CN105198970A - VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein - Google Patents
VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as preparation method and application of VP0 recombinant protein Download PDFInfo
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Abstract
The invention discloses a VP (viral protein)0 recombinant protein of DHAV (duck hepatitis A virus)-1 as well as a preparation method and an application of the VP0 recombinant protein and belongs to the technical field of bio-medicine. The preparation method of the VP0 recombinant protein comprises the following steps: a), target fragment cloning of a whole VP0 gene and expression vector construction; b), expression of the VP0 recombinant protein; c), purification of the VP0 recombinant protein. The invention further provides research on the function of the VP0 recombinant protein in detection and immune protection. A novel way is provided for detection, prevention and treatment of duck viral hepatitis, an indirect ELISA (enzyme linked immunosorbent assay) method based on the VP0 recombinant protein is established, and the method has the characteristics of good repeatability and high specificity.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein and its preparation method and application.
Background technology
Duck hepatitis has another name called duck viral hepatitis (Duckviralhepatitis, DVH), by duck hepatitis virus (Duckhepatitisvirus, DHV) a kind of high degree in contact caused and the duck viral transmissible disease of lethality, it is characterized by morbidity urgency, propagate soon, the course of disease is short, case fatality rate is high, sick duck cuts open the visible hepatomegaly of inspection, surface is dendroid hyperemia or haemorrhagic puncta, and this disease was found in the U.S. early than 1945.DHV divides three serotypes (DHV-1, DHV-2 and DHV-3) at first, DHV-2 and DHV-3 was incorporated into Astroviridae afterwards, without antigen dependency between three serotypes.Present DHV-1 has been named as DHAV (DuckhepatitisAvirus, DHAV), the fowl hepatitis virus being classified as Picornaviridae belongs to (Avihepatovirus), comprise three genotype (DHAV-1,2 and 3), wherein 1 type duck hepatitis A virus (HAV) (DHAV-1) be distribute the widest, endanger maximum DHAV.
DHAV-1 belongs to Picornaviridae fowl hepatovirus, and virus particle is spherical in shape or class is spherical, diameter 20 ~ 40nm.Such viral capsid is icosahedral symmetry, comprises 32 capsomeres, does not have cyst membrane and projection, and rate of propagation is fast, and fecundity is strong, copies a generation and is about 2h.DHAV-1 is single strand RNA virus, geneome RNA length 7kb ~ 8kb, and molecular weight is 8.6 × 10
6da, it has stronger infectivity.DHAV-1 viability is extremely strong, and it can resist the impact of external environment.Under different conditions, the viability of this virus is different.As under physical environment, DHAV-1 can be survived for a long time and breed, and shady and cool environment, as more than the 37d that can survive in muck, can be survived at 0 ~ 4 DEG C more than 2 years, and can survive at-20 DEG C and reach 9 years.DHAV-1 can tolerate strong acid, all insensitive to organic solvents such as chloroform, methyl alcohol, ether, and also has obvious resistibility to common sterilizing agent such as formaldehyde, formalin etc., also has certain tolerance effect to heat.The red corpuscle of any animal of DHAV-1 not aggegation, does not adsorb any red corpuscle yet.Research due to DHAV-1 starts more late, and the virus panel of Picornaviridae has similar gene replication, functional mechanism, so, understand the structure of other picornaviruss, gene function equimolecular biological characteristics, the research that can be DHAV-1 molecular structure and biology aspect provides better thinking and innovation.
Picornavirus is a class minimum in RNA viruses, distribution is very extensive, can be classified as enterovirus genus (enterovirus), aphthovirus genus (Aphthovirus), cardiovirus (Cardiovirus), Liposcelis entomophila (Hepatovirus), the lonely Tobamovirus (Parechovirus) of secondary intestines, equine rhinoviruses genus (Erbovirus), ridge Tobamovirus (Kobuvirus), prompt Shen Tobamovirus (Teschovirus), fowl hepatitis virus genus (Avihepatovirus) etc.Picornavirus is rounded, and virus particle diameter is 20 ~ 40nm, and without cyst membrane, the structural protein (capsid protein) of virus generally comprise VP1, VP2, VP3 and VP4.Wherein VP1, VP2, VP3 are exposed to virus surface, form a subunit.VP2 albumen is positioned at capsid surface, containing epitope and some epitope can induce body produce neutralizing antibody, exist between capsid protein VP2 and pro apoptotic protein/proapoptotic protein Siva and interact, the VP2 albumen of Coxsackie virus B3 may be attached on proapoptotic protein Siva specifically, thus affects apoptotic induction, the diffusion of virus and the pathology process that causes of virus; VP4 is embedded in virus particle inside, the inside being in protein coat is connected with geneome RNA, the conformational change occurred when the outside exposure of VP4 albumen under physiological temp and virion adherent cell film has and directly contacts, and may take part in the relevant dependent event of cell entry cell processes after virion adherent cell film.The protein coat of picornavirus has self very strong dynamic change, instead of as crystalline structure figure reflects, be in a stationary state.The research of antibody and virus structure conformation relation is to the design of ideal vaccine and prevent the propagation of picornavirus significant better.But it is relatively little to the research of DHAV capsid protein.Bioinformatic analysis and supposition show, the capsid protein of DHAV-1 may be VP1, VP3 and VP0, instead of be VP1, VP2, VP3 and VP4 as most picornavirus, therefore DHAV-1 is VP0 or VP2 and VP4 on earth, whether they are positioned at or are exposed to virus surface has no report at present, and its antigenic research report is less.
Therefore prokaryotic expression is carried out for DHAV-1VP0 gene, and the effect of VP0 albumen in detection and immunoprotection of prokaryotic expression is studied, become the focus of those skilled in the art's research, exist in prior art and carry out for DHAV-1VP0 gene the technology that prokaryotic expression obtains VP0 albumen, but this VP0 albumen still exists and is difficult to expression and purification, protein-active is low, detect duck viral hepatitis antibody precision not high, the shortcoming that detection efficiency is low, as can be seen here, the prokaryotic expression method of DHAV-1VP0 gene is made further improvements, make the VP0 expression of recombinant proteins amount that obtains large, purifying is easy, protein-active is good, have and detecting the advantage that in duck viral hepatitis antibody, precision is high, find the detection of duck viral hepatitis and the effective way of control, become those skilled in the art's problem demanding prompt solution.
Summary of the invention
An object of the present invention overcomes the problems of the prior art, provides a kind of VP0 recombinant protein and its production and use.
For solving the problems of the technologies described above, the technical scheme of employing comprises:
A kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, its aminoacid sequence is as shown in SEQIDNO:1, and its DNA sequence dna is as shown in SEQIDNO:2.
A preparation method for above-mentioned 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, comprises the steps:
Step one: the clone of 1 type duck hepatitis A virus (HAV) VP0 full genome: analyze and determine 1 type duck hepatitis A virus (HAV) VP0 full genome and restriction enzyme site, according to the DHAV-1X strain complete genome sequence that Genbank accession number is JQ316452.1, the Auele Specific Primer of design amplification 1 type duck hepatitis A virus (HAV) VP0 gene DNA fragment, add upper BamH I and Xho I two restriction enzyme sites at 5 ' end of primer simultaneously, extract the RNA of 1 type duck hepatitis A virus (HAV), utilize Auele Specific Primer to carry out RT-PCR amplification, obtain the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome;
Step 2: build recombinant expression vector pET-32a (+)/VP0: the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out T clone and obtain pMD18-T/VP0, cultivate the bacterial strain containing recombinant plasmid pMD18-T/VP0 and expression plasmid pET-32a (+) respectively, extract plasmid, and with BamH I and Xho I, double digestion is carried out to it, analyze, reclaim, after purifying, the object fragment obtained with 1 type duck hepatitis A virus (HAV) VP0 full genome of sticky end is connected with double digestion process process LAN plasmid pET-32a (+), connection product is carried out transforming and identifying,
Step 3: prepare 1 type duck hepatitis A virus (HAV) VP0 recombinant protein: be transformed into by recombinant expression vector pET-32a (+)/VP0 in Ecoli.BL21 expressive host bacterium, screening, by the bacterial strain containing recombinant expression vector pET-32a (+)/VP0 after screening through cultivating abduction delivering, by expression product after qualification, purifying, obtain 1 type duck hepatitis A virus (HAV) VP0 recombinant protein.
Further, described Auele Specific Primer is:
Upstream primer P1:5 ' GGATCCGAAATGGATACTCTTACCAAAAAT3 ',
Downstream primer P2:5 ' CTCGAGTCCCTGATTGTCAAATGGTC3 '.
Further, in described step 2, the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out the method for T clone and is: the object fragment of the 1 type duck hepatitis A virus (HAV) VP0 full genome described step one obtained carries out purifying recovery, purified product end adds A, be connected with pMD18-T carrier and obtain connecting product, by described connection product conversion to competent cell, bacterium liquid PCR screening positive clone, carries out enzyme by the pMD18-T/VP0 obtained and cuts qualification.
Further, the ligation system in described step 2 is, with the object fragment 5.5 μ l of 1 type duck hepatitis A virus (HAV) VP0 full genome of sticky end, and expression vector pET-32a (+) 2 μ l, 2 × T
4dNALigaseMix7.5 μ l.Wink from, mixing, 16 DEG C of connections are spent the night.
Further, the abduction delivering condition of described 1 type duck hepatitis A virus (HAV) VP0 recombinant protein is: be transformed into by recombinant expression vector pET-32a (+)/VP0 in Host Strains BL21,0.4mmol/LIPTG, 37 DEG C of induction 6h;
Further, in described step 3, purification process is Ni
2+-NTA affinitive layer purification.
1 type duck hepatitis A virus (HAV) VP0 recombinant protein is used for the detection reagent as detecting 1 type duck viral hepatitis antibody.
A kind of ELISA kit for detecting 1 type duck viral hepatitis antibody, comprise elisa plate, PBST damping fluid, confining liquid, ELIAS secondary antibody, nitrite ion and stop buffer, described ELISA kit also comprises 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1, the reaction conditions of described ELISA is the bag of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein as antigen is 1.67 μ g/ml by concentration, best bag is that 37 DEG C of 1h turn 4 DEG C and spend the night by condition, serum dilution is 1:160, the degree of releasing of the goat-anti duck IgG of HRP mark is 1:400, confining liquid is the gelatin of 5%, off-period is 0.5h, developing time is 10min.
1 type duck hepatitis A virus (HAV) VP0 recombinant protein is used for the genetic engineering subunit vaccine as 1 type duck viral hepatitis.
Beneficial effect of the present invention comprises:
1. successfully constructing recombinant expression vector pET-32a (+)/VP0 also can at expression in escherichia coli 1 type duck hepatitis A virus (HAV) VP0 recombinant protein; Easy acquisition meets the recombinant protein of application requiring, uses Ni
2+the method of-NTA affinitive layer purification obtains higher, the active recombinant protein preferably of purity; 1 type duck hepatitis A virus (HAV) VP0 recombinant protein of preparation can be used for detection and the control of duck viral hepatitis;
2. recombinant protein can DHAV-1 sero-reaction anti-with rabbit, has good reactionogenicity, can be used for the detection of 1 type duck hepatitis A virus (HAV) antibody; Successfully establish the indirect ELISA method based on VP0 recombinant protein, the method has reproducible and feature that is high specificity; Wherein high for duck viral hepatitis antibody test precision, make envelope antigen with the recombinant protein of 1.67 μ g/ml and can detect 2
12the serum to be checked of dilution;
3. pair VP0 recombinant protein carries out Analysis of Immunogenicity, and result shows, recombinant protein can stimulate body to produce antibody and cause the change of cytokine content, can be used for the control of 1 type duck viral hepatitis.
Accompanying drawing explanation
Figure 1 shows that the RT-PCR Product Identification figure of VP0 full genome of the present invention.
Figure 2 shows that the PCR qualification figure of pMD18-T/VP0 plasmid of the present invention.
Figure 3 shows that the enzyme of pMD18-T/VP0 plasmid of the present invention cuts qualification figure.
Figure 4 shows that the PCR qualification figure of pET-32a (+) of the present invention/VP0 plasmid.
Figure 5 shows that the enzyme of pET-32a (+) of the present invention/VP0 plasmid cuts qualification figure.
Figure 6 shows that the present invention 1 type duck hepatitis A virus (HAV) VP0 recombinant protein western blot figure.
Figure 7 shows that the electrophorogram before and after the present invention 1 type duck hepatitis A virus (HAV) VP0 recombinant protein purification.
Figure 8 shows that the optimization figure of IPTG concentration of the present invention.
Figure 9 shows that the optimization figure of abduction delivering time of the present invention.
Figure 10 shows that the optimization figure of abduction delivering temperature of the present invention.
Figure 11 shows that VP0 recombinant protein soluble analysis figure of the present invention.
Figure 12 shows that VP0 recombinant protein ELISA of the present invention detects the antibody horizontal figure of immune duckling.
Figure 13 shows that the ELISA detected result of VP0 recombinant protein of the present invention immunity duckling cytokine: the content ELISA detected result figure of Figure 13 A:VP0 recombinant protein immunity duckling CD4T lymphocyte subgroup; The content ELISA detected result figure of Figure 13 B:VP0 recombinant protein immunity duckling CD8T lymphocyte subgroup; The content ELISA detected result figure of Figure 13 C:VP0 recombinant protein immunity duckling IL-4; The content ELISA detected result figure of Figure 13 D:VP0 recombinant protein immunity duckling INF-γ.
The susceptibility of Figure 14 VP0 protein ELISA method detects analysis chart.
Embodiment
Hereafter will describe the specific embodiment of the invention in detail in conjunction with concrete accompanying drawing.It should be noted that the combination of technical characteristic or the technical characteristic described in following embodiment should not be considered to isolated, they can mutually be combined thus be reached better technique effect.
Trizolplus, PrimeScript
tMrT-PCR kit, PrimeSTARPCRMix, DL2000Marker, DL15000Marker, ligationmix connect test kit, BamHI, XhoI restriction enzyme, and the reagent such as IPTG are purchased from precious biotechnology (Dalian) company limited;
TMB nitrite ion, DAB colouring reagents box etc. are purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
Plasmid extraction test kit, DNA gel recovery test kits etc. are purchased from OMEGA;
Tris alkali, glycine, acrylamide, N, N'-methylene-bisacrylamide, ammonium persulphate, TEMED, R250, BSA, imidazoles etc. are purchased from Shanghai Sheng Gong biotechnology company limited;
Low molecular weight protein Marker, pre-dyed albumen Marker, Agarose (agarose), pvdf membrane, Ni
2+-NTA sepharose, etc. be purchased from Bio-rad;
The goat-anti duck IgG of HRP mark is purchased from KPL company;
Freund's complete adjuvant is purchased from Sigma;
IL-4, CD4+, CD8+, IFN-γ duck Virus monitory test kit is purchased from R & D company.
1 type duck hepatitis A virus (HAV) of the present invention, genome sequence has been disclosed in GenBank database, and registration number is JQ316452.1.
1 type duck hepatitis A virus (HAV) of the present invention is for obtain with reference to the Isolation and ldentification method described in open paper " research of duck viral hepatitis--pathogen separation, qualification and low virulent strain are cultivated ", " research of duck viral hepatitis--pathogen separation, qualification and low virulent strain are cultivated " was disclosed on the 19th volume 1 phase in 1993 " Chinese Veterinary Journal ", and the start-stop page number is 3 ~ 4 pages.Patent applicant ensured to provide 1 type duck hepatitis A virus (HAV) of the present invention to the public in Two decades years from the applying date.
Embodiment 1:
A kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, its aminoacid sequence is as shown in SEQIDNO:1, and its DNA sequence dna is as shown in SEQIDNO:2.
A preparation method for 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, comprises the steps:
Step one: the clone of 1 type duck hepatitis A virus (HAV) VP0 full genome: analyze and determine 1 type duck hepatitis A virus (HAV) VP0 full genome and restriction enzyme site, according to the DHAV-1X strain complete genome sequence that Genbank accession number is JQ316452.1, the Auele Specific Primer of design amplification 1 type duck hepatitis A virus (HAV) VP0 gene DNA fragment, add upper BamH I and Xho I two restriction enzyme sites at 5 ' end of primer simultaneously, extract 1 type duck hepatitis A virus (HAV) RNA, utilize Auele Specific Primer to carry out RT-PCR amplification, obtain the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome.
Wherein Auele Specific Primer is:
Upstream primer P1:5 ' GGATCCGAAATGGATACTCTTACCAAAAAT3 ' (SEQIDNO:3),
Downstream primer P2:5 ' CTCGAGTCCCTGATTGTCAAATGGTC3 ' (SEQIDNO:4).
Wherein said RT-PCR amplification adopts two-step approach, first synthesizes cDNA, then carries out pcr amplification:
1, reverse transcription:
According to the PrimeScript of TaKaRa
tMrT-PCR kit specification sheets carries out the process of template, primer, then prepares following reaction solution: template 2 μ l, random primer 3 μ l, dNTPmix (2.5mM) 4 μ l, adds ddH
2o to 10 μ l.65 DEG C of 5min, rapidly at more than chilling 2min on ice; Brief centrifugation makes the denaturing soln of template ribonucleic acid/primer be gathered at the bottom of pipe, adds in above-mentioned template ribonucleic acid/primer denaturing soln: 5 × PrimeScriptBuffer4 μ l, RNaseInhibitor0.5 μ l, PrimeScriptRTase1 μ l, adds ddH
2o to 20 μ l.Reaction conditions: 30 DEG C of 10min, 42 DEG C of 30min, 95 DEG C of 5min; After 70 DEG C of insulation 15min, cooled on ice ,-20 DEG C of preservations.
2, pcr amplification:
Add reagent by reagent specification sheets and carry out pcr amplification, amplification system: cDNA1 μ L, 2 × PCRmix10 μ L, each 1 μ l of primer (10pm), adds ddH
2o to 20 μ l.Pcr amplification program is: 95 DEG C of 5min, (94 DEG C of 40s, 55 DEG C of 40s, 72 DEG C of 1min) × 35cycles, 72 DEG C of 10min, 16 DEG C of 2h.Terminate rear 4 DEG C of preservations.
3, the detection of RT-PCR product:
Get after 10 μ LPCR products mix with 1 μ L sample-loading buffer, 1% agarose gel electrophoresis 30min, observes amplified fragments under ultraviolet lamp.
Detected result: with the virus genome RNA extracted for template, RT-PCR obtains the gene fragment meeting expection clip size, the object fragment length of 1 type duck hepatitis A virus (HAV) VP0 full genome is 768bp, result as shown in Figure 1, the RT-PCR product of M:DL2000Marker, 1:VP0 full genome in Fig. 1.
Step 2: build recombinant expression vector pET-32a (+)/VP0: the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out T clone and obtain pMD18-T/VP0, cultivate the bacterial strain containing recombinant plasmid pMD18-T/VP0 and expression plasmid pET-32a (+) respectively, extract plasmid, and with BamH I and Xho I, double digestion is carried out to it, analyze, reclaim, after purifying, the object fragment obtained with 1 type duck hepatitis A virus (HAV) VP0 full genome of sticky end is connected with double digestion process process LAN plasmid pET-32a (+), connection product is carried out transforming and identifying.
Wherein the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out T clone and obtains the method for pMD18-T/VP0 and step is:
1, the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome that namely obtains of RT-PCR product and the connection of pMD18-T carrier:
Identify that correct object fragment is pressed DNA glue and reclaimed the recovery of test kit specification sheets purifying, purified product end adds A: common 2 × Taq enzyme Mix2.5 μ L, and glue reclaims product 7.5 μ L, mixing, and 72 DEG C extend 30min; Add the linked system that 10 μ L set up by following reagent in order successively: 2 × T
4dNAligaseMix5 μ L, pMD18-T carrier 1 μ L, adds the object fragment 4 μ L after A.After mixing, gentle centrifugation is at the bottom of pipe.16 DEG C of water-bath 2 ~ 4h or spend the night.
2, the conversion of recombinant DNA molecules:
(1) preparation of competent cell:
Under aseptic condition, this laboratory of streak inoculation is prepared and is stored in the escherichia coli DH5a of-70 DEG C of Ultralow Temperature Freezers, is inverted overnight incubation for 37 DEG C; Next day chooses bacterium, and in inoculation LB liquid nutrient medium, constant temperature jolting is spent the night; According to 1:100 enlarged culturing to OD
600nmbe 0.4 ~ 0.5, ice bath 1h; Under aseptic condition, bacterium liquid 4 DEG C, the centrifugal 10min of 4000r/min, abandon most supernatant liquor; The aseptic CaCl of 0.1mol/L of 10mL precooling
2the resuspended precipitation of solution, ice bath 30min, 4 DEG C of centrifugal 10min of 4000r/min, abandon most supernatant liquor; Ice precooling 0.1mol/L is containing the aseptic CaCl of 15% glycerine
2solution 1mL is resuspended, and after mixing, with 50 μ L/ pipe packing ,-70 DEG C save backup.
(2) conversion of product is connected:
Add in DH5a competent cell and connect product 5 μ L, slowly softly blow and beat mixing, place 30min on ice; 42 DEG C of heat shock 90s, ice bath 2min, add 445 μ LLB liquid nutrient mediums, the centrifugal 2min of 37 DEG C of 200 ~ 250r/min shaking culture 1h, 4000r/min, mixing; Bacterium liquid is spread evenly across containing on antibiotic LB agar plate, treats that bacterium liquid is absorbed rearmounted 37 DEG C of thermostat container incubated overnight and occurs to single bacterium colony; The single bacterium colony of picking spends the night in 37 DEG C of 300r/min shaking culture respectively.
(3) bacterium liquid PCR screening positive clone:
To become the bacterium liquid of muddiness into template, pcr amplification VP0 gene.Product 1% agarose gel electrophoresis checks amplification.
(4) enzyme of recombinant plasmid cuts qualification:
Carry out plasmid extraction according to plasmid extraction test kit operation steps, through BamH I and the qualification of Xho I double digestion, enzyme cuts system: 10 × buffer1 μ l, BamH I 0.5 μ l, Xho I 0.5 μ l, recombinant plasmid 8 μ l.37 DEG C of enzymes cut 1.5h, and digestion products 1% agarose electrophoresis is identified.
Qualification result:
After VP0 full genome is connected to pMD18-T, cut through PCR and enzyme the band all obtaining after qualification expecting, in pMD18-T/VP0, the length of VP0 gene is 768bp, as shown in Figures 2 and 3.M:DL2000Marker, 1:PCR product in Fig. 2; M:DL2000Marker, 1:BamH I/Xho I digestion products in Fig. 3.
Wherein the extraction of recombinant plasmid pMD18-T/VP0 and expression plasmid pET-32a (+) and the method for double digestion are: cultivate the bacterial strain containing recombinant plasmid pMD18-T/VP0 and expression plasmid pET-32a (+) respectively, extract plasmid, and with BamH I and Xho I, double digestion is carried out to it, method is the same, carry out electrophoretic analysis enzyme with 1.0% sepharose and cut result, cut glue and reclaim and purifying goal gene and linearizing plasmid vector.
Method wherein with object fragment and expression vector pET-32 (a)+be connected of 1 type duck hepatitis A virus (HAV) VP0 gene DNA full genome of sticky end is: be connected with the expression vector of double digestion process by the goal gene with sticky end obtained, ligation system: goal gene fragment 5.5 μ l, expression vector 2 μ l, 2 × T
4dNALigaseMix7.5 μ l.Wink from, mixing, 16 DEG C of connections are spent the night.
The conversion that this place described connects product is identical with the method for qualification with the conversion in T cloning process with authentication method.Finally to check order confirmation.
Qualification result: by VP0 full gene cloning to pET-32 (a)+carrier, the band all obtaining after qualification expecting is cut through PCR and enzyme, in recombinant plasmid pET-32 (a) +/VP0, the length of VP0 full genome is 768bp, as shown in Figure 4 and Figure 5, M:DL2000Marker, 1:PCR product in Fig. 4; M:DL15000Marker, 1:BamH I/Xho I digestion products in Fig. 5.PET-32 (a) +/VP0 sequencing result is shown by Blastn sequence analysis analysis on NCBI, and the gene fragment size inserted in carrier is 768bp, consistent with expection, shows that recombinant expression vector successfully constructs.
Step 3: prepare 1 type duck hepatitis A virus (HAV) VP0 recombinant protein: be transformed into by recombinant expression vector pET-32a (+)/VP0 in Ecoli.BL21 expressive host bacterium, screening, by the bacterial strain containing recombinant expression vector pET-32a (+)/VP0 after screening through cultivating abduction delivering, by expression product after qualification, purifying, obtain 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, purification process is Ni
2+-NTA affinitive layer purification.
Wherein the screening method of target protein expression bacterium is: be transformed into by expression plasmid pET-32a (+)/VP0 in Ecoli.BL21 expressive host bacterium, transformed bacteria is through 37 DEG C of incubated overnight, and next day is about 2.5h by 1:100 enlarged culturing, bacterium liquid OD
600nmadding IPTG when reaching 0.6 to concentration is 0.4mmol/L37 DEG C of induction 4h, bacterium will be expressed with the centrifugal 10min of 12000r/min, precipitation 20mmol/LTris-HCl (pH8.0) suspends by 1:10 bacterium liquid, the centrifugal 10min of 12000r/min after ice-bath ultrasonic fragmentation, supernatant is got 80ul and is added 20ul5 × SDSloadingbuffer, precipitation 1ml8M urea dissolves, and the centrifugal 10min of 8000r/min, gets this lysate of 80ul and add 20ul5 × SDSloadingbuffer equally.Sample boils 10min, centrifugal 10min, the SDS-PAGE electrophoresis detection of 10000r/min.
To the expression condition optimization of target protein, concrete optimization step is shown in embodiment 2, wherein the best abduction delivering condition of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein is: be transformed in Host Strains BL21 by recombinant expression vector pET-32 (a) +/VP0,0.4mmol/LIPTG, 37 DEG C of induction 6h, recombinant protein size is about 50KD.On the VP0 recombinant protein of expressing, cleer and peaceful inclusion body has existence.
Wherein said authentication method is Western-blot identification method: detect according to the protein immunoblot handbook of Bio-rad company: SDS-PAGE electrophoresis; Turning sandwich according to installing electricity from the order of positive pole → negative pole: sponge, 3 filter paper, pvdf membrane, PAGE glue, 3 filter paper, sponge, putting into the electrophoresis chamber filling transferring film damping fluid, electrophoresis chamber being put into frozen water; 60 ~ 80V transferring film, 1 ~ 2h, takes out pvdf membrane, PBST rinsing 3 times, 5min/ time; With the rabbit anti-DHAV-1 sera incubation pvdf membrane of confining liquid dilution, act on 1h under room temperature, wash film 3 times with PBST, each 10min ~ 15min; The goat anti-rabbit igg diluting HRP mark with confining liquid 1:1000 hatches 1h; PBST liquid washs 3 times again, each 10min ~ 15min; Add DAB substrate nitrite ion, jog colour developing 15min, treat that object band is high-visible, ultrapure water color development stopping, thieving paper suck dry moisture, keeps in Dark Place.
Qualification result: the immunoblot results of albumen shows as shown in Figure 6, M in Fig. 6: pre-dyed albumen Marker, 1:VP0 recombinant protein.The recombinant protein of expressing can be combined with DHAV-1 antibodies specific, shows that the recombinant protein of expressing by the identification of DHAV-1 serum, can have good reactionogenicity.
Wherein the purification process of recombinant protein comprises:
1, the great expression of target protein
Under the suitableeest IPTG concentration, optimum temperuture and best induction time condition, expressive host bacterium is incubated in LB/Amp liquid nutrient medium, sample preparation, upper cleer and peaceful inclusion body-20 DEG C is saved backup.
2, the affinitive layer purification of target protein
By the product of great expression according to affinity chromatography specification sheets purification process purification of recombinant proteins, the albumen of purifying is carried out dialysis concentrated after, get the appropriate concentration detecting purifying protein, residue is placed in-20 DEG C and saves backup.
3, target protein washing inclusion body purification
By great expression bacterium according to after the method process of sample preparation, the urea washes of the inclusion body different concns of collection, centrifuging and taking supernatant sample preparation after washing, the effect of SDS-PAGE electrophoresis detection purifying.
4, expressing protein cuts glue purification
By great expression bacterium according to after method for making sample sample preparation, the electrophoresis chamber adopting Bio-rad company to buy carries out cutting glue and reclaims, sample preparation after concentrated, the effect of SDS-PAGE electrophoresis detection purifying.
Through the trial of three kinds of purification process, find Ni
2+the best results of-NTA affinitive layer purification.Through Ni
2+-NTA affinitive layer purification, obtains the albumen that purity, concentration are all higher, as shown in Figure 7, in Fig. 7 before 1:1 type duck hepatitis A virus (HAV) VP0 recombinant protein purification, after 2:1 type duck hepatitis A virus (HAV) VP0 recombinant protein purification.
The optimization of embodiment 2 expression of recombinant proteins condition
Experimental technique:
1, the optimization of IPTG concentration
Inoculated in 4 LB/Amp substratum test tubes according to 1:100 by expression bacterium, 37 DEG C are cultured to OD
600nmto 0.6 time, adding IPTG respectively to final concentration is: 0mmol/L, 0.4mmol/L, 0.8mmol/L and 1.2mmol/L inducing culture about 4h, sample preparation, SDS-PAGE electrophoresis.
2, the optimization of induction time
Expression bacterium is inoculated in 5 LB/Amp substratum test tubes according to 1:100, OD to be cultured to
600nmafter reaching 0.6, add best IPTG concentration, induce 4h, 6h, 8h, 10h and 12h respectively, process the same.
3, the optimization of inducing temperature
Expression bacterium is inoculated in 3 LB/Amp substratum test tubes according to 1:100, OD to be cultured to
600nmafter reaching 0.6, add best IPTG concentration, inducing culture about 4h under 20 DEG C, 30 DEG C and 37 DEG C of conditions, processes the same respectively.
Experimental result:
Recombinant plasmid pET-32 (a) +/VP0 is transformed in Host Strains BL21, optimizing its optimum condition of the expression is 0.4mmol/LIPTG, 37 DEG C of induction 6h, as shown in figs. 8-10, the size of recombinant protein is about 50KD, on the VP0 recombinant protein of expressing, cleer and peaceful inclusion body has existence, as shown in figure 11.Wherein in Fig. 8, M: albumen Marker, 1 ~ 4 is respectively: the expression product of the IPTG concentration of 0mmol/L, 0.4mmol/L, 0.8mmol/L and 1.2mmol/L; In Fig. 9, M: albumen Marker, 1 ~ 5 is respectively: the expression product of 4h, 6h, 8h, 10h and 12h; In Figure 10,1 ~ 3 is respectively: the expression product of 37 DEG C, 30 DEG C and 20 DEG C; In Figure 11, M: albumen Marker, 1: inclusion body expression product, 2: supernatant expression product.
Embodiment 3 is based on the foundation of the ELISA method of recombinant protein
Test method:
1, based on the optimization of the ELISA method reaction conditions of recombinant protein
Conventionally operate ELISA reaction conditions, the optimization of ELISA method reaction conditions is specific as follows:
(1) antigen coated concentration: VP0 recombinant protein (1mg/ml) does 1:100,1:200,1:300,1:400,1:600,1:800,1:1200,1:1600 and 1:2400 nine extent of dilution;
(2) optimum dilution degree of serum: DHAV-1 male/female serum is 1:10 respectively; 1:20,1:40,1:80,1:160,1:320 and 1:640 seven extent of dilution;
(3) optimum dilution degree of enzyme labelled antibody: 1:200,1:400,1:800,1:1600 and 1:3200 five extent of dilution are set;
(4) bag of albumen is by condition: 37 DEG C of 1h turn 4 DEG C spend the night, 37 DEG C of 4h turn 4 DEG C spend the night, direct 4 DEG C spend the night and 37 DEG C of 2h, tetra-conditions;
(5) selection of confining liquid: 1%BSA, 5%BSA, 1% skim-milk, 5% skim-milk, 1% gelatin and 5% gelatin, six kinds of confining liquids are set;
(6) off-period: 0.5h, 1h, 1.5h and 2h tetra-gradients are set;
(7) developing time: 5min, 10min, 15min, 20min, 25min and 30min six gradients are set.
2, the determination of positive threshold value:
56 parts of negative serum OD are detected with the top condition of ELISA method optimization
450nm/ OD
630nmvalue, calculates cut-off value=average+3 × standard deviation (Vcut-off=X+3 × SD).
3, the specific detection of ELISA method
(1) specific cross experiment
With the positive serum that the ELISA method set up detects Salmonella anatis, E. coli isolated from ducks, duck swell Mo Shi bacillus in head septicaemia virus, avian influenza virus (H5), duck plague virus and duck, DHAV-1 positive serum, negative serum control are set.
(2) specific inhibition experiment
Be antigen 1 0:1 positive serum by volume ratio, in 37 DEG C He after 1h, be diluted to best serum dilution, detect by the ELISA method set up, calculate blocking-up rate.
4, the variation coefficient
Get the enzyme plate (bar) of the recombinant protein antigen bag quilt with a collection of purifying, according to the top condition operation detection 6 parts of known positive serums of the ELISA method set up, every part of serum arranges 6 repetitions, calculates variation within batch coefficient; Get the antigen coated enzyme plate (bar) of different batches purifying, detect 6 parts of identical positive serums, every part of serum arranges 6 repetitions, calculates interassay coefficient of variation, variation coefficient CV=standard deviation/mean value (SD/M × 100%).
5, ELISA method susceptibility detects
Get after 5 parts of known duck hepatitis A virus (HAV) positive serum 1:10 dilute, then with 2 times of doubling dilutions, detect by the ELISA method set up, determine to detect positive maximum serum dilution
Test-results:
1, based on the optimization of the ELISA method reaction conditions of recombinant protein:
(1) the best bag of antigen is by the optimum dilution degree of concentration and serum
Each antigen diluent degree arranges 2 repetitions, averages, and selects P/N value the maximum to be top condition.As shown in Table 1, the best bag of VP0 recombinant protein antigen is 1.67 μ g/ml by concentration, and serum dilution is 1:160.
Table 1VP0 recombinant protein antigen bag is by concentration and an antiserum(antisera) optimum dilution degree
(2) optimum dilution degree of enzyme labelled antibody
As shown in Table 2, the optimum dilution degree of the goat-anti duck IgG of HRP mark is 1:400.
The best effort concentration of table 2 ELIAS secondary antibody
| ELIAS secondary antibody extent of dilution | 1:200 | 1:400 | 1:800 | 1:1600 | 1:3200 |
| Positive | 0.836 | 0.474 | 0.233 | 0.096 | 0.039 |
| Negative | 0.141 | 0.059 | 0.022 | 0.003 | 0.003 |
| P/N | 5.945 | 8.092 | 10.415 | 28.324 | 14.808 |
(3) bag of recombinant protein is by condition
As shown in Table 3, the best bag of VP0 recombinant protein is that 37 DEG C of 1h turn 4 DEG C and spend the night by condition.
Table 3VP0 recombinant protein bag is by the optimization of condition
| Bag is by condition | 37 DEG C of 1h turn 4 DEG C and spend the night | 37 DEG C of 4h turn 4 DEG C and spend the night | 4 DEG C are spent the night | 37℃2h |
| Positive | 1.232 | 1.079 | 1.058 | 0.841 |
| Negative | 0.256 | 0.272 | 0.274 | 0.212 |
| P/N | 5.352 | 3.767 | 3.784 | 3.886 |
(4) selection of best confining liquid
As shown in Table 4, best confining liquid is the gelatin of 5%.
The selection of the best confining liquid of table 4
| Confining liquid | 1％BSA | 5％BSA | 1% gelatin | 5% gelatin | 1% skim-milk | 5% skim-milk |
| Positive | 1.001 | 0.891 | 0.966 | 0.910 | 0.905 | 0.887 |
| Negative | 0.299 | 0.264 | 0.251 | 0.227 | 0.245 | 0.228 |
| P/N | 3.347 | 3.377 | 3.858 | 4.013 | 3.698 | 3.890 |
(5) best off-period
As shown in Table 5, best off-period is 0.5h.
The optimization of table 5 off-period
| Off-period | 0.5h | 1h | 1.5h | 2h |
| Positive | 0.984 | 0.954 | 0.912 | 0.899 |
| Negative | 0.229 | 0.221 | 0.229 | 0.208 |
| P/N | 4.319 | 4.209 | 3.963 | 4.272 |
(6) selection of developing time
As shown in Table 6, best developing time is 10min.
The optimization of the best developing time of table 6
| Developing time | 5min | 10min | 15min | 20min | 25min | 30min |
| Positive | 0.750 | 0.824 | 0.927 | 0.936 | 0.936 | 0.892 |
| Negative | 0.197 | 0.177 | 0.208 | 0.208 | 0.227 | 0.219 |
| P/N | 4.266 | 4.836 | 4.299 | 4.303 | 4.268 | 4.019 |
2, the determination of positive threshold value
As shown in Table 7 ,+3 × standard deviation of averaging is as positive threshold value, then property threshold value is 0.120+3 × 0.085=0.375.
The OD of table 756 part negative serum
450nm/ OD
630nmvalue
| 0.147 | 0.120 | 0.050 | 0.056 | 0.127 | 0.094 | 0.152 | 0.147 |
| 0.022 | 0.057 | 0.224 | 0.091 | 0.092 | 0.035 | 0.285 | 0.022 |
| 0.150 | 0.364 | 0.039 | 0.085 | 0.107 | 0.035 | 0.213 | 0.150 |
| 0.182 | 0.102 | 0.176 | 0.161 | 0.067 | 0.095 | 0.317 | 0.182 |
| 0.086 | 0.390 | 0.084 | 0.099 | 0.319 | 0.087 | 0.086 | 0.086 |
| 0.133 | 0.253 | 0.123 | 0.154 | 0.073 | 0.048 | 0.044 | 0.133 |
| 0.094 | 0.090 | 0.077 | 0.054 | 0.051 | 0.054 | 0.039 | 0.094 |
3, specificity experiments
(1) specific cross experiment
Learnt by table 8, detecting DHAV-1 positive serum based on VP0 recombinant protein indirect ELISA method is strong positive reaction, and present negative reaction with the positive serum of other cause of diseases of bird, namely OD value is all lower than positive threshold value, illustrates that the method all has good specificity.
Table 8VP0 recombinant protein ELISA method specific detection
(2) specific inhibition experiment
Learnt by table 9, it is 94% that this indirect ELISA method detects the blocking-up rate of VP0 recombinant protein to DHAV-1 positive serum obtained, and shows that recombinant protein can specific binding good with DHAV-1 positive serum.
Table 9VP0 recombinant protein blocking experiment
| Before blocking-up | 1.325 |
| After blocking-up | 0.080 |
| Blocking-up rate | 94％ |
4, the variation coefficient
The results are shown in Table 10, enzyme plate (bar) the variation within batch coefficient of recombinant protein antigen bag quilt is at 0.82%-2.94%, and interassay coefficient of variation, at 1.25%-5.84%, is all less than 10%.
In table 10 batch and interassay coefficient of variation
5, ELISA method susceptibility detects
Susceptibility detected result is shown in Figure 14, detects 5 parts of positive serums, according to positive threshold determination serum-dilution to OD during 1:5120
450nm/ OD
630nm>0.375, namely the sensitivity of VP0 albumen is 1:5120, shows that ELISA method all has higher susceptibility.
The indirect ELISA method of embodiment 4VP0 recombinant protein is to the detection of duck serum sample
Detect 60 parts of duck serum samples with the indirect ELISA method based on VP0 albumen, 50 parts of serum are positive as a result; Detect 60 parts of identical serum with the ELISA method based on DHAV-1 simultaneously, the relatively detected result of two kinds of ELISA method, as shown in Table 11, positive rate based on the indirect ELISA method of VP0 albumen is 92.3%, negative recall rate is 75%, is 90% with the positive coincidence rate of the ELISA method of DHAV-1.
Table 11 is with the detection of the ELISA method coincidence rate of VP0 albumen and DHAV-1
Embodiment 5VP0 recombinant protein is to the immunogenic test experience of duckling
Experimental technique:
1, laboratory animal grouping and immunity
Carry out being grouped into VP0 recombinant protein group and blank group according to table 12, carry out immunity according to the immunizing dose in table and approach.
The grouping of table 12 animal and immunity
| Group | Size of animal | Immunization time | Immunization route and dosage |
| VP0 recombinant protein+Freund's complete adjuvant | 10 | 1 age in days | Neck is subcutaneous, 0.1mg albumen |
| Blank group | 10 | 1 age in days | Subcutaneous, physiological saline |
2, duck blood specimen collection is tested
2 groups of ducklings are respectively at 3d, 7d, 15d and 21d jugular vein blood collection before immunity and after immunity, and only, the heparin sodium anti-freezing of half blood is used for the detection of t lymphocyte subset group to 1ml/, half blood without the need to anti-freezing, for the detection of humoral immunity level and cytokine.
3, the detection of immune cell factor
The level of CD4, CD8, IL-4 and INF-γ 4 kinds of immune cell factors is measured according to cytokine ELISA kit specification sheets.
4, duckling antibody titer detects
The ELISA method based on VP0 recombinant protein set up according to embodiment 3 detects the antibody titer of serum sample.
Experimental result:
1, the detection of antibody horizontal
That detects different time points immune duck serum antibody based on VP0 recombinant protein ELISA method the results are shown in Figure 12, compared with control group, VP0 recombinant protein immune group antibody horizontal 7d after immunity all slightly raises, 15d peaks, after immunity, 21d declines but still maintains certain level, and antibody do not detected during control group 21d.
2, the level detection of immune cell factor
Cytokines measurement the results are shown in Figure 13, after the immunity of VP0 recombinant protein, t lymphocyte subset group and cytokine content all have rising in various degree, after immunity, 7d peaks, and significant difference (P<0.05) is there is compared with control group, the content of cytokine reduces rapidly afterwards, and 15 ~ 21d content is close to control group.
According to bioinformatic analysis and supposition, VP0 of the present invention has good antigenicity, to the prokaryotic expression of VP0 gene to seem very meaningful to the research of follow-up function.
About the vivoexpression of VP0 albumen, have bibliographical information to cross protokaryon and eukaryotic expression, wherein the expression amount of eucaryon is low, and cost is high, is not suitable for large-scale production practice.And during escherichia coli prokaryotic expression, it is generally acknowledged the native conformation of albumen closer to albumen of solubility expression, using value is higher, and inclusion body is the aggregate of non-activity, can affect the biologic activity of albumen.Recombinant expression vector pET-32a (+)/VP0 of this research and establishment expresses with inclusion bodies in e. coli bl21; can protected protein from the degraded of proteolytic enzyme; so easily obtain a large amount of expressing proteins; key is renaturation, again folding thus have good antigenicity in vitro after making how to make solubilization of inclusion bodies; the VP0 recombinant protein of expressing with inclusion bodies is after obtaining by preparation method of the present invention, and the no matter antigen coated concentration of indirect ELISA set up with it, sensitivity are all better than the VP0 recombinant protein of the solubility expression of existing report.Therefore the present invention can easily obtain the expressing protein meeting production practice and application in a large number, and can obtain the high and expressing protein that purity is large of concentration by centrifugal inclusion body, protein purification cost is low, simple to operate.
Although the VP0 recombinant protein also having to express detects the report of the indirect ELISA method of DHAV-1 antibody as envelope antigen before, but recombinant protein is higher as antigen coated concentration, 8 μ g/ml are needed as during envelope antigen after the VP0 recombinant protein purification that inclusion body is expressed, as also needing 4 μ g/ml during envelope antigen after the VP0 recombinant protein purification of solubility expression, because its recombinant protein is mainly expressed with the form of inclusion body, lower and the protein purification poor effect of solubility expression amount, therefore obtains the VP0 recombinant protein meeting service requirements comparatively difficult.This test establishes the indirect ELISA method detecting DHAV-1 antibody using the VP0 recombinant protein of prokaryotic expression as envelope antigen, recombinant protein is only 1.67 μ g/ml as the concentration of envelope antigen, for existing VP0 inclusion body expression and purification albumen bag by concentration 1/4 less than, solubility expression purifying protein bag by concentration 1/2 less than, show that the VP0 recombinant protein that this research is expressed is better as the antigen of the indirect ELISA method detecting DHAV-1 antibody, because the VP0 recombinant protein in the present invention is mainly with inclusion bodies great expression, purer target protein is obtained after affinity chromatography purifying, solve desired recombinant protein in prior art to be difficult to express, be difficult to the key issue of purifying, in addition, namely the VP0 recombinant protein of bibliographical information cannot detect most of positive serum as the indirect ELISA method of envelope antigen after 3 or 4 doubling dilutions, can detect 2 than virus as the indirect ELISA of envelope antigen
10the positive serum gap of more than diluting is very large, and the indirect ELISA that the VP0 recombinant protein of this research is set up can detect 1:5120 and (is greater than 2
12) positive serum that dilutes, reach the sensitivity of the indirect ELISA using virus as envelope antigen completely, swell to Salmonella anatis, E. coli isolated from ducks, duck the detection of positive serum of a septicaemia virus, avian influenza virus, duck plague virus and riemerella anatipestifer, present method demonstrates good specificity, by detection and the application of the enzyme plate of the VP0 recombinant protein antigen bag quilt with a collection of and different batches purifying, present method demonstrates good circulation ratio, 1 type duck hepatitis A virus (HAV) VP0 recombinant protein of the present invention is applied to the detection reagent as detecting duck viral hepatitis antibody.VP0 recombinant protein of the present invention also can be applicable to the genetic engineering subunit vaccine as duck viral hepatitis.VP0 recombinant protein also can be used for using in the ELISA kit of detection duck viral hepatitis antibody, described test kit comprises elisa plate, PBST damping fluid, confining liquid, ELIAS secondary antibody, nitrite ion and stop buffer, described ELISA kit also comprises 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1, 1 type duck hepatitis A virus (HAV) VP0 recombinant protein is 1.67 μ g/ml as the bag of antigen by concentration, best bag is that 37 DEG C of 1h turn 4 DEG C and spend the night by condition, serum dilution is 1:160, the degree of releasing of the goat-anti duck IgG of HRP mark is 1:400, confining liquid is the gelatin of 5%, off-period is 0.5h, developing time is 10min.
Although give some embodiments of the present invention, it will be understood by those of skill in the art that without departing from the spirit of the invention herein, can change embodiment herein.Above-described embodiment is exemplary, should using embodiment herein as the restriction of interest field of the present invention.
Claims (10)
1. a 1 type duck hepatitis A virus (HAV) VP0 recombinant protein, it is characterized in that, its aminoacid sequence is as shown in SEQIDNO:1, and its DNA sequence dna is as shown in SEQIDNO:2.
2. a preparation method for 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1, is characterized in that, comprise the steps:
Step one: the clone of 1 type duck hepatitis A virus (HAV) VP0 full genome: analyze and determine 1 type duck hepatitis A virus (HAV) VP0 full genome and restriction enzyme site, according to the DHAV-1X strain complete genome sequence that Genbank accession number is JQ316452.1, the Auele Specific Primer of design amplification 1 type duck hepatitis A virus (HAV) VP0 gene DNA fragment, add upper BamH I and Xho I two restriction enzyme sites respectively at 5 ' end of upstream and downstream primer simultaneously, extract the RNA of 1 type duck hepatitis A virus (HAV), utilize Auele Specific Primer to carry out RT-PCR amplification, obtain the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome;
Step 2: build recombinant expression vector pET-32a (+)/VP0: the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out T clone and obtain pMD18-T/VP0, cultivate the bacterial strain containing recombinant plasmid pMD18-T/VP0 and expression plasmid pET-32a (+) respectively, extract plasmid, and with BamH I and Xho I, double digestion is carried out to it, analyze, reclaim, after purifying, the object fragment obtained with 1 type duck hepatitis A virus (HAV) VP0 full genome of sticky end is connected with double digestion process process LAN plasmid pET-32a (+), connection product is carried out transforming and identifying,
Step 3: prepare 1 type duck hepatitis A virus (HAV) VP0 recombinant protein: be transformed into by recombinant expression vector pET-32a (+)/VP0 in Ecoli.BL21 expressive host bacterium, screening, by the bacterial strain containing recombinant expression vector pET-32a (+)/VP0 after screening through cultivating abduction delivering, by expression product after qualification, purifying, obtain 1 type duck hepatitis A virus (HAV) VP0 recombinant protein.
3. the preparation method of a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 2, it is characterized in that, described Auele Specific Primer is:
Upstream primer P1:5 ' GGATCCGAAATGGATACTCTTACCAAAAAT3 ',
Downstream primer P2:5 ' CTCGAGTCCCTGATTGTCAAATGGTC3 '.
4. the preparation method of a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 2, it is characterized in that, in described step 2, the object fragment of 1 type duck hepatitis A virus (HAV) VP0 full genome is carried out the method for T clone and is: the object fragment of the 1 type duck hepatitis A virus (HAV) VP0 full genome described step one obtained carries out purifying recovery, purified product end adds A, be connected with pMD18-T carrier and obtain connecting product, by described connection product conversion to competent cell, bacterium liquid PCR screening positive clone, carries out enzyme by the pMD18-T/VP0 obtained and cuts qualification.
5. the preparation method of a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 2, it is characterized in that, ligation system in described step 2 is, with the object fragment 5.5 μ l of 1 type duck hepatitis A virus (HAV) VP0 full genome of sticky end, expression vector pET-32a (+) 2 μ l, 2 × T
4dNALigaseMix7.5 μ l.Wink from, mixing, 16 DEG C of connections are spent the night.
6. the preparation method of a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 2, it is characterized in that, the abduction delivering condition of described 1 type duck hepatitis A virus (HAV) VP0 recombinant protein is: be transformed into by recombinant expression vector pET-32a (+)/VP0 in Host Strains BL21,0.4mmol/LIPTG, 37 DEG C of induction 6h.
7. the preparation method of a kind of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 2, it is characterized in that, in described step 3, purification process is Ni
2+-NTA affinitive layer purification.
8. 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1 is used for the detection reagent as detecting 1 type duck viral hepatitis antibody.
9. one kind for detecting the ELISA kit of 1 type duck viral hepatitis antibody, it is characterized in that, comprise elisa plate, PBST damping fluid, confining liquid, ELIAS secondary antibody, nitrite ion and stop buffer, described ELISA kit also comprises 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1, the reaction conditions of described ELISA is the bag of 1 type duck hepatitis A virus (HAV) VP0 recombinant protein as antigen is 1.67 μ g/ml by concentration, best bag is that 37 DEG C of 1h turn 4 DEG C and spend the night by condition, serum dilution is 1:160, the degree of releasing of the goat-anti duck IgG of HRP mark is 1:400, confining liquid is the gelatin of 5%, off-period is 0.5h, developing time is 10min.
10. 1 type duck hepatitis A virus (HAV) VP0 recombinant protein according to claim 1, is characterized in that, for the genetic engineering subunit vaccine as 1 type duck viral hepatitis.
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| CN110283836A (en) * | 2019-06-24 | 2019-09-27 | 四川农业大学 | 3 type duck hepatitis A virus mutated gene ISA-T1142A of one kind and construction method |
| CN110283834A (en) * | 2019-06-24 | 2019-09-27 | 四川农业大学 | 3 type duck hepatitis A virus mutated gene ISA-A117C of one kind and construction method |
| CN110295149A (en) * | 2019-06-24 | 2019-10-01 | 四川农业大学 | A kind of CH-P60-117C plants of 3 type duck hepatitis A virus of mutant strain and construction method |
| CN110684781A (en) * | 2019-06-24 | 2020-01-14 | 四川农业大学 | Type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A and construction method thereof |
| CN117069866A (en) * | 2023-10-16 | 2023-11-17 | 东北农业大学 | Duck hepatitis A virus type 3 recombinant immunogen and application thereof |
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| CN110095607A (en) * | 2019-04-16 | 2019-08-06 | 东北农业大学 | For detecting universal indirect ELISA reagent kit and its application of 1 type and 3 type duck hepatitis A virus serum antibodies |
| CN110095607B (en) * | 2019-04-16 | 2021-08-10 | 东北农业大学 | Universal indirect ELISA kit for detecting 1-type and 3-type duck hepatitis A virus serum antibodies and application thereof |
| CN110283836A (en) * | 2019-06-24 | 2019-09-27 | 四川农业大学 | 3 type duck hepatitis A virus mutated gene ISA-T1142A of one kind and construction method |
| CN110283834A (en) * | 2019-06-24 | 2019-09-27 | 四川农业大学 | 3 type duck hepatitis A virus mutated gene ISA-A117C of one kind and construction method |
| CN110295149A (en) * | 2019-06-24 | 2019-10-01 | 四川农业大学 | A kind of CH-P60-117C plants of 3 type duck hepatitis A virus of mutant strain and construction method |
| CN110684781A (en) * | 2019-06-24 | 2020-01-14 | 四川农业大学 | Type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A and construction method thereof |
| CN110684781B (en) * | 2019-06-24 | 2021-05-28 | 四川农业大学 | Type 3 duck hepatitis A virus mutant gene ISA-A117C-T1142A and construction method thereof |
| CN110283836B (en) * | 2019-06-24 | 2021-07-06 | 四川农业大学 | Type 3 duck hepatitis A virus mutant gene ISA-T1142A and construction method |
| CN110295149B (en) * | 2019-06-24 | 2022-06-24 | 四川农业大学 | Mutant strain 3 type duck hepatitis A virus CH-P60-117C strain and construction method thereof |
| CN117069866A (en) * | 2023-10-16 | 2023-11-17 | 东北农业大学 | Duck hepatitis A virus type 3 recombinant immunogen and application thereof |
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Application publication date: 20151230 |