CN1800376A - Recombinant T4 phage for displaying influenza virus non-structural protein NS1 and its uses - Google Patents
Recombinant T4 phage for displaying influenza virus non-structural protein NS1 and its uses Download PDFInfo
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Abstract
The invention relates to a recombination T4 phase of showing influenza virus non-structural protein NS1 which is an admixture protein of T4 phase of showing influenza virus non-structural protein NS1 and T4 phase. It also relates to the application of the recombination T4 phase. The recombination T4 phase is easy to purify; the NS1 protein showed on the T4 phase surface has integrated space constitution; the NS1 protein which is admixture expressed with SOC has high copying number; the T4 phase is the carrier of the NS1 protein.
Description
Technical field
The present invention relates to the genetically engineered field, more specifically, relate to structure, expression and the application of the recombinant T 4 bacteriophage of displaying influenza virus non-structural protein NS 1.The invention still further relates to the application of this recombinant T 4 bacteriophage in detecting influenza antibodies, particularly adopt this recombinant T 4 bacteriophage to distinguish virus infection fowl group and vaccine immunity fowl group.
Background technology
(Avian Influenza AI) is a kind of infection and/or the disease syndromes of the bird (poultry and wild fowl) that caused by the A of orthomyxovirus section type influenza virus in bird flu.Occur on one's body birds such as chicken, duck, goose, pigeon and the birds, but mainly encroach on turkey and chicken, Mammals such as pig, horse, sea dog, people etc. also can infect.According to avian influenza virus (Avian Influenza Virus, AIV) strain is pathogenic, the kind of fowl, environment, factor such as feeding and management condition and concurrent disease, metainfective fowl only shows as symptomless infection, slight upper airway symptoms, egg productivity drops to acute various ways such as pathogenic death, directly influence health and products thereof the quality of chicken body, it is one of modernized aviculture important fowl upper transmissible disease of being difficult to tackle, with Marek, infectious bursal disease, leukemia, RE hyperplasia diseases etc. are the same, also are the important immunosuppressive diseases of serious threat poultry.Classified as the category-A animal epidemic by OIE, China classifies it as class animal epidemic.
At present adopt " immunity " hold concurrently measure of " slaughtering " to control bird flu more.China's Mainland, Mexico etc. adopt countries and regions this measure to control the popular of bird flu effectively.Adopt the method for inactivated vaccine immune control bird flu more and more to come into one's own, the method for immunity is adopted in more and more countries and area.But problem of the consequent is that immunity has caused the interference to epidemic monitoring.Because the serological method of traditional monitoring bird flu mainly is two: the one, agar diffusion test (AGP), the one, hemagglutination-inhibition test (HI).These two kinds of methods all can not be distinguished the antibody that produces behind immune antibody that produces of avian influenza vaccine and the avian influenza.
Avian influenza virus (AIV) is the sub-thread minus-stranded rna virus, belongs to the A of orthomyxovirus section type Influenza Virus, and A type influenza virus gene is made up of 8 discontinuous viral RNA sections.Fragment 8 is that the NS gene is the shortest in 8 fragments, and the coding Nonstructural Protein has two reading frames, and 2 kinds of protein of codified are NS1 and NS2, and both molecular weight are respectively 25kDa and 14kDa.These two kinds of protein are present in the cells infected in a large number, and mainly in nuclear, mainly in cytoplasm, there is minor N S2 protein ingredient in NS2 to NS1 in virus particle.Synthesized in a large number at the early stage NS1 that infects, and NS2 just can synthesize production in the later stage.Early stage at cell infection can find to have in the nucleus a large amount of NS1 to assemble, and also can find in tenuigenin.As the non-constituent of virus, this proteic appearance only is confined to the virus infection phase, and can not appear in the sophisticated avian influenza virus, thereby can not produce corresponding antibody when using the inactivated vaccine immunity.Therefore, only may come across among the wild virus infection group at the proteic antibody of NS1.Thereby can be with NS1 and antibody thereof as a mark of virus infection body in order to distinguish the immunity group and to infect group's differential diagnosis.
The effect of NS1 has been subjected to investigators' interest greatly recently.Many scholars attempt to differentiate that as antigen AIV infects fowl group and immune fowl group with the expression product of NS1.In order to obtain the NS1 albumen of large-scale purification, generally adopt engineered method to express the NS1 gene.The method that adopts generally is to express NS1 albumen in escherichia expression system at present.The present invention adopts a kind of new expression method---and display technique of bacteriophage obtains a large amount of active NS1 albumen that has.
Phage display technique (phage display) is a new biotechnology (Smith who grows up the eighties, 1985), it can be illustrated in phage surface with the form of fusion rotein with allogenic polypeptide and protein, and keeps relatively independent space conformation and biological activity.The typical member of T4 Phagus A group A2 subgroup in tailed phages, its grown form structure is the directly about 95nm of head length, transverse diameter is about 65nm.T4 phage capsid must be formed by capsid protein by 3 kinds: major capsid protein gp23 and 2 less important capsid protein gp24, gp20.Wherein molecular weight is that the gp23 of 45kDa has 960 copies in each virion, and all the other 2 less important capsid protein copy numbers are less, and gp24 has 55 copies, and gp20 has only 12 copies.In addition, at the appearance bread of capsid by 2 kinds of nonessential coat protein: molecular weight is that SOC and the molecular weight of 9kDa is the HOC of 40kDa, and the two is distributed in phage icosahedron surface at a distance of 7nm with symmetric form.SOC and HOC only provide phage extra stabilizing power, just are assembled into the capsid surface after the assembling of phage capsid is finished, and their disappearance does not influence breeding and the infection of T4 phage.The SOC or the HOC albumen of foreign protein and T4 phage surface are formed fusion rotein, thereby can obtain to have the recombinant T 4 bacteriophage of foreign protein.Adopt such method, expression HIV (human immunodeficiency virus) (HIV) gp120 V3 peptide, poliovirus VP1 (Ren et al have been obtained, 1996), meningitis Salmonella PorA (Jiang et al how, 1997) antigen protein, infectious bursal disease virus VP2 (Cao Yongchang etc., 2003), bird flu H5 hypotype and H9 subtype HA protein (Lv Yingzi etc., 2002), the recombinant T 4 bacteriophage of foot and mouth disease VP1 albumen (Shi Quancheng etc., 2002), newcastle disease virus F albumen and N albumen (Liu You etc., 2004).
Summary of the invention
(1) technical problem that will solve
The objective of the invention is to obtain to show the recombinant T 4 bacteriophage of avian influenza virus non-structural protein NS 1, and adopt this recombinant T 4 bacteriophage as antigen, distinguish the antibody of inactivated avian influenza vaccine immune animal and avian influenza animal, promptly distinguish vaccine immunity animal and virus infection animal.
(2) technical scheme
The present invention is a kind of recombinant T 4 bacteriophage of displaying influenza virus non-structural protein NS 1, is that influenza virus non-structural protein NS 1 and T4 phage SOC albumen are formed fusion rotein, is positioned at the recombinant T 4 bacteriophage head surface.
In the recombinant T 4 bacteriophage of the present invention, influenza virus non-structural protein NS 1 is the protein with sequence shown in the SEQ IDNO:2; Or have sequence shown in the SEQ ID NO:2 and still have the active protein of influenza virus non-structural protein NS 1 through changing, lacking or increase one or several amino-acid residues; Or the homology more than 80% is arranged and have the active protein of influenza virus non-structural protein NS 1 with the aminoacid sequence shown in the SEQ ID NO:2.
In the recombinant T 4 bacteriophage of the present invention, the nucleotides sequence of encoding influenza virus non-structural protein NS 1 is classified SEQ ID NO:1 as; Or SEQ ID NO:1 still expresses and has the active proteinic sequence of influenza virus non-structural protein NS 1 through changing, lack or increase one or several Nucleotide; Or with nucleotide sequence shown in the SEQID NO:1 in section more than continuous 200 bases homology more than 80% is arranged, and its expression product still has the active nucleotide sequence of influenza virus non-structural protein NS 1.
The present invention also provides the application of described recombinant T 4 bacteriophage, with recombinant T 4 bacteriophage as antigen, direct immunization or with the T4 phage with make the vaccine immunity animal after adjuvant cooperates, obtain the specific antibody of anti-NS1.
The another kind of recombinant T 4 bacteriophage of the present invention is used, be as the avian influenza specific antibody in the Detection of antigen animal serum with recombinant T 4 bacteriophage, distinguish the antibody of avian influenza vaccine immune animal and avian influenza animal, promptly distinguish influenza infection animal and inactivated influenza virus vaccine immune animal.
Application described in the present invention, employed detection method is an enzyme linked immunosorbent assay.
(1) at first adopts reverse transcription-polymerase chain reaction (RT-PCR) amplification NS1 gene fragment.Extracting avian influenza virus RNA, be primer with F1 (5 '-ATG GAT TCC AAC ACT G-3 ') and F2 (5 '-TCA AAC TTC TGA CTC-3 '), synthesizes the cDNA of NS1 gene under the effect of AMV ThermoScript II, acquisition RT product.The RT product is as the template of pcr amplification NS1 gene fragment, with P1 (5 '-ATG AAT TCT ATG GAT TCC AAC ACT G-3 ') and P2 (5 '-ATG AATTCG TCA AAC TTC TGA CTC-3 ') is primer, PCR method amplification NS1 gene routinely, reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 40s, 50 ℃ of annealing 90s, 72 ℃ are extended 90s, circulate 30 times; Last 72 ℃ are extended 10min.The PCR product detects with the 10g/L agarose gel electrophoresis.
(2) adopt pR plasmid (Ren et al, 1996; Ren Zhaojun, 2004, CN1523114A) construction recombination plasmid pR-NS1.With the PCR purified product of EcoR I at 37 ℃ of digestion NS1 genes, with EcoR I and CIAP at 37 ℃ of digested plasmid pR, then under the effect of T4 ligase enzyme, NS1 PCR product after ligase enzyme cuts back to close and plasmid pR connect product and are transformed into bacillus coli DH 5 alpha.Method for transformation is conventional CaCl2 method.Through penbritin (Amp) resistance screening, obtained to insert the recon of NS1 gene.Use two couples of Auele Specific Primer SOC-S (GAA TCA TAT GGC TAG TCT CGCGG)/P2 and P1/P2 to carry out the PCR screening then.Picking list colony inoculation was cultivated 16-20 hour with the 200r/m jolting at 37 ℃ in 3mL LA/Amp nutrient solution.To PCR screening male bacterium, centrifugal collection thalline, the extracting plasmid is identified and sequencing through digestion with restriction enzyme again, obtains recombinant plasmid pR-NS1.
(3) with recombinant plasmid pR-NS1 and defective T4 phage T4-Z1 (Ren et a1,1996; Ren Zhaojun, 2004, CN1523114A) carry out homologous recombination, obtain to show the proteic recombinant T 4 bacteriophage of NS1.
With recombinant plasmid pR-NS1 transformed into escherichia coli DH5 α, the intestinal bacteria of acquisition are designated as E-NS1 earlier.Carry out following operation then:
In the vessel of 500 μ L SC nutrient solutions of packing into, add 1 μ L acillin (Amp), add 10 μ L E-NS1 then, be cultured to optical density value OD600 at 37 ℃ and be approximately at 0.5 o'clock, add 50 μ LT4-Z1, be cultured to bacterial debris 35 ℃ of 200r/m joltings and occurred.
At 1 through autoclaving (15lpf/in
2, add 600 μ L cultivation in test tube 20min) respectively and surpass 12 hours fresh bacillus coli DH 5 alpha, do not add N,O-Diacetylmuramidase, will be at the autoclaving (15lpf/in of 2-10 ℃ of storage
2, 20min) the SC top layer substratum of Guoing is placed in the microwave oven and dissolves, and treats that getting 2mL when the agar temperature drops to about 55 ℃ pours in the test tube that bacillus coli DH 5 alpha is housed.With mixed solution mixing on the vortex oscillator, be poured on the plate immediately.By the time during the agar total condensation, on SC top layer substratum, put 10 μ L respectively and cultivate E-NS1 bacterial lysate more than 12 hours, wait to absorb finish after, be placed on 37 ℃ and cultivate more than 16 hours.
If reorganization has taken place in plasmid among the E-NS1 and T4-Z1, successfully the NS1 gene is changed in the T4 phage, then on the plate that does not add N,O-Diacetylmuramidase, can grow plaque.Again spread the SC plate culture medium, with the sterilization toothpick plaque that grows is put the plum blossom spot on the SC plate then.Being placed on 37 ℃ cultivates more than 12 hours.Well-grown plum blossom spot peels it with the blade that disinfects, and is immersed in the phosphoric acid buffer more than 6 hours.
Adopt PCR screening T4 phage transformant.With the Auele Specific Primer P1/P2 specific band that increases from positive plum blossom spot soak solution, then explanation has obtained the proteic recombinant T 4 bacteriophage of displaying NS1.
The SC nutrient solution is prepared by following method: contain 10g Tryptone and 5g NaCl among every 1000mL, add distilled water and be settled to 1000mL, 15lpf/in
24 ℃ of preservations behind the autoclaving 20min.
SC bottom substratum is prepared by following method: contain 10g Tryptone among every 1000mL, and 5gNaCl and 12g agar powder, distilled water is settled to 1000mL, 15lpf/in
2Autoclaving 20min is cooled to 50 ℃, adds the 50mL 1mol/L Tris-HCl (pH7.5) and the 10mL 25% Trisodium Citrate 2H of sterilization
2O solution, it is dull and stereotyped to shake up the back, 4 ℃ of preservations.
SC top layer substratum is prepared by following method: contain 10g Tryptone among every 1000mL, and 5gNaCl and 7g agar powder, distilled water is settled to 1000mL, 15lpf/in
2Autoclaving 20min, 4 ℃ of preservations.The time spent heating for dissolving is cooled to 50 ℃, adds the 50mL 1mol/L Tris-HCl (pH7.5) and the 10mL 25% Trisodium Citrate 2H of sterilization
2O solution, it is standby to shake up the back.
(3) beneficial effect
The present invention has tangible advantage and effect.Recombinant T 4 bacteriophage provided by the invention is easy to purifying, and the NS1 albumen that is illustrated in the T4 phage surface has complete space conformation.Because the SOC site has 960 copies on T4 bacteriophage head surface, thus with the proteic copy number height of the NS1 of SOC amalgamation and expression.Because NS1 albumen is distributed in T4 bacteriophage head surface, thereby the T4 phage itself served as the proteic carrier of NS1, and when recombinant T 4 bacteriophage provided by the invention during as antigen, the immunogenicity of NS1 is stronger than independent NS1 albumen.When using as the antigen of vaccine or as the antigen of immunodetection, compare with the NS1 albumen that other protein expression system is expressed, recombinant T 4 bacteriophage of the present invention has tangible advantage.
Description of drawings
Fig. 1 is the pcr amplification product electrophorogram of NS1 gene.M is dna molecular amount standard DL2000DNA Marker; 1-4 is a NS1 gene PCR amplified production;
Fig. 2 is the The selection result of pR-NS1 recon.The PCR product size of NS1 gene is 678bp; The about 900bp of PCR product size of SOC-NS1 fusion gene.M is DL2000 DNA Marker, and 1,5 is same transformant, and 2,6 is same transformant, 3,7 is same transformant, 4,8 is same transformant, and wherein 1,2,3,4 is the PCR product of P1/P2 primer amplified, and 5,6,7,8 is the PCR product of SOC-S/P2 primer amplified;
Fig. 3 is the structure synoptic diagram of reorganization integrated plasmid pR-NS1.PR represents integrated plasmid, and pR-NS1 has represented to insert the recombination and integration plasmid of NS1 gene.Amp
rExpression acillin resistance, Kp
rThe expression kalamycin resistance.E, soc, den V are the genes on the T4 phage.EcoR I, BamH I, Nde I, HindIII, Pst I, Pvu I are the restriction endonuclease sites on the plasmid;
Fig. 4 is that NS1 albumen is showed mode chart in recombinant T 4 bacteriophage SOC site.A is reorganization integrated plasmid pR-NS1; B is defective type T4 phage T4-Z1; C is for carrying the recombinant T 4 bacteriophage T4-NS1 of goal gene NS1 after recombinating;
Fig. 5 is the PCR qualification result figure of recombinant T 4 bacteriophage.Swimming lane 1-8 is the PCR product that contains the recombinant phage of NS1 gene, and M is a molecular weight marker;
Fig. 6 is that the immunoblotting (Western blot) of recombinant phage T4-NS1 detects figure.M is prestained protein molecular weight standard; 1 is recombinant phage T4-NS1.
Embodiment
Extract the RNA of H 5 N 1 avian influenza subtype virus according to RizolLS Reagent specification sheets (GibcoBRL company product), with F1 (5 '-ATG GAT TCC AAC ACT G-3 ') and F2 (5 '-TCA AACTTC TGA CTC-3 ') is primer, the cDNA of synthetic NS1 gene obtains the RT product under AMV ThermoScript II (TaKaRa company product) effect.The RT reaction conditions is as follows: 42 ℃ of 60min, 95 ℃ of 3min then.The RT product is as the template of pcr amplification NS1 gene fragment, with P1 (5 '-ATG AATTCT ATG GAT TCC AAC ACT G-3 ') and P2 (5 '-ATG AAT TCG TCA AAC TTCTGA CTC-3 ') is primer, PCR method amplification NS1 gene routinely, reaction conditions is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 40s, 50 ℃ of annealing 90s, 72 ℃ are extended 90s, circulate 30 times; Last 72 ℃ are extended 10min.The PCR product detects with the 10g/L agarose gel electrophoresis, observes specific band (Fig. 1).The result shows that successfully amplification has obtained the gene fragment of NS1.
The structure of embodiment 2 recombinant plasmid pR-NS1
Employing pR plasmid (Ren Zhaojun, 2004, CN1523114A) construction recombination plasmid pR-NS1, its building process is as shown in Figure 3.With the PCR product of EcoR I at 37 ℃ of digestion NS1 genes, use EcoR I and CIAP at 37 ℃ of digested plasmid pR, then under the effect of T4 ligase enzyme, connect PCR product and the plasmid pR of the NS1 of digestion.To connect product transformed into escherichia coli DH5 α.Method for transformation is conventional CaCl2 method.Through Amp
rResistance screening, the recon of NS1 gene has been inserted in acquisition.Use two couples of Auele Specific Primer SOC-S (GAATCA TATGGCTAGTCTCGCGG)/P2 and P1/P2 to carry out PCR screening (Fig. 2) then.Picking list colony inoculation is in 3mL LA substratum, 37 ℃, the 200r/m jolting was cultivated 16-20 hour, to PCR screening male bacterium, the centrifugal collection thalline of 8000r/m with E.Z.N.A. Plasmid Minipreps Kit (Omega company product) extracting plasmid, is identified and sequencing through digestion with restriction enzyme again, obtain recombinant plasmid, identify correct recombinant plasmid called after pR-NS1.
The acquisition of embodiment 3 recombinant phage T4-NS1
With recombinant plasmid pR-NS1 and defective T4 phage T4-Z1 (Ren Zhaojun, 2004, CN1523114A) carry out homologous recombination, obtain to show the proteic recombinant T 4 bacteriophage of NS1.Its building process as shown in Figure 4.Earlier recombinant plasmid pR-NS1 is transformed into bacillus coli DH 5 alpha, the intestinal bacteria of acquisition are designated as E-NS1.Carry out following operation then:
In the vessel of 500 μ L SC nutrient solutions of packing into, add 1 μ L acillin (Amp), add 10 μ L E-NS1 then, be cultured to optical density value OD at 37 ℃
600Be approximately at 0.5 o'clock, add 50 μ LT4-Z1, be cultured to bacterial debris 35 ℃ of 200r/m joltings and occurred.
At 1 through autoclaving (15lpf/in
2, add 600 μ L cultivation in test tube 20min) respectively and surpass 12 hours fresh bacillus coli DH 5 alpha, do not add N,O-Diacetylmuramidase, will be at the autoclaving (15lpf/in of 2-10 ℃ of storage
2, 20min) the SC top layer substratum of Guoing is placed in the microwave oven and dissolves, and treats that getting 2mL when the agar temperature drops to about 55 ℃ pours in the test tube that bacillus coli DH 5 alpha is housed.With mixed solution mixing on the vortex oscillator, be poured on immediately on the plate that has been covered with SC bottom substratum.By the time during the agar total condensation, on SC top layer substratum, put 10 μ L respectively and cultivate E-NS1 bacterial lysate more than 12 hours, wait to absorb finish after, be placed on 37 ℃ and cultivated 16 hours.
If reorganization has taken place in plasmid among the E-NS1 and T4-Z1, successfully the NS1 gene is changed in the T4 phage, then on the plate that does not add N,O-Diacetylmuramidase, can grow plaque.Again spread the SC plate culture medium, with the sterilization toothpick plaque that grows is put the plum blossom spot on the SC plate then.Being placed on 37 ℃ cultivates more than 12 hours.Well-grown plum blossom spot peels it with the blade that disinfects, and is immersed in the phosphoric acid buffer 6 hours.
Adopt PCR screening T4 phage transformant.With the Auele Specific Primer P1/P2 specific band (Fig. 5) that increases from positive plum blossom spot soak solution, then explanation has obtained the proteic recombinant T 4 bacteriophage of displaying NS1.
The SC nutrient solution is prepared by following method: contain 10g Tryptone and 5g NaCl among every 1000mL, add distilled water and be settled to 1000mL, 15lpf/in
24 ℃ of preservations behind the autoclaving 20min.
SC bottom substratum is prepared by following method: contain 10g Tryptone among every 1000mL, and 5gNaCl and 12g agar powder, distilled water is settled to 1000mL, 15lpf/in
2Autoclaving 20min is cooled to 50 ℃, adds the 50mL 1mol/L Tris-HCl (pH7.5) and the 10mL 25% Trisodium Citrate 2H of sterilization
2O solution, it is dull and stereotyped to shake up the back, 4 ℃ of preservations.
SC top layer substratum is prepared by following method: contain 10g Tryptone among every 1000mL, and 5gNaCl and 7g agar powder, distilled water is settled to 1000mL, 15lpf/in2 autoclaving 20min, 4 ℃ of preservations.The time spent heating for dissolving is cooled to 50 ℃, adds the 50mL 1mol/L Tris-HCl (pH7.5) and the 10mL 25% Trisodium Citrate 2H of sterilization
2O solution, it is standby to shake up the back.
30 mouse are divided into 3 groups, 10 every group, raise in shield retaining the drinking-water and the feed of the sterilization of feeding every day.Virus inoculation in the time of about 8 ages in week.Wherein use the H5N1 subtype avian influenza virus of living through the intramuscular injection infecting mouse for one group, (viral HA titre is 1: 2 to every mouse 0.2ml live virus
8); Infected the back first the 9th day, the virus infection of using same dosage again once.Another organizes by same dosage, (viral HA titre is 1: 2 to same mode with the H9N2 subtype avian influenza virus infecting mouse of living
9).Inoculate inactivated avian influenza vaccine (the agricultural bio tech ltd product of the big China in Zhaoqing) by same dosage, same mode for the 3rd group.Inoculate back 15 days first, mouse is carried out the eyeball blood sampling, ordinary method prepares serum, adopts blood clotting to suppress experiment (HI) and measures its antibody horizontal, and HI tires and is 8log
2More than.℃ preservation of serum sample packing postposition-20 is standby.
The immunoblotting of embodiment 5 recombinant phages (Western Blot) is analyzed
A large amount of recombinant phage T4-NS1 that cultivate, centrifugal, method concentrates with PEG/NaCl routinely, when phage titre reaches 10
11During pfu/mL, carry out SDS-PAGE and detect.The SDS-PAGE method is as follows: get each 100 μ L of recombinant phage T4-NS1 and T4 phage SOC disappearance strain T4-Z1, add 2 * sample-loading buffer, 100 μ L then, behind the mixing, sample is distinguished freeze thawing three times in-80 ℃ of refrigerators and 37 ℃ of baking ovens." protein handbook method prepares SDS-polyacrylamide gel (SDS-PAGE) with reference to volume such as Wang Jiazheng (2000).Spacer gel concentration is 5%, and resolving gel concentration is 12%.Sample to be checked is boiled 5min, get 30 μ L and add in the gel point sample hole, under electric field action, carry out electrophoresis.Electrophoretic buffer is the Tfis-glycine.Voltage is 80v/cm, and voltage is elevated to 160v/cm when treating tetrabromophenol sulfonphthalein arrival spacer gel bottom.When treating tetrabromophenol sulfonphthalein, stop electrophoresis, take off gel near the gel bottom.Then carry out immunoblotting (WesternBlot) analysis.
Western Blot analytical procedure is as follows: the SDS-PAGE electrophoresis does not dye to gel after finishing.Cut nitrocellulose filter and two filter paper big or small together, at soaking at room temperature 15min in transfering buffering liquid, by the order of sponge-filter paper-gel-nitrocellulose filter-filter paper-sponge transfer printing is installed and presss from both sides with the gel after the electrophoresis with gel phase.To a side joint positive pole of film be arranged in the transfer printing folder, and under the voltage of 15V, shifted 1.5 hours.Film after the transfer spends the night with 4 ℃ of sealings of 5% skim-milk, discards confining liquid, washes film 3 times with TBS.Discard TBS, add the mice serum (is anti-) with the avian influenza of 1: 100 times of dilution, gentle jolting 3 hours with 1% skim-milk/PBS.Discard one and resist, wash film 3 times with TBS, about at every turn 10min.Discard TBS, add two anti-(the mountain sheep anti-mouse igg antibody of horseradish peroxidase-labeled, Zhuhai hundred companies difficult to understand) with 1: 1000 times of dilution, gentle jolting at least 1 hour with 1% skim-milk/PBS.Discard two and resist, wash film 3 times with TBS, about at every turn 30min.Discard TBS, add the colour developing of DAB solution, the jog nitrocellulose filter treats that colour developing reaches to a certain degree, washes the reaction of film color development stopping with distilled water.The visible specific band (Fig. 6) in the 39kDa position.
Recombinant phage is prepared into oil emulsion vaccine, and every ml contains 1 * 10
10The recombinant phage that pfu is above.10 mouse are raised in shield retaining feed every day clean drinking-water and feed.In the time of about 8 ages in week with recombinant phage oil emulsion vaccine immune mouse, every mouse 0.2ml, first immunisation is after 14 days, the vaccine booster immunization of using same dosage again is once.Behind the booster immunization 14 days, mouse is carried out the eyeball blood sampling, ordinary method prepares serum, and ℃ preservations of the serum sample packing postposition-20 of acquisition is standby, and with NS1 antibody horizontal in the ELISA detection mice serum.
With the recombinant T 4 bacteriophage is envelope antigen, detects NS1 antibody with ELISA.The ELISA program is as follows: is 1 * 10 with recombinant T 4 bacteriophage with PBST (PBS, pH7.2 contain 0.05% polysorbas20) dilution
8Pfu/ml, every hole adds the recombinant T 4 bacteriophage of 100 μ L dilution, places more than 12 hours for 4 ℃.After washing 3 times with cleaning buffer solution (PBS, pH7.2 contain 0.05% polysorbas20), every hole adds skim-milk/PBS of 100 μ L5%, places 2 hours for 37 ℃.After washing 3 times with cleaning buffer solution, every hole adds the mice serum of 100 μ L with 100 times of 1% skim-milks/PBS dilution, 37 ℃ of reactions 1 hour.After washing 3 times with cleaning buffer solution again, every hole adds 100 μ L and dilutes the mountain sheep anti-mouse igg antibody (Zhuhai hundred companies difficult to understand) of 1000 times horseradish peroxidase-labeled with 1% skim-milk/PBS, and 37 ℃ were reacted 1 hour.After washing 3 times with cleaning buffer solution, every hole adds colour developing liquid (10mg TMB is dissolved in the 10ml dehydrated alcohol) 100 μ L, and room temperature is observed colour-change.Reach the colour developing peak when 20min, every hole adds 100 μ L stop buffer (1mol/L H
2SO
4), OD is read in the color development stopping reaction on microplate reader
450Value.The ratio P/N of calculation sample detected value (P) and negative serum detected value (N), the P/N value was judged to be the positive greater than 2.1 o'clock.
Detect mice serum with this method, find the mice serum and the recombinant phage oil emulsion vaccine mice immunized serum of avian influenza, its P/N value is judged to be the positive all greater than 2.1; And the P/N value of inactivated avian influenza vaccine mice immunized serum is judged to be feminine gender (table 1) less than 2.1.
Table 1 mice serum ELISA detected result
| Antiserum(antisera) | OD 450Value | P/N |
| The mice serum that infects the H5N1 avian influenza virus infects the mice serum |
1.122 1.058 0.462 1.213 0.303 | 3.7 3.5 1.5 4.0 -- |
The ELISA program is as follows: is 1 * 10 with recombinant T 4 bacteriophage with PBST (PBS, pH7.2 contain 0.05% polysorbas20) dilution
8Pfu/ml, every hole adds the recombinant T 4 bacteriophage of 100 μ L dilution, places more than 12 hours for 4 ℃.After washing 3 times with cleaning buffer solution (PBS, pH7.2 contain 0.05% polysorbas20), every hole adds skim-milk/PBS of 100 μ L 5%, places 2 hours for 37 ℃.After washing 3 times with cleaning buffer solution, every hole adds the chicken serum of 100 μ L with 100 times of 1% skim-milks/PBS dilution, 37 ℃ of reactions 1 hour.After washing 3 times with cleaning buffer solution again, every hole adds 100 μ L and dilutes the anti-chicken igg antibody of goat (U.S. Sigma company) of 1000 times horseradish peroxidase-labeled with 1% skim-milk/PBS, and 37 ℃ were reacted 1 hour.After washing 3 times with cleaning buffer solution, every hole adds colour developing liquid (10mg TMB is dissolved in the 10ml dehydrated alcohol) 100 μ L, and room temperature is observed colour-change.Reach the colour developing peak at 20min, every hole adds 100 μ L stop buffer (1mol/L H
2SO
4), OD is read in the color development stopping reaction on microplate reader
450Value.The ratio P/N of calculation sample detected value (P) and negative serum detected value (N), the P/N value was judged to be the positive greater than 2.1 o'clock.
This method has good specificity.Detect newcastle disease (ND) specific serum, avian encephalomyelitis (AE) specific serum, chicken infectious bronchitis (IB) specific serum, infectious laryngotracheitis of chicken (ILT) specific serum, avian infectious capsule disease (IBD) specific serum, no cross reaction with this method.Detect picking up from 84 parts of on-the-spot chicken serum samples, detect positive 66 parts, negative 18 parts (table 2).
Table 2 chicken serum ELISA detected result
| Serum number | P/N | The result | Serum number | P/N | The result | Serum number | P/N | The result | Serum number | P/N | The |
| 1 2 3 4 5 6 | 2.24 2.67 2.33 2.44 2.70 3.23 | + + + + + + | 22 23 24 25 26 27 | 2.20 2.32 2.75 2.79 2.77 2.66 | + + + + + + | 43 44 45 46 47 48 | 2.83 2.27 2.63 2.76 2.68 2.89 | + + + + + + | 64 65 66 67 68 69 | 1.11 1.74 2.16 2.32 2.03 1.87 | - - + + - - |
| 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 | 3.06 3.16 2.92 3.06 2.57 2.49 2.52 2.39 2.93 2.52 2.64 2.89 2.81 2.53 2.51 | + + + + + + + + + + + + + + + | 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 | 2.36 2.84 2.00 3.16 2.30 2.75 2.62 2.78 2.54 3.10 3.19 3.14 2.90 2.94 2.79 | + + - + + + + + + + + + + + + | 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 | 2.92 3.05 3.06 3.20 2.48 2.67 3.01 2.24 2.16 2.74 1.97 2.02 1.79 2.17 2.45 | + + + + + + + + + + - - - + + | 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 | 1.87 2.08 2.62 1.72 1.72 1.57 1.96 1.87 2.01 2.77 2.01 2.82 2.16 1.49 2.22 | - - + - - - - - - + - + + - + |
Sequence table
＜110〉Agricultural University Of South China
＜120〉a kind of recombinant T 4 bacteriophage of displaying influenza virus non-structural protein NS 1 and application thereof
<130>SEQ ID NO：1
<160>1
<170>PatentIn version 3.3
<210>1
<211>654
<212>DNA
<213>Avian influenza virus
<400>1
atggattcca acactgtgtc aagtttccag gtagactgct ttctttggca tgtccgcaaa 60
cgatttgcag accaagaact gggtgatgcc ccatttctag accggcttcg ccgagatcag 120
aagtccctaa aaggaagagg cagcactctt ggtctggaca tcagaaccgc cactcgtgaa 180
ggaaagcata tagtggagcg gattctggag gaagagtcag atgaggcact taaaatgact 240
actgcttcag tgccagctcc acgctaccta actgacatga ctcttgaaga aatgtcaaaa 300
gattggttaa tgctcattcc caaacagaaa gtgacagggt ccctttgcat tagaatggac 360
caagctatag tggataaaaa catcacattg aaagcaaact tcagtgtgat ttttaatcga 420
ctggaagctc taatactact tagagctttt acagacgaag gaacaatagt gggtgaaatc 480
catactgatg aggatgtcaa aaattcacca ttaccttctc ttccaggagc aattggggtc 540
ctcatcggag gatttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600
ttcgcttgga gaaacagcga tgagaatggg agacctccac tccctccaaa gtag 654
＜110〉Agricultural University Of South China
＜120〉a kind of recombinant T 4 bacteriophage of displaying influenza virus non-structural protein NS 1 and application thereof
<130>SEQ ID NO：2
<160>1
<170>PatentIn version 3.3
<210>1
<211>217
<212>PRT
<213>Avian influenza virus
<400>1
Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe
20 25 30
Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser
35 40 45
Thr Leu Gly Leu Asp Ile Arg Thr Ala Thr Arg Glu Gly Lys His Ile
50 55 60
Val Glu Arg Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Thr Ala Ser Val Pro Ala Pro Arg Tyr Leu Thr Asp Met Thr Leu Glu
85 90 95
Glu Met Ser Lys Asp Trp Leu Met Leu Ile Pro Lys Gln Lys Val Thr
100 105 110
Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Val Asp Lys Asn Ile
115 120 125
Thr Leu Lys Ala Asn Phe Ser Val Ile Phe Asn Arg Leu Glu Ala Leu
130 135 140
Ile Leu Leu Arg Ala Phe Thr Asp Glu Gly Thr Ile Val Gly Glu Ile
145 150 155 160
His Thr Asp Glu Asp Val Lys Asn Ser Pro Leu Pro Ser Leu Pro Gly
165 170 175
Ala Ile Gly Val Leu Ile Gly Gly Phe Glu Trp Asn Asp Asn Thr Val
180 185 190
Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Asn Ser Asp Glu
195 200 205
Asn Gly Arg Pro Pro Leu Pro Pro Lys
210 215
Claims (7)
1, a kind of recombinant T 4 bacteriophage of displaying influenza virus non-structural protein NS 1 is characterized in that albumen NS1 and T4 phage SOC albumen composition fusion rotein, is positioned at the recombinant T 4 bacteriophage head surface.
2, recombinant T 4 bacteriophage according to claim 1 is characterized in that influenza virus non-structural protein NS 1 is:
1) has the protein of sequence shown in the SEQ ID NO:2;
2) has sequence shown in the SEQ ID NO:2 through changing, lack or increase one or several amino acid
Residue still has the active protein of influenza virus non-structural protein NS 1; Or
3) homology more than 80% is arranged and have the active protein of influenza virus non-structural protein NS 1 with the aminoacid sequence shown in the SEQ ID NO:2.
3, recombinant T 4 bacteriophage according to claim 1 and 2 is characterized in that the nucleotides sequence of encoding influenza virus non-structural protein NS 1 is classified as:
1)SEQ ID NO：1；
2) SEQ ID NO:1 still expresses and has the active proteinic sequence of influenza virus non-structural protein NS 1 through changing, lack or increase one or several Nucleotide; Or
3) with shown in the SEQ ID NO:1 in the nucleotide sequence the above section of continuous 200 bases homology more than 80% is arranged, and its expression product still has the active nucleotide sequence of influenza virus non-structural protein NS 1.
4, a kind of method for preparing the specific antibody of anti-NS1 is characterized in that recombinant T 4 bacteriophage with any claim 1-3 as antigen, direct immunization or with the T4 phage with make the vaccine immunity animal after adjuvant cooperates, obtain the specific antibody of anti-NS1.
5, method according to claim 4 is characterized in that being used for immune animal is rabbit, chicken, mouse, goat.
6, the method for avian influenza specific antibody in a kind of vitro detection animal serum is characterized in that the recombinant T 4 bacteriophage of any claim 1-3 is reacted as antigen and animal serum, and the detection reaction result estimates whether antibody exists in the serum then.
7, method according to claim 6, wherein detection method adopts enzyme-linked immunosorbent assay.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510126416 CN1800376A (en) | 2005-12-08 | 2005-12-08 | Recombinant T4 phage for displaying influenza virus non-structural protein NS1 and its uses |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044344A (en) * | 2015-06-25 | 2015-11-11 | 中牧实业股份有限公司 | Qualitative and quantitative foot and mouth disease virus antigen detection method |
| CN116555197A (en) * | 2023-06-25 | 2023-08-08 | 中国科学院深圳先进技术研究院 | Salmonella engineering membrane penetrating phage and construction method and application thereof |
-
2005
- 2005-12-08 CN CN 200510126416 patent/CN1800376A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044344A (en) * | 2015-06-25 | 2015-11-11 | 中牧实业股份有限公司 | Qualitative and quantitative foot and mouth disease virus antigen detection method |
| CN116555197A (en) * | 2023-06-25 | 2023-08-08 | 中国科学院深圳先进技术研究院 | Salmonella engineering membrane penetrating phage and construction method and application thereof |
| CN116555197B (en) * | 2023-06-25 | 2023-09-01 | 中国科学院深圳先进技术研究院 | Salmonella engineering membrane penetrating phage and construction method and application thereof |
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