression carrier, then recombinant plasmid is imported to Pichiapastoris expression strain and obtain recombinant yeast pichia pastoris, obtain the genetic engineering bacterium of the neuraminidase of secreting, expressing Influenza virus H1N1 hypotype, after cultivation, obtain thering is bioactive neuraminidase, in reaction system, add this brachymemma neuraminidase, substrate 4-MUNANA and inhibitor to be screened, the variation of fluorescence intensity under the exciting light of 360nm and the utilizing emitted light of 460nm after incubation.
Wherein, the preferred approach of the genetic engineering bacterium of the neuraminidase of acquisition secreting, expressing Influenza virus H1N1 hypotype as described above, the nucleotide sequence of the Neuraminidase Gene of described Influenza virus H1N1 hypotype is following any one: the 1) nucleotide sequence shown in SEQ ID No.1 in sequence table, 2) nucleotide sequence of encoding amino acid sequence albumen as shown in SEQ ID No.2 in sequence table; The preferred pichia spp KM71 of described pichia spp or SMD1168 expression strain.And compound 4-MUNANA (4-methylumbelliferyl-N-acetyl-α-D-neuralminic acid) is the specific substrate of neuraminidase, the meta-bolites 4-MU (4-methylumbelliferone) producing under neuraminidase effect is excited and can produce 460nm fluorescence by 360nm light irradiation, and the variation of fluorescence intensity can be reacted neuraminic acid enzymic activity delicately.The present invention optimizes the conditions such as enzyme amount, concentration of substrate, reaction times, pH value of reaction system, calcium ion concn and the temperature of reaction of neuraminidase, thereby sets up a kind of high flux screening model of neuraminidase inhibitor.Preferably described reaction system is 32.5mM pH6.5 MES damping fluid, 4mM CaCl
2; Substrate is preferably 100 μ M4-MUNANA; Preferably 37 ℃ of heated culture temperatures.
In the present invention, above-mentioned optimum condition can arbitrary combination on the basis that meets this area general knowledge, obtains the preferred embodiments of the invention.
The raw material that the present invention is used or reagent except special instruction, all commercially available obtaining.
Than prior art, beneficial effect of the present invention is as follows: pichia spp secreting, expressing Influenza virus H1N1 hypotype neuraminidase for the present invention, obtained and there is bioactive neuraminidase, avoided loaded down with trivial details consuming time and there is safety problem prepare the operating process of NA by totivirus.82 amino acid whose brachymemma neuraminidases specifically having removed NA full length sequence N end, the gene of brachymemma only contains functional area, has removed part that may be irrelevant with catalytic activity, and the secreting, expressing efficiency of albumen is high.The present invention also utilizes this enzyme to build Influenza virus H1N1 hypotype neuraminidase inhibitor screening model, have that mechanism of drug action is clear and definite, material usage is few, breakneck acceleration is fast, highly sensitive, can realize the advantages such as high flux screening.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, feature of the present invention and beneficial effect are described.
Fig. 1 is the agarose gel electrophoresis figure of amplification brachymemma NA gene, wherein, and 1,2: brachymemma NA gene (primer NA_F and NA_R), 3,4: brachymemma NA gene (primer NA_F2 and NA_R2), M: molecular weight Marker DL2000.
Fig. 2 is that the enzyme of recombinant plasmid is cut evaluation electrophorogram, wherein, M: molecular weight Marker DL2000,1: the recombinant plasmid pPICZ alpha A-NAS that enzyme is not cut, 2: through the pPICZ α A-NAS of Xho I and Xba I double digestion, 3: through the pPICZ α A-NAS2 of EcoR I and Not I double digestion.
Fig. 3 is the enzyme activity determination result of restructuring bacterial strain inducing 96h fermented supernatant fluid, wherein, 1: empty carrier pPICZ α A transforms the recombinant bacterial strain of KM71, 2～4: recombinant plasmid pPICZ alpha A-NAS transforms the recombinant bacterial strain of KM71, 5～7: recombinant plasmid pPICZ alpha A-NAS2 transforms the recombinant bacterial strain of KM71, 8～10: recombinant plasmid pPICZ alpha A-NA transforms the recombinant bacterial strain of KM71, 11: empty carrier pPICZ α A transforms the recombinant bacterial strain of SMD1168, 12～14: recombinant plasmid pPICZ alpha A-NAS transforms the recombinant bacterial strain of SMD1168, 15～17: recombinant plasmid pPICZ alpha A-NAS2 transforms the recombinant bacterial strain of SMD1168, 18～20: recombinant plasmid pPICZ alpha A-NA transforms the recombinant bacterial strain of SMD1168.
Fig. 4 is Oseltamivir, Zha Na meter Wei, amantadine and the Rimantadine inhibition experimental result to brachymemma NA.
Embodiment
Further illustrate the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer." room temperature " described in embodiment refers to the temperature of the operation room of testing, and is generally 25 ℃.
The structure of embodiment 1 recombinant plasmid pPICZ alpha A-NAS
1) obtain the neuraminic acid enzyme amino acid sequence of 1629 Influenza virus H1N1 hypotypes of report in year March in April, 2009 to 2010 from NCBI website, after CluxtalX software compare of analysis, choose most representative virus strain A/Wisconsin/629-S0247/2009 (H1N1) (GenBank:CY051377) as research object, the nucleic acid total length of this gene is 1407bp, 469 amino acid of encoding.The sequence of this gene being carried out to codon optimized, codon optimized NA full-length gene according to the expression custom of pichia spp is shown in shown in sequence table SEQ ID NO.1.Adopt the method for chemosynthesis to carry out full gene to the gene after optimizing and synthesize, synthetic full-length gene is connected to the upper DCRP carrier pUC57-NA of cloning vector pUC57, sequencing checking entirely true (entrusting Shanghai to dodge brilliant biosynthesizing).
2) be designed for the primer of pcr amplification brachymemma NA gene, upstream primer NA_F5 '-GC
cTCGAGaAAAGAGTTAAGTTGGCTGG-3 ' (as shown in SEQ ID No.3 in sequence table), downstream primer NA_R 5 '-GCTCTAGACCCTTGTCAATGGTGAATGG-3 ' (as shown in SEQ ID No.4 in sequence table), this brachymemma NA gene has been removed 82 amino acid whose encoding sequences of its N end, and the aminoacid sequence of brachymemma NA genes encoding is shown in SEQ ID NO.2.
3) take the cloning vector pUC57-NA that contains codon optimized total length NA gene as template, utilize primer NA_F and NA_R at LA Taq
(TaKaRa) under catalysis, carry out pcr amplification, obtain the brachymemma NA gene fragment of 1.2kb.The program of amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1.5min, totally 30 circulations; 72 ℃ are extended 10min again), amplification is as shown in Figure 1.
4) respectively brachymemma NA gene fragment and the yeast expression vector pPICZ α A (purchased from Invitrogen) of amplification are carried out to double digestion with XhoI and XbaI, agarose gel electrophoresis reclaims object fragment, utilize T4 ligase enzyme (TaKaRa) in 16 ℃ of connections of spending the night, connect product Transformed E .coli DH5 α and coat and contain 25mg/L Zeocin
tMless salt LB agar plate, 37 ℃ cultivate 12～16h.
5) 10 the inoculation 5mL of single bacterium colony that grow in picking resistant panel contain 25mg/L zeocin
tMless salt LB liquid nutrient medium, incubated overnight under 37 ℃ of conditions of 200rpm, extract plasmid, carry out double digestion evaluation with XhoI and XbaI, obtain the recombinant plasmid pPICZ alpha A-NAS that contains brachymemma NA gene, enzyme is cut to correct plasmid and check order, restriction enzyme digestion and electrophoresis result as shown in Figure 2.
The structure of embodiment 2 recombinant pichia yeast strains and the abduction delivering of brachymemma NA
1) prepare KM71 and SMD1168 competence, concrete steps comprise:
A. KM71 (purchased from Invitrogen) and the single bacterium colony of SMD1168 (purchased from Invitrogen) of the dull and stereotyped growth of YPD at 30 ℃ of pickings, inoculate 30 ℃ of incubated overnight of 20mL YPD liquid nutrient medium 250rpm.
B. get 0.1～0.5mL overnight culture, the 2L shaking flask that inoculation contains 500mL fresh culture, incubated overnight is to OD600=1.3～1.5.
C. at 4 ℃, the centrifugal 5min collecting cell of 1500g, with the aqua sterilisa suspension cell of 500mL precooling.
D. as above centrifugal, with the aqua sterilisa suspension cell of 250mL precooling.
E. as above centrifugal, with the 1M sorbyl alcohol suspension cell of 20mL precooling.
F. as above centrifugal, with the 1M sorbyl alcohol suspension cell of 1mL precooling, to the about 1.5mL of final volume.
2) linearizing pPICZ α A-NAS electricity transforms KM71 and SMD1168, and concrete steps comprise:
A. with SacI, expression vector pPICZ α A-NAS is carried out to linearizing, linearizing fragment reclaims through ethanol precipitation.
B. mix 80 μ L competent cells and approximately 10 μ g linearizing pPICZ α A-NAS fragment and be transferred to the 0.2cm electric shock cup of 0 ℃ of precooling, leave standstill 5min on ice.
C. transform according to BioRad electricity conversion instrument operational manual electricity, shock parameters is: voltage 2.5KV, resistance 200 Ω, electric capacity 20kF.
D. electric shock adds at once the ice-cold 1M sorbyl alcohol of 1mL to electric shock cup and is transferred in centrifuge tube after finishing.
E. centrifuge tube is statically placed in to 30 ℃ of incubation 60～90min.
F. coat 100mg/L Zeocin with 100-300 μ L/ flat board
tMyPDS flat board, 30 ℃ are cultured to single bacterium colony and form.
3) utilize primer 5 ' AOX1 (5 '-GACTGGTTCCAATTGACAAGC-3 ') and 3 ' AOX1 (5 '-GCAAATGGCATTCTGACATCC-3 ') to carry out bacterium colony PCR evaluation, the template of bacterium colony PCR is the thalline through freeze-thaw method processing, and amplification condition is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 3min, totally 30 circulations; 72 ℃ are extended 10min again.
4) picking PCR identifies and the correct single bacterium colony that checks order respectively, is inoculated in 25mL BMGY substratum, cultivates, until cell concn reaches OD600=2～6 in the shaking table of 28 ℃ of 250rpm with 500mL triangular flask.Centrifugal 5min under room temperature 4000rpm condition, collecting cell, outwells after supernatant with 20mL BMMY substratum re-suspended cell, abduction delivering under 28 ℃ of 250rpm, it is 0.5% continuation induction that every 24h adds methyl alcohol to final concentration, respectively at 24h, 48h, 72h and 96h sampling.
Embodiment 3NA determination of activity and inhibition experiment
0.5% methanol induction recombinant yeast pichia pastoris secreting, expressing brachymemma NA, the bacterium liquid of abduction delivering 96h centrifugal 5min under 12000rpm, getting supernatant liquor tests for NA determination of activity as crude enzyme liquid, utilize ultrafiltration and concentration centrifuge tube by concentrated approximately 10 times of crude enzyme liquid simultaneously, inhibition experiment for Oseltamivir, Zha Na meter Wei, amantadine and Rimantadine to brachymemma NA, laboratory apparatus is the multi-functional microplate reader of BioTek and 96 hole black enzyme plates.
NA determination of activity: get 30 μ l fermented supernatant fluids, (final concentration is 32.5mM MES damping fluid, pH6.5,4mM CaCl to add 75 μ l reaction substrates
2, 100 μ M substrate 4-MUNANA), the variation of dynamic method fluorescence intensity under the exciting light of 360nm and the utilizing emitted light of 460nm.With doubling dilution to the 4-MU of proper concn as standard substance drawing standard curve the enzymic activity for quantitative brachymemma NA, the enzyme work of a unit is defined as per minute catalytic substrate 4-MUNANA at 37 ℃ and generates the enzyme amount of a micromole 4-MU.Determination of activity result as shown in Figure 3.
Oseltamivir, Zha Na meter Wei, amantadine and Rimantadine suppress experiment: by four kinds of reagent water doubling dilutions, get 15 μ l, add 30 μ l NA crude enzyme liquids, 75 μ l reaction substrates, at 37 ℃, react 30min, after mixing under the exciting light of 360nm and the utilizing emitted light of 460nm fluorescence intensity.Suppress experimental result as shown in Figure 4.
The determination of activity of structure, conversion and the brachymemma NAS2 of embodiment 4 (comparative example) recombinant vectors pPICZ α A-NAS2
1) design primer (upstream primer NA_F2 5 '-GC
gAATTCgTTAAGTTGGCTGGTAACTCC-3 ' and downstream primer NA_R25 '-AAT
gCGGCCGCcTTGTCAATGGTGAATG-3 ') for pcr amplification brachymemma NAS2 gene, this brachymemma NAS2 gene has been removed 82 amino acid of neuraminidase N end equally, and restriction enzyme site is respectively EcoR I and Not I (underscore place).Because the restriction enzyme site adopting is different from embodiment 1, build plasmid with this product to primer amplification and transform pichia spp, the albumen of recon secreting, expressing does not have natural N end, can have more 6 amino acid (EAEAEF), these 6 occurrences of amino acid directly cause the neuraminidase of expressing there is no catalytic activity.
2) take the cloning vector pUC57-NA that contains codon optimized NA gene as template, utilize primer NA_F2 and NA_R2 at LA Taq
(TaKaRa) under catalysis, carry out pcr amplification, obtain the brachymemma NA gene fragment of 1.2kb.The program of amplification is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 1.5min, totally 30 circulations; 72 ℃ are extended 10min again), electrophoresis result is as shown in Figure 1.
3) respectively brachymemma NA gene fragment and the yeast expression vector pPICZ α A of amplification are carried out to double digestion with EcoR I and Not I, agarose gel electrophoresis reclaims object fragment, utilize T4 ligase enzyme (TaKaRa) in 16 ℃ of connections of spending the night, connect product Transformed E .coli DH5 α and coat and contain 25mg/LZeocin
tMless salt LB agar plate, 37 ℃ cultivate 12～16h.
4) 10 the inoculation 5mL of single bacterium colony that grow in picking resistant panel contain 25mg/L zeocin
tMless salt LB liquid nutrient medium, incubated overnight under 37 ℃ of conditions of 200rpm, extracts plasmid, carries out double digestion evaluation with EcoR I and Not I, obtains the recombinant plasmid pPICZ alpha A-NAS2 that contains brachymemma NA gene, enzyme is cut to correct plasmid and check order.
5) recombinant plasmid pPICZ alpha A-NAS2 transforms KM71 and SMD1168 competence, and screening recombinant bacterial strain is cultivated and induces recombinant bacterial strain to express NAS2, measures NAS2 activity, and concrete steps are with embodiment 1,2 and 3, and determination of activity result as shown in Figure 3.
The determination of activity of structure, conversion and the total length NA of embodiment 5 (comparative example) recombinant vectors pPICZ α A-NA
1) respectively the cloning vector pUC57-NA that contains codon optimized total length NA gene and yeast expression vector pPICZ α A are carried out to double digestion with EcoR I and Not I, agarose gel electrophoresis reclaims object fragment, utilize T4 ligase enzyme (TaKaRa) in 16 ℃ of connections of spending the night, connect product Transformed E .coliDH5 α and coat and contain 25mg/L Zeocin
tMless salt LB agar plate, 37 ℃ cultivate 12～16h.
2) 10 the inoculation 5mL of single bacterium colony that grow in picking resistant panel contain 25mg/L zeocin
tMless salt LB liquid nutrient medium, incubated overnight under 37 ℃ of conditions of 200rpm, extracts plasmid, carries out double digestion evaluation with EcoR I and Not I, obtains the recombinant plasmid pPICZ alpha A-NA that contains brachymemma NA gene, enzyme is cut to correct plasmid and check order.
3) recombinant plasmid pPICZ alpha A-NA transforms KM71 and SMD1168 competence, and screening recombinant bacterial strain is cultivated and induces recombinant bacterial strain to express total length NA, measures NA activity, and concrete steps are with embodiment 1,2 and 3, and determination of activity result as shown in Figure 3.
Claims (8)
1. a Neuraminidase Gene for Influenza virus H1N1 hypotype, is characterized in that, its nucleotide sequence is: the nucleotide sequence of encoding amino acid sequence albumen as shown in SEQ ID No.2 in sequence table.
2. contain the recombinant vectors of the Neuraminidase Gene of Influenza virus H1N1 hypotype as claimed in claim 1.
3. contain the transformant of recombinant vectors as claimed in claim 2.
4. a neuraminidase for the Influenza virus H1N1 hypotype of restructuring, is characterized in that, its aminoacid sequence is in sequence table shown in SEQ ID No.2.
5. a method of preparing the genetic engineering bacterium of the neuraminidase of secreting, expressing Influenza virus H1N1 hypotype, is characterized in that, comprises the following steps:
1) synthesize through codon optimized nucleotide sequence the Neuraminidase Gene as shown in SEQ ID No.1 in sequence table;
2) Neuraminidase Gene is cloned into construction recombination plasmid in Yeast expression carrier, then recombinant plasmid is imported to Pichiapastoris expression strain and obtain recombinant yeast pichia pastoris, must express the genetic engineering bacterium of neuraminidase, described Neuraminidase Gene is take the Neuraminidase Gene shown in SEQ ID No.1 in sequence table as template, and utilizing primer amplification to obtain encoding amino acid sequence is the Neuraminidase Gene of the brachymemma of albumen shown in SEQ ID No.2 in sequence table; Described primer is: the sequence of upstream primer NA_F is as shown in SEQ ID No.3 in sequence table, and the sequence of downstream primer NA_R is as shown in SEQ ID No.4 in sequence table; Described Yeast expression carrier is plasmid pPICZ α A; Described Pichiapastoris expression strain is pichia spp KM71 or SMD1168.
6. the genetic engineering bacterium of the neuraminidase of the secreting, expressing Influenza virus H1N1 hypotype that method as claimed in claim 5 prepares.
7. the construction process of a neuraminidase inhibitor screening model, it is characterized in that, comprise the following steps: the Neuraminidase Gene of Influenza virus H1N1 hypotype as claimed in claim 1 is cloned into construction recombination plasmid in Yeast expression carrier, then recombinant plasmid is imported to Pichiapastoris expression strain and obtain recombinant yeast pichia pastoris, obtain the genetic engineering bacterium of the neuraminidase of secreting, expressing Influenza virus H1N1 hypotype, after cultivation, obtain thering is bioactive neuraminidase, in reaction system, add this neuraminidase, substrate 4-MUNANA and inhibitor to be screened, the variation of fluorescence intensity under the exciting light of 360nm and the utilizing emitted light of 460nm after incubation.
8. construction process claimed in claim 7, is characterized in that, described reaction system is 32.5mM pH6.5 MES damping fluid, 4mM CaCl
2; Substrate is 100 μ M 4-MUNANA; Heated culture temperature is 37 ℃.
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