CN105198969B

CN105198969B - The B cell epitope and its identification method of a kind of 1 type duck hepatitis A virus VP3 albumen and application

Info

Publication number: CN105198969B
Application number: CN201510732218.0A
Authority: CN
Inventors: 程安春; 汪铭书; 沈友林; 杨乔; 朱德康; 陈孝跃
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2015-11-02
Filing date: 2015-11-02
Publication date: 2019-03-08
Anticipated expiration: 2035-11-02
Also published as: CN105198969A

Abstract

The invention belongs to technical field of bioengineering, B cell epitope and its identification method and application more particularly to a kind of 1 type duck hepatitis A virus VP3 albumen, identification method is the following steps are included: obtaining VP3 target fragment, building recombinant expression plasmid pGEX-VP3, preparing VP3 recombinant protein, prepare VP3 recombinant protein polyclonal antibody, measure DHAV-1 virus to the median lethal dose ELD of chicken embryo or duck embryos₅₀, the measurement neutralization titer of VP3 recombinant protein polyclonal antibody, B cell antigen epi-position identification on VP3 albumen.Integrated use bioinformatics software of the present invention predicts the Linear B Cell Epitopes and epitope synthesis peptide of VP3 albumen, artificial epitope peptide is identified in conjunction with indirect ELISA method, can for the carrying out in a deep going way of subsequent DHAV-1 immunological investigation, establish new diagnostic preparation and exploitation polypeptide vaccine provides reference.

Description

The B cell epitope and its identification method of a kind of 1 type duck hepatitis A virus VP3 albumen and Using

Technical field

The invention belongs to technical field of bioengineering, and in particular to a kind of B cell table of 1 type duck hepatitis A virus VP3 albumen Position and its identification method and application.

Background technique

Duck hepatitis also known as duck virus hepatitis (Duck viral hepatitis, DVH), are by duck hepatitis virus (Duck Hepatitis virus, DHV) caused by a kind of duck viral infectious disease of high degree in contact and lethal, it is characterized in that morbidity Anxious, propagation is fastly, the course of disease is short, case fatality rate is high, and the visible hepatomegaly of sick duck dissect, surface are in dendroid congested or haemorrhagic puncta, is somebody's turn to do Disease was most found in the U.S. earlier than 1945.DHV initially divides three serotypes (DHV-1, DHV-2 and DHV-3), later DHV-2 and DHV-3 is incorporated into Astroviridae, nonantigenic correlation between three serotypes.Present DHV-1 has been named as duck A type liver Scorching virus (Duck hepatitis A virus, DHAV), is classified as the fowl hepatitis virus category of Picornaviridae It (Avihepatovirus), include three genotype (DHAV-1,2 and 3), wherein 1 type duck hepatitis A virus (DHAV-1) is distribution Most wide, the maximum duck hepatitis A virus of harm.DHAV can be proliferated in the chorioallantoic cavity of duck embryos, chicken embryo and goose embryo, can also Through chick embryo fibroblast, chicken embryo Foetus renal cells, duck embryonic fibroblast, duck embryos fetal liver cell, duck embryos Foetus renal cells etc. Various kinds of cell culture.DHAV resistance is strong, has more significant resistance to common virus inactivator and disinfectant, and be resistant to The acidic environment of pH3.0 also has stronger resistance to heat.DHAV in cell culture is 48min in 50 DEG C of half-life period, one Determine NaCl, Na of concentration₂SO₄、MgCl₂Or MgSO₄It can prevent virus from inactivating at 50 DEG C, but 62 DEG C of 30min can make its whole go out It is living.Virus in infected chicken blastochyle can survive half a year in 4 DEG C, set -20 DEG C and be then able to maintain vigor for 9 years.In natural environment, Virus can survive at least 10w in unwashed pollution incubator, and can survive 37d or more in muck in the cool.DHAV Duckling can be caused to fall ill throughout the year (under natural conditions), and can be dead in 3~4d after infection.Without clinical condition after adult kind duck infection Shape, and its egg laying performance is not influenced.Virus is mainly excreted by alimentary canal, then further through food or drinking-water through respiratory tract Subinfection, sick duck, adult are main ergophore with malicious duck and rehabilitation duck again with alimentary canal, can be caused by excrement toxin expelling The infection of other susceptible ducks groups, the virus without ovum vertical transmission to offspring, but obviously can the horizontal transmission in duck group.It should Virus is very high to the lethality of duckling, and the duckling death rate within 1 week old is up to 95%, and the death rate is down to 50% in 1~3 week old Hereinafter, can be more lower to 4~5 week old death rates.

Epitope is to play the antigenic structure and function unit of antigen molecule, they are small small peptide, is not surpassed generally Cross 20aa.Linear epitope and conformational epitope can be divided into from structure, the existing t cell epitope of linear epitope also has B cell epitope, And conformational epitope is specific to B cell epitope.Specific site in B cell epitope can be known by bone-marrow-derived lymphocyte specificity It not and combines, induces humoral immunity of organism response, to effectively remove cause of disease.Research to epitope is medical diagnosis on disease, determines Point engineered protein kind biological product and the basis for preventing, treating disease, the research of especially current epiposition vaccine is all with table Based on the research of position.

Currently, the method for research epitope mainly has monoclonal antibody technique, display technique of bacteriophage, cracking both at home and abroad Method, intermeshing polypeptide method, peptide chips high throughput scan method etc., and many test results are shown, individually or more than comprehensive utilization It is feasible that technology, which carries out epitope research,.With the development of bioinformatics technique, the research of B cell epitope can be more at present Over-borrowing helps software prediction, and its accuracy is also higher and higher.The prediction of most B cell epitopes is all with primary structure Based on, and to the accuracy of comformational epitope prediction not as good as linear epitope.Combined Hydrophilic, flexibility, surface accessibility, antigen Property and secondary structure can analyze prediction Linear B Cell Epitopes, VP3 is the main structural proteins of picornavirus, it is numerous research Prove that there are numerous B cell epitopes on the VP3 albumen of picornavirus, and the B cell epitope of the VP3 albumen of DHAV is not yet The report for seeing research and confirmation, theoretically speculates, can also have a large amount of B cell epitopes on VP3 albumen, therefore excavate its epitope Information has researching value.

For the detection of 1 type duck hepatitis A virus and preventive means, it is still necessary to improve and perfect, current detection method masters at present Still traditional Serologic detection based on totivirus or the molecular Biological Detection based on virus genome sequence are wanted, therefore anxious Novel effective diagnostic reagent need to be researched and developed.Prevent aspect antibiotic can only pre- bacteriological protection secondary infection, immunity inoculation is still anti- The main means of control.The prevention of 1 type duck hepatitis A virus mainly realized by immune attenuated live vaccines, treatment mainly pass through by Dynamic inoculation DHAV antibody, the above measure still have the not ideal enough problem of efficiency, therefore, how for VP3 albumen Antigen epitope polypeptide constructs new generation vaccine, establishes more accurate sensitive diagnostic method and its diagnostic reagent, becomes this field skill Art personnel technical problem urgently to be resolved.

Summary of the invention

The present invention in order to solve above-mentioned technical problem, provide a kind of 1 type duck hepatitis A virus VP3 albumen B cell epitope and Its identification method and application.Integrated use bioinformatics software of the present invention predicts the Linear B Cell Epitopes of VP3 albumen and people Work synthesizes epitope peptide, is identified in conjunction with indirect ELISA method artificial epitope peptide, can be subsequent DHAV-1 immunological investigation Carry out in a deep going way, new diagnostic preparation and the exploitation offers reference such as polypeptide vaccine be provided.The present invention is reflected using indirect ELISA method Determine B cell epitope, this method is practicable as the result is shown.

In order to reach above-mentioned technical effect, technical solution of the present invention includes:

A kind of B cell epitope of 1 type duck hepatitis A virus VP3 albumen, amino acid sequence as shown in SEQ ID NO:4 or Shown in SEQ ID NO:5.

A kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope, comprising the following steps:

Step 1: it obtains VP3 target fragment: determining that 1 type duck hepatitis A virus VP3 truncation gene restriction enzyme site and specificity draw Object, the upstream primer are 5'-GGATCCGCAATGGACAATCAGGGAAAGAGA-3 ', see SEQ ID NO:2, downstream primer For 5'-CTCGAGAACTGGCTAATCATCCCCTA-3 ', SEQ ID NO:3 is seen；It is proliferated 1 type duck hepatitis A virus strain, and extracts 1 The RNA of type duck hepatitis A virus strain is that template carries out after cDNA synthesis with spy to the RNA of 1 type duck hepatitis A virus using random primer Specific primer carries out PCR amplification, obtains VP3 target fragment；

Step 2: the VP3 target fragment T-A building recombinant expression plasmid pGEX-VP3: is cloned into pMD19-T (Simple) in carrier, building recombination T plasmid pMD19-VP3 distinguishes double digestion empty plasmid pGEX-4T-1 with BamH I and Xho I With the correct positive recombinant plasmid pMD19-VP3 of identification, recycling, connection, identification obtain recombinant expression plasmid pGEX-VP3；

Step 3: preparation VP3 recombinant protein: the recombinant expression plasmid pGEX-VP3 is converted to Escherichia coli In BL21DE3, recombinant protein inducing expression is carried out, after, by bacterium solution purifying, identification, obtain 1 type duck hepatitis A virus VP3 weight Histone, the VP3 recombinant protein amino acid sequence is as shown in SEQ ID NO:1；

Step 4: preparation VP3 recombinant protein polyclonal antibody detects serum titer and identifies the special of polyclonal antibody Property；

Step 5: median lethal dose ELD of the measurement DHAV-1 virus to chicken embryo or duck embryos₅₀；

Step 6: the neutralization titer of measurement VP3 recombinant protein polyclonal antibody；

Step 7: B cell antigen epi-position is identified on VP3 albumen: prediction VP3 protein B cell antigen epitope, artificial synthesized table Position peptide carries out indirect ELISA with VP3 recombinant protein polyclonal antibody and negative serum.

Further, the VP3 recombinant protein Anti-TNF-α preparation step includes: to be exempted from the VP3 recombinant protein of purifying Epidemic disease hero rabbit prepares antiserum, with the serum titer of agar gel diffusion test detection last time 7d after immune, obtains VP3 recombinant protein Polyclonal antibody.

Further, the immune program includes: the first VP3 recombinant protein solution and isometric Freund exempted from 0.5mg Freund's complete adjuvant emulsification is after Water-In-Oil state, the nape of the neck is intradermal, rabbit is subcutaneously injected；Head exempts from the latter 11 days VP3 with 1mg and recombinates egg White solution and isometric incomplete Freund's adjuvant emulsify after Water-In-Oil state, to carry out two in the nape of the neck subcutaneous injection of rabbit Exempt from；Two exempt from the latter 22 days VP3 recombinant proteins with 1mg to emulsify with isometric incomplete Freund's adjuvant to be nape after Water-In-Oil state Portion subcutaneous injection rabbit carries out three and exempts from；Head exempt from after 34 and 47 days, with the VP3 recombinant protein auricular vein of 0.5mg inject rabbit into Row four exempts to exempt from five.

Further, median lethal dose ELD of the measurement DHAV-1 virus to chicken embryo₅₀Step includes: that DHAV-1 is weak The virus liquid of strain makees continuous 10 times and dilutes and be inoculated with 9 age in days chick embryo allantoic cavities, shines egg observation, system daily to the chicken embryo after inoculation Each group chicken embryo death situation is counted, the ELD of virus liquid is calculated using Reed-Muench method₅₀。

Further, the neutralization titer step of the measurement VP3 recombinant protein polyclonal antibody includes: with surveying ELD₅₀'s The virus liquid of DHAV-1 low virulent strain is imitated by the neutralization of fixed virus-diluted blood heat-clearing method measurement VP3 recombinant protein polyclonal antibody Valence calculates its neutralization titer with Reed-Muench method.

Further, the measurement B cell antigen epi-position authentication step includes: first respectively to VP3 protein amino acid sequence Flexibility, surface accessibility and hydrophily analyzed；Further forecasting sequence antigenicity, prediction obtain VP3 recombinant protein B cell epitope, then epitope synthesis peptide fragment sample makees antigen with the VP3 recombinant protein of purifying, VP3 recombinant protein is more Clonal antibody selects positive hole OD value 1.0 or so, feminine gender right as detection reagent using indirect ELISA by square matrix titration According to OD value 0.1 or so antigen coat concentration as best antigen diluent degree, each antigen table is finally diluted with best antigen concentration Position peptide, makees primary antibody with the rabbit-anti VP3 recombinant protein polyclonal antibody of doubling dilution, and negative rabbit anteserum control, HRP- is arranged in parallel Goat anti-rabbit igg makees secondary antibody, identifies according to indirect ELISA method artificial synthesized epitope peptide fragment.

In above-mentioned identification method, 1 type duck hepatitis A virus strain in the step 1 is H plants of 1 type duck hepatitis A virus, tiny RNA Viraceae (Picornaviridae), fowl hepatovirus (Avihepatovirus), scientific name are 1 type duck hepatitis A virus (Duck Hepatitis A Virus type 1, DHAV-1), H plants of the 1 type duck hepatitis A virus is preserved in China typical culture collection Center, deposit number are CCTCC NO.V201539.

The DHAV-1 low virulent strain, the culture are protected on November 20th, 2012 by being deposited in Chinese Typical Representative culture Hiding center receives, and registers on the books.Deposit number is CCTCC NO.V201248, and is the biology authorized in patent gazette Material, Patent No. ZL201310011872.3.

The B cell epitope of the 1 type duck hepatitis A virus VP3 albumen can be applied to the infection of 1 type duck hepatitis A virus of detection or 1 In the kit of type duck hepatitis A virus antibody test.

The B cell epitope of the 1 type duck hepatitis A virus VP3 albumen is used as the infection of 1 type duck hepatitis A virus of detection or 1 type duck Antigen in the reagent constituents of hepatitis A virus antibody test.

The B cell epitope of the 1 type duck hepatitis A virus VP3 albumen prevents 1 type duck hepatitis A virus genetic engineering epidemic disease in preparation It is applied in seedling.

The beneficial effect comprise that

1, a kind of B cell epitope of 1 type duck hepatitis A virus VP3 albumen provided by the invention is used as 1 type of detection The detection antigen of DHAV antibody and the sequence of genetic engineering subunit vaccine；

2, the B cell epitope of a kind of 1 type duck hepatitis A virus VP3 albumen provided by the invention can be used for detecting after coupling 1 type duck hepatitis A virus antibody during duck blood is clear establishes the examination of the infection of 1 type duck hepatitis A virus and 1 type duck hepatitis A virus antibody test Agent box and ELISA detection method；

3, the method for epitope in the prior art mainly has monoclonal antibody technique, display technique of bacteriophage, cracking Method, intermeshing polypeptide method, peptide chips high throughput scan method etc. or need specific apparatus, reagent or technology, and the present invention only adopts It is identified with conventional polyclonal antibody, it is easy, quick.

Biomaterial preservation

H plants of 1 type duck hepatitis A virus is preserved in China typical culture collection center preservation in the present invention, and deposit number is CCTCC NO.V201539, address are located at Wuhan, China Wuhan University, the deposit date is on August 31st, 2015, classification naming 1 H plants of type duck hepatitis A virus.

Detailed description of the invention

Fig. 1 show duck hepatitis A virus genome structure ideograph of the present invention.

Fig. 2 show the analysis chart of the VP3 recombinant protein expression of the embodiment of the present invention 1.

Fig. 3 show the optimizing detection figure of 2 recombinant protein of embodiment of the present invention expression IPTG concentration.

Fig. 4 show the optimizing detection figure of 2 recombinant protein induced expression temperature of the embodiment of the present invention.

Fig. 5 show the optimizing detection figure of 2 recombinant protein induced expression time of the embodiment of the present invention.

Fig. 6 show the RT-PCR amplified production electrophoresis detection figure of the VP3 gene of the embodiment of the present invention 1.

Fig. 7 show the pMD-VP3 recombination T plasmid identification figure of the embodiment of the present invention 1.

Fig. 8 show the pGEX-VP3 recombinant expression plasmid qualification figure of the embodiment of the present invention 1.

Fig. 9 show the pGEX-VP3 recombinant expression plasmid sequencing result sequence alignment figure of the embodiment of the present invention 1.

Figure 10 show the VP3 recombinant protein purification detection figure of the embodiment of the present invention 1.

Figure 11 show the VP3 recombinant protein immunoblotting analysis analysis chart of the embodiment of the present invention 1.

Figure 12 show the qualification figure of VP3 recombinant protein polyclonal antibody in the embodiment of the present invention 3.

Figure 13 show enzyme labelled antibody working concentration figure in the embodiment of the present invention 4.

Figure 14 show VP3 protein B cell epitope identification analysis chart in the embodiment of the present invention 3.

Figure 15 show ELISA detection method sensitivity analysis figure in the embodiment of the present invention 4.

Figure 16 show ELISA detection method cross reactivity analysis chart in the embodiment of the present invention 4.

Figure 17 show DHAV-1 positive serum in the embodiment of the present invention 4 and blocks analysis chart.

Figure 18 show DHAV-3 positive serum in the embodiment of the present invention 4 and blocks analysis chart.

Figure 19 show the comparative analysis figure of epitope peptide and VP3 recombinant protein in antibody test in the embodiment of the present invention 4.

Specific embodiment

Below in conjunction with specific attached drawing the present invention is described in detail specific embodiment.It should be noted that in following embodiments The combination of the technical characteristic or technical characteristic of description is not construed as isolated, they can be combined with each other to reaching To superior technique effect.

H plants of 1 type duck hepatitis A virus of the present invention, sequence is had been disclosed in GenBank database, and registration number is JQ301467。

As shown in Figure 1, carrying out bioinformatic analysis supposition referring to other picornavirus, the albumen of DHAV is by gene Polyprotein (polyprotein) precursor of the ORF expression of long 2249nt is processed by self-cleavage in group.ORF Tri- areas P1, P2 and P3 can be divided into, the area P1 encodes VP0 (1AB), three kinds of structural proteins of VP3 (1C) and VP1 (1D), the area P2 and P3 Area is separately encoded 2A (containing 2A1,2A2 and 2A3), seven kinds of 2B, 2C and 3A, 3B, 3C, 3D non-structural proteins.Virion enters thin It after born of the same parents, is translated first, 2A, 3C (3CD) protease of generation have hydrolysis function, then start to carry out protein precursor Cutting.It is cut into mature albumen and needs to carry out two steps, the first step is to cut P2-P3 connection and P1- by 3C (3CD), 2A P2 connection generates tri- P1 (or P1-2A), P2 and P3 poly precursor proteins.Second step then completes remaining step by 3C (3CD) Suddenly.

Embodiment 1

A kind of purposes of the B cell epitope of above-mentioned 1 type duck hepatitis A virus VP3 albumen can be applied to 1 type duck hepatitis A of detection In the kit of poison infection or vaccine antibody level.

Further, the B cell epitope of the 1 type duck hepatitis A virus VP3 albumen is used as detection 1 type duck hepatitis A infection Or the antigen in the reagent constituents of vaccine antibody level.

A kind of purposes of the B cell epitope of above-mentioned 1 type duck hepatitis A virus VP3 albumen, the 1 type duck hepatitis A virus VP3 egg White B cell epitope is applied in preparation 1 type duck hepatitis A virus vaccine of prevention.

Step 1: it obtains VP3 target fragment: determining that 1 type duck hepatitis A virus VP3 truncation gene restriction enzyme site and specificity draw Object, the upstream primer are 5'-GGATCCGCAATGGACAATCAGGGAAAGAGA-3 ', downstream primer 5'-CTCGAGAACTGGCTAATCATCCCCTA-3'；

1, DHAV-1 Strain is proliferated: after DHAV-1 virus liquid is made appropriate dilution with physiological saline, 1:100 (v/v) adds Enter dual anti-and 37 DEG C of incubation 1h, is inoculated with allantoic cavity of 11 ages in days without DHAV-1 maternal antibody duck embryos, 0.2mL/ pieces.37 DEG C of routines are incubated Change culture, abandons embryo dead in for 24 hours.Dead embryo in for 24 hours~96h is collected, collects allantoic fluid after 4 DEG C of preservations overnight, -20 DEG C freeze It is spare.

2, the RNA:(1 of DHAV-1 Strain is extracted) it takes in 200 μ L to 1.5mL EP pipe of viral suspension, RNAiso is added 1000 μ L of Plus, is mixed by inversion, is placed at room temperature for 10min；(2) 200 μ L chloroforms are added, covers tightly EP pipe lid, firmly shakes, room temperature Place 10min；(3) 4 DEG C, 12000rpm is centrifuged 15min, and supernatant is taken to be transferred to another 1.5mL import without (cutting in RNase EP pipe Avoid the dynamic white interphase of suction).(4) it is added and sucts clear isometric isopropanol, gently reverse EP pipe to be to mix well liquid, 4 DEG C of precipitating 20min in centrifuge.(5) 4 DEG C, 12000r/min is centrifuged 15min, and all supernatants are carefully sucked with sample loading gun.(6) It is gently washed one time with 75% ethyl alcohol that 1mL DEPC water is prepared, 4 DEG C, 12000r/min centrifugation 10min are carefully sucked with sample loading gun All supernatants, stand 5min in super-clean bench, make its natural air drying.(7) plus appropriate RNase Free water dissolves sediment, and -20 DEG C save.

3, the RT-PCR amplification of VP3 gene: being that template carries out cDNA using RNA of the random primer to 1 type duck hepatitis A virus PCR amplification is carried out with specific primer after synthesis, obtains VP3 target fragment；

System (20 μ L) is added by the PrimeScript Reverse Transcript specification of precious biology, reverse transcription is closed Specific as follows at cDNA: 1.5mL import is without addition RNA 2 μ L, Random Primers (10 μm of ol/L) 5 μ in RNase EP pipe Chilling 2min, centrifugation 5s are backward on ice rapidly after L, dNTP Mixture (2.5mmol/L each) 4 μ L, 65 DEG C of upper heat preservation 5min 5 × PrimeScript Buffer, 4 μ L, PrimeScript Reverse Transcript Mix, 1 μ L is added in pipe, finally 4 μ L of RNase free water is added.It adds cold on ice after 30 DEG C of 10min, then 42 DEG C of 40min, last 70 DEG C of heat preservations 15min after system But, it -20 saves backup.

Explanation referring next to EmeraldAmp PCR Master Mix and to PCR reaction condition grope determine ginseng Number expands target fragment with high fidelity enzyme again after condition understands, amplification system and program are shown in Table 1.

Table 1PCR amplification system and program

10 × Loading the buffer for taking 4.5 μ L PCR products that 0.5 μ L is added after amplification is solidifying in 1% agarose Electrophoresis in glue observes and records result in gel imaging system.As a result as shown in Figure 6, discovery (actual size at about 750bp 774bp) there is specific band, is consistent with expected results.In Fig. 6, M:DL2000 molecular weight standard；1:VP3 gene RT-PCR Product.

Step 2: the VP3 target fragment T-A building recombinant expression plasmid pGEX-VP3: is cloned into pMD19-T (Simple) in carrier, building recombination T plasmid pMD19-VP3, specific steps include: that will identify that correct PCR product is coagulated by DNA The operating procedure of plastic recovery kit carries out glue recovery purifying, and adds A (7.5uL recycling+2.5 μ L of segment to recovery product end PCR Master Mix, 72 DEG C of 30min), after agarose gel electrophoresis estimates concentration, is calculated and added by the molar ratio of two segments Linked system is shown in Table 2, connects pMD19-T (Simple) carrier, 16 DEG C overnight.

Table 2T-A clones linked system

Component	Volume
		PMD19-T (Simple) carrier	0.03pmol
VP3 target fragment	0.18pmol
		SolutionⅠ	5.0μL
Total volume	10μL

Above-mentioned 7.5 μ L of connection product is taken to be added in 100 μ L DH5 α competence, ice bath 30min after soft mixing, then 42 DEG C of water-bath heat shock 90s are set immediately, continue ice bath 3min after taking-up, 800 μ L LB culture solutions are added in 37 DEG C of shaken cultivation lh, 3000r/min 2min centrifugation, abandons supernatant, is applied on LB plate (Amp+) after staying about 250 μ L to mix precipitating, 37 DEG C of culture 16h. Random picking single colonie, is inoculated into 5mL LB culture medium (Amp+), and 37 DEG C of shaken cultivations are stayed overnight, and carries out bacterium solution PCR after muddy Identification, it is optionally a to identify that correct bacterium illustrates to extract plasmid, further enzyme referring to Omega company small amount plasmid extraction agent box Cut (37 DEG C, 1.5h) identifications.Bacterium solution PCR identification system (10 μ L): 5 μ L of PCR Master Mix, bacterium solution 1 μ L, P1, P2 (10mmol/L) each 0.5 μ L, 3 μ L of sterile purified water.(10 μ L) is identified in digestion: plasmid 8 μ L, BamH I, Xho I each 0.5 μ L, 10 × K buffer 1 μ L, 37 DEG C of digestion 1.5h.

As a result, positive colony is named as pMD19-VP3, expansion takes the detection digestion of 1% agarose gel electrophoresis after cultivating A part of 30% glycerine physiological saline conservation, -70 DEG C freeze.

Double digestion empty plasmid pGEX-4T-1 is distinguished with BamH I and Xho I and identifies correct positive recombinant plasmid pMD19- VP3, recycling, connection, identification, obtains recombinant expression plasmid pGEX-VP3；Specific steps include: digestion system: pMD19-VP3 or PGEX-4T-148 μ L, BamH I, each 6 μ L of 3 μ L, 10 × K buffer of Xho I (15U/ μ L).After having added digestion system, 20 μ L/ Pipe packing, 37 DEG C of digestion 2h.To 1% agarose gel electrophoresis of product after digestion, cut respectively under uv analyzer VP3 and The gel purpose band of pGEX-4T-1 is purified with DNA glue recovery purifying kit.Electrophoresis estimates concentration, calculates and adds Linked system is shown in Table 3,16 DEG C overnight.

Table 3 is subcloned linked system

Component	Volume
		PGEX-4T-1 endonuclease bamhi	0.03pmol
VP3 glue recovery product	0.18pmol
		Solution I	7.5μL
Total volume	15μL

Above-mentioned 7.5 μ L of connection product is taken to be added in 100 μ L DH5 α competence, ice bath 30min after soft mixing, then 42 DEG C of water-bath heat shock 90s are set immediately, continue ice bath 3min after taking-up, 800 μ L LB culture solutions are added in 37 DEG C of shaken cultivation lh, 3000r/min 2min centrifugation, abandons supernatant, is applied on LB plate (Amp+) after staying about 250 μ L to mix precipitating, 37 DEG C of cultures.Next day It chooses the identification method that single colonie is cloned by T and carries out bacterium solution PCR identification and digestion identification respectively, correct bacterium solution send Invitrogen Company (Shanghai) sequencing, is compared sequencing result, and name positive plasmid is pGEX-VP3.

The band that the bacterium solution PCR identification and digestion identification of pMD-VP3 recombination T plasmid obtain expected size see Fig. 7 (M1, M2:DL15000 and DL2000 molecular weight standard；1:BamH I and the identification of I double digestion of Xho；The PCR of 2:pMD-VP3 is identified). The band that pGEX-VP3 recombinant expression plasmid also obtains expected size through bacterium solution PCR identification and digestion identification see Fig. 8 (M1, M2: DL15000 and DL2000 molecular weight standard；The identification of I single endonuclease digestion of 1:BamH；2:BamH I and the identification of I double digestion of Xho；3:pGEX- The PCR of VP3 is identified).To sequencing result by Blastn sequence analysis on NCBI analysis shows, the gene piece being inserted into carrier Duan great little is 774bp, and the VP3 gene order cloned and original segments homology are 100%, as a result sees Fig. 9, in bracket Region is VP3 gene order.Show that recombinant expression carrier constructs successfully, is named as pGEX-VP3.

Step 3: preparation VP3 recombinant protein: the recombinant expression plasmid pGEX-VP3 is converted to e. coli bl21 (DE3) in, recombinant protein inducing expression is carried out, after, by bacterium solution purifying, identification, obtain 1 type duck hepatitis A virus VP3 recombination Albumen.

The recombinant protein expression induction step includes: the e. coli bl21 of picking pGEX-VP3 containing recombinant plasmid (DE3) single bacterium drops down onto 37 DEG C of activation in 50mL LB liquid medium and stays overnight, and next day is by bacterium solution and LB culture volume ratio 1:100 Ratio be inoculated in 100mL LB liquid medium expand culture, 200r/min, 37 DEG C, culture 3~4h to A600 be 0.5~ 0.7, IPTG to final concentration of 0.2mmol/L, 37 DEG C of induction 8h is added.

The purification step includes: that the bacterium solution 8000r/min centrifugation 5min after expression containing recombinant protein is collected thallus Precipitating, and the Tris-Cl that 20mmol/L pH is 8.0 is added by Tris-Cl solution and bacterium solution volume ratio 1:10 and suspends, Yu Bingshui Ultrasonic disruption thalline in bath, ultrasonic 60s are spaced 60s, repeatedly until bacterium solution is clear and transparent, the bacterium solution after ultrasonication 10000rpm is centrifuged 10min, collects precipitating and with the sample preparation after completely dissolution of 8mol/L urea, and albumen Vertial electrophorestic tank electrophoresis is simultaneously cut Lower purpose band places electrodialysis in albumen buffer, collects protein liquid, is concentrated by ultrafiltration, obtains recombinant protein after purification.It takes Part carries out SDS-PAGE detection, as shown in Figure 10 it is found that the obtained purity of protein of this method is higher, M in Figure 10: low molecular weight Protein standards, 1: unpurified VP3 recombinant protein, 2: VP3 recombinant protein after purification.

The Western blot of recombinant protein is analyzed: the sample of the recombinant protein containing VP3 being carried out SDS-PAGE, electrophoresis is tied fastly The film of suitable size is cut when beam as required and filter paper and is impregnated.According to positive "-three layers of filter paper-pvdf membrane-gel-three of fiber mat The sequence installation electricity of layer filter paper-fiber mat " cathode turns sandwich, and 80V transfers 90min, after pvdf membrane is taken out, seal up and close Liquid is incubated for 1h in 37 DEG C of vibrations, takes out after being then incubated for 1h with 37 DEG C of vibrations of the diluted rabbit-anti DHAV-1IgG of 1:100, PBS is washed It washs 3 times, each 5min, 1h is then incubated in 37 DEG C of vibrations with the diluted HRP- goat anti-rabbit igg of 1:3000, presses DAB after PBS washing Colour reagent box specification colour developing, with distilled water flushing color development stopping when purpose band is high-visible.Pvdf membrane drying is kept away Light saves.

Make antigen with the sample of the recombinant protein containing VP3, rabbit-anti DHAV-1 antibody makees primary antibody, carries out Western blot mirror It is fixed, the special sexual imprinting of expected size is as a result produced, and negative rabbit anteserum control has no specific band as shown in Figure 11, demonstrate,proves The VP3 recombinant protein of bright expression can be identified there is good reactionogenicity, Tu11Zhong, M: pre-dyed egg by rabbit-anti DHAV-1 antibody White Marker；1: the sample incubation rabbit-anti DHAV-1 antibody of the recombinant protein containing VP3；2: the sample incubation rabbit of the recombinant protein containing VP3 Negative serum.

1, the preparation of polyvalent antibody:

Formaldehyde fumigation disinfection is carried out to animal house in advance, after closed stifling 3d, windowing ventilation 2d, the male rabbit adaptability bought back 1d is raised, and in a small amount of negative blood of immune the previous day acquisition, separates serum, -20 DEG C save backup.Estimated in advance with nucleic acid-protein instrument The concentration of VP3 recombinant protein is surveyed, and the protein content according to needed for each immune stage determines required amount of antigen, then by such as lower section Method emulsifies antigen: after adjuvant and recombinant protein are mixed in equal volume, the ultrasonic emulsification in ice bath, until antigen drop will be emulsified in ice Occurs the round oil droplet not scattered in complete and 10min in water.Immune program includes: the first VP3 recombination exempted from 0.5mg Protein solution and the emulsification of isometric Freund's complete adjuvant are after Water-In-Oil state, the nape of the neck is intradermal, rabbit is subcutaneously injected；After head exempts from After the 11 days VP3 recombinant protein solution with 1mg and the emulsification of isometric incomplete Freund's adjuvant are Water-In-Oil state, in the neck of rabbit Dorsal sc injection carries out two and exempts from；Two, which exempt from the latter 22 days VP3 recombinant proteins with 1mg with the emulsification of isometric incomplete Freund's adjuvant, is After Water-In-Oil state, the nape of the neck subcutaneous injection rabbit carries out three and exempts from；Head exempts from 34 and 47 days afterwards, with the VP3 recombinant protein ear of 0.5mg Edge intravenous injection rabbit carries out four and exempts to exempt from five.

2, polyvalent antibody fine jade expand potency measurement: four exempt from 6d after rabbit auricular vein take a blood sample on a small quantity, carry out agar gel diffusion test. The VP3 recombinant protein of 0.2~0.3mg/mL is added dropwise in the interstitial hole of plum blossom hole, and negative rabbit anteserum, 1:2,1 is added in holes around one by one: 4, precipitation line is observed in the diluted immune rabbit anteserum of 1:8,1:16 and 1:32,50 holes μ L/, 37 DEG C of wet box incubations afterwards for 24 hours.Antiserum effect Otherwise arteria carotis bloodletting when valence is greater than 1:16 is reinforced exempting from once, until reaching requirement again.Rear neck artery bloodletting is immunized in last time (guarantee sterile working as far as possible), after collecting 37 DEG C of standing lh of blood, 4 DEG C of refrigerator overnights are divided serum is precipitated for second day 10min is centrifuged with 5000r/min from, blood clot, separation again is precipitated serum, the 3mL/ bottles of penicillin bottles for being sub-packed in 5mL, and -20 It DEG C saves backup.

3, the specificity identification of polyvalent antibody:

Making antigen with VP3 recombinant protein, more anti-(1:100) of preparation make primary antibody, the goat anti-rabbit igg of HRP label (1: 3000) make secondary antibody, by above-mentioned Western blot method, identify VP3 recombinant protein polyclonal antibody specificity.

Step 5: median lethal dose ELD of the measurement DHAV-1 Strain to chicken embryo or duck embryos₅₀: ELD₅₀Determination step such as Under: (1) in penicillin bottle or centrifuge tube with sterile saline the CH60 strain virus liquid of DHAV-1 is made into continuous 10 times dilution, From 10^-4~10^-8.(2) the 9 age in days chick embryo allantoic cavity of virus inoculation that will have been diluted, 0.2mL/ pieces, is sealed with paraffin by 5 pieces/dilution Mouthful, embryo dead within for 24 hours is abandoned in 37 DEG C of cultures, and chicken embryo dead later sets 4 DEG C of preservations for 24 hours.Continuous culture 5d, records result. (3) normal chicken embryo control (replacing venom with sterile saline, be inoculated with 5 pieces, 0.2mL/ pieces according to the above method) is set.(4)Reed- Muench method calculated result.

Step 6: the neutralization titer of measurement VP3 recombinant protein polyclonal antibody: the measurement of polyvalent antibody neutralization titer uses Fixed virus-diluted blood heat-clearing method, it is specific as follows: (1) VP3 polyvalent antibody sterile saline to be made into continuous twice of doubling dilution (l:2~1:256).It (2) is 200TCID with normal saline dilution by the CH60 virus stock solution used for having surveyed virulence₅₀After/0.1mL, with The serum to be checked of each dilution mixes well in equal volume, sets 37 DEG C of effect 1h, shakes 1~2 time, is neutralized therebetween.(3) will The mixed liquor with after is inoculated with 9 age in days chick embryo allantoic cavities among the above, and 5 pieces/dilution, 0.2mL/ pieces, 37 DEG C are continued to be incubated for.(4) it sets Following control: blank control group (substitutes neutralizer, step operation as above with sterile saline, chicken embryo must all be good for work)；Disease (100ELD is applied alone in poison control₅₀As above step is inoculated with for the CH60 strain of/0.2mL, and chicken embryo must be all dead).(5) it is observed continuously 5d records death condition, Reed-Muench method calculated result.

Step 7: B cell antigen epi-position is identified on VP3 albumen:

1, the prediction of epitope and the synthesis of peptide fragment:

According to the B cell Antigen Epitope Prediction of VP3 as a result, synthesizing corresponding epitope peptide fragment, the packing of 1mg/ pipe is respectively designated as VP3a(¹³¹FNTGRYQMSWYPIADGEQSL¹⁵⁰)、VP3b(²⁰⁰VNSSAPSNID²⁰⁹).A pipe is respectively taken to be diluted to 1mg/mL, it will be dilute Release liquid be placed in -20 DEG C freeze it is spare.

2, the determination of the best diluted concentration of epitope peptide:

Make antigen with the VP3 recombinant protein of purifying, the rabbit-anti VP3 recombinant protein polyvalent antibody of aforementioned preparation makees primary antibody, HRP- goat anti-rabbit igg makees secondary antibody, and rabbit negative serum control is arranged, and indirect ELISA is carried out with square matrix titration, to determine antigen most Good working concentration.Specific step is as follows:

(1) it is coated with: VP3 recombinant protein is subjected to 1:20,1:40,1:80,1:160,1 with carbonate buffer solution (pH9.6): 320,1:640,1:1280 and 1:2560 gradient dilution and coated elisa plate, 100 holes μ L/, 4 DEG C overnight.

(2) board-washing: taking out ELISA Plate, abandons antigen liquid, and washing buffer (pH7.4) board-washing 4 times, each 5min is patted dry anti- Answer plate (board-washing process is similarly hereinafter).

(3) it closes: 1% gelatin solution, 200 holes μ L/, 37 DEG C of incubation 90min being added into micro-reaction hole.

(4) it is loaded: taking out the ELISA Plate closed, abandon confining liquid, board-washing pats dry.By VP3 polyclonal serum and negative blood Sorting does not carry out the continuous doubling dilution of 1:80,1:160,1:320,1:640,1:1280,1:2560 and 1:5120, is separately added into micro- Quantitative response hole, 100 holes μ L/, 37 DEG C of incubation 90min.

(5) add ELIAS secondary antibody: taking out ELISA Plate, abandon serum dilution in hole, board-washing pats dry.It is diluted that 1:3000 is added HRP- goat anti-rabbit igg secondary antibody, 100 holes μ L/, 37 DEG C of effect 45min.

(6) it develops the color: taking out ELISA Plate, abandon liquid in hole, board-washing pats dry, and TMB working solution, 100 holes μ L/, room is then added After temperature is protected from light effect 10min, 2mol/L H is added in 50 holes μ L/₂SO₄Reaction is terminated, with microplate reader with 450nm-630nm dual wavelength Measure OD value.

(7) the OD value for selecting positive hole is 1.0 or so, the antigen diluent degree of the OD value 0.1 or so of negative control is as work Make concentration.

3, the identification of epitope:

Each epitope peptide is diluted with the above-mentioned optimum concentration determined, rabbit-anti VP3 polyclonal antibody is made primary antibody and (set in parallel Set negative rabbit anteserum control), carry out continuous twice of the doubling dilution of 1:20~1:2560.HRP- goat anti-rabbit igg makees secondary antibody, between progress It meets ELISA to detect, the ELISA step that the indirect ELISA detection method is determined with the best diluted concentration of above-mentioned epitope peptide, Artificial synthesized epitope peptide fragment is identified.

The optimization of the expression analysis and expression condition of 2 recombinant protein of embodiment

Test method:

1, the expression analysis of recombinant protein

E. coli bl21 (DE3) positive bacteria inoculation LB (Amp+) the liquid training that recombinant expression plasmid pGEX-VP3 is converted Base is supported, 37 DEG C of activation are overnight.The bacterium solution of activation is taken to be inoculated with LB (Amp+) fluid nutrient medium, 37 DEG C of expansions according to 1:100 volume ratio 3~4h is cultivated, until A600 about 0.6.Final volume 0.4mmol/L IPTG, 37 DEG C of inducing expression 4h are added, after, bacterium solution 8000r/min is centrifuged 5min and collects bacterial sediment, and 20mmol/L is added in 1:10 (Tris-Cl solution: bacterium solution) by volume Tris-Cl (pH8.0) solution suspension, the ultrasonic disruption thalline in ice-water bath, ultrasonic 60s are spaced 60s, repeatedly until bacterium solution It is clear and transparent.Bacterium solution 10000rpm after ultrasonication is centrifuged 10min, collects supernatant precipitating respectively, and precipitating is urinated with 8mol/L Plain sufficiently dissolution.The dissolved each 80 μ L of precipitating of broken supernatant is taken respectively, with 20 μ 5 × SDS of L Loading Buffer mixing, SDS-PAGE is detected after boiling 10min." empty plasmid pGEX-4T-1 induction ", " recombination pGEX-VP3 are set simultaneously Do not induce " and " recombination pGEX-VP3 induction " control.

2, the optimization of recombinant protein expression condition

BL21 (DE3) single bacterium of picking pGEX-VP3 containing recombinant plasmid drops down onto 37 in 50mL LB liquid medium (Amp+) DEG C activation overnight.Next day, 1:100 (bacterium solution: LB culture medium) ratio was inoculated in 100mL LB liquid medium (Amp+) by volume Middle expansion culture, 200r/min, 37 DEG C of culture 3~4h to A600 about 0.6.Expression condition optimization is done referring next to document.

(1) optimization of IPTG concentration: be separately added into IPTG to final concentration of: 0mmol/L, 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L and 1.5mmol/L, Fiber differentiation 4h, thalline were collected by centrifugation, surpasses Precipitating is dissolved after sound is broken, SDS-PAGE detection is carried out to the precipitating of dissolution.

(2) optimization of time: with (1) method, the IPTG of optium concentration is added, respectively induce 0h, 2h, 4h, 6h, 8h, 19h and 22h carries out SDS-PAGE detection to the precipitating of dissolution after being crushed.

(3) optimization of inducing temperature: with the method for (2), being added the IPTG of optium concentration and use best induction time, point The inducing expression not at 16 DEG C, 25 DEG C, 30 DEG C and 37 DEG C carries out SDS-PAGE detection to the precipitating of dissolution after being crushed.

Test result:

1, the expression analysis of recombinant protein

Supernatant precipitating of the positive restructuring expression bacterium after inducing expression, ultrasonication containing pGEX-VP3 carries out SDS- PAGE detection, as shown in Fig. 2, 1~5 be respectively empty plasmid induction, recombinant plasmid do not induce full bacterium, recombinant plasmid induce full bacterium, Recombinant plasmid induces supernatant recombinant plasmid induction inclusion body precipitating；It was found that there is purpose band in 54kD or so, show VP3 base Because being expressed, and almost all exists with inclusion bodies.

2, the optimization of recombinant protein expression condition

The optimization of recombinant protein expression condition as seen in figures 3-5,1~8 difference IPTG concentration 0.1 in Fig. 3,0.2,0.4, 0.6,0.8,1.0,1.5 and 0mmol/L；1~4 is respectively 16 DEG C, 25 DEG C, 30 DEG C and 37 DEG C in Fig. 4；1~7 is respectively in Fig. 5 22h, 19h, 8h, 6h, 4h, 2h and 0h.IPTG concentration, inducing temperature, induction time are optimized respectively, induced as the result is shown The optimum condition of VP3 expression is 0.2mmol/L IPTG, 37 DEG C of induction 8h.

A kind of identification experiment result of 1 type duck hepatitis A virus VP3 protein B cell epitope of embodiment 3

1, the preparation and titration of VP3 recombinant protein polyclonal antibody

Prepare antiserum with the immune male rabbit of the VP3 recombinant protein of purifying, specific steps: head exempts from the VP3 recombination egg with 0.5mg After white solution and the emulsification of isometric Freund's complete adjuvant are Water-In-Oil state, the nape of the neck is intradermal, subcutaneous multi-point injection rabbit；Head exempts from The 11 days VP3 recombinant protein solution with 1mg and the emulsification of isometric incomplete Freund's adjuvant are the nape of the neck skin after Water-In-Oil state afterwards Lower multi-point injection rabbit carries out two and exempts from；Head exempts from the latter 22 days VP3 recombinant proteins with 1mg and isometric incomplete Freund's adjuvant emulsifies After Water-In-Oil state, the subcutaneous multi-point injection rabbit of the nape of the neck carries out three and exempts from；Head exempts from 34 and 47 days afterwards, is recombinated with the VP3 of 0.5mg Albumen auricular vein injection rabbit carries out four and exempts to exempt from five.With the serum effect of agar gel diffusion test detection last time 7d after immune Valence, discovery 1:16 have an obvious sediment line, 1:32 also visible faint lines, and rabbit negative serum is without precipitation line.

2, the specificity identification of VP3 recombinant protein polyclonal antibody

The VP3 recombinant protein of the VP3 recombinant protein polyclonal antibody of acquisition and SDS-PAGE are subjected to Western blot React the specificity of the standby polyclonal antibody of system of identification.As a result VP3 polyclonal antibody can be reacted with VP3 recombinant protein, be generated pre- The special sexual imprinting of phase size, as shown in figure 12, and rabbit negative serum occurs without special sexual imprinting, it was demonstrated that the more of preparation anti-have Preferable specificity, meets the requirements.Wherein in Figure 12, M: pre-dyed albumen Marker；It is mostly anti-that 1:VP3 recombinant protein is incubated for VP3；2: VP3 recombinant protein is incubated for negative rabbit anteserum.

3, the neutralization activity detection of VP3 recombinant protein polyclonal antibody

(1) CH60 plants of chicken embryo median lethal dose (ELD of DHAV-1 weak poison₅₀) measurement

CH60 virus liquid is made into continuous 10 times of dilutions and is inoculated with 9 age in days chick embryo allantoic cavities, the chicken embryo after inoculation is shone daily Egg observation, statistics each group chicken embryo death situation is as shown in table 4, and the ELD of this batch of virus liquid is calculated using Reed-Muench method₅₀For 10^-7 _. ³, i.e., the virus liquid make 10^-7 _. ³Dilution, it is dead that inoculation 0.2mL can be such that half chicken embryo occurs.

Table 4 surveys ELD₅₀Chicken embryo death situation statistical result

Distance proportion=(percentage -50% higher than 50%)/(percentage-higher than 50% is lower than 50% percentage)

=(62.5%-50%)/(62.5%-25%)=0.3

lg(ELD₅₀Logarithm+distance proportion × coefficient of dilution logarithm of 50% viral dilution of)=be higher than

=-7+0.3 × (- 1)=- 7.3；That is ELD₅₀=10^-7.3

(2) measurement of VP3 recombinant protein polyclonal antibody neutralization titer

With surveying ELD₅₀Virus liquid, using the neutralization titer that " fixed virus-dilute serum " method measurement VP3 is mostly anti-, system The results are shown in Table 5 for meter, and calculating its neutralization titer with Reed-Muench method is 2^-5.29, i.e., 2^-5.29The diluted serum can be protected It protects 50% chicken embryo and death does not occur.Show that VP3 recombinant protein can be used for preparing DHAV-1 neutralizing antibody and can be used as subunit Vaccine.

The chicken embryo death situation statistical result of the survey neutralization titer of table 5

=(72.7%-50%)/(72.7%-40%)=0.694

Lg (neutralization titer)=higher than logarithm-distance proportion × coefficient of dilution logarithm of 50% serum dilution

=-1.8-0.694 × (- 0.3)=- 1.59, neutralization titer=10^-1.59=1:39=2^-5.29, 0.2mL

4, VP3 protein B cell antigen epitope is identified

(1) prediction of VP3 protein B cell antigen epitope and the synthesis of peptide fragment

VP3 protein amino acid sequence is carried out respectively using Karplus&Schulz, Emini and Parker method flexible Property, surface accessibility and hydrophily are analyzed.Further combined with the Protean program in DNAstar7.1, use Jameson-Wolf method forecasting sequence antigenicity, comprehensive each factor are predicted to obtain possible B cell epitope to be 131~150 (FNTGRYQMSWYPIADGEQSL is named as VP3a), 200~209 (VNSSAPSNID is named as VP3b).It is given birth to by gill Change (Shanghai) Co., Ltd. and synthesizes corresponding epitope peptide fragment.

(2) determination of the best diluted concentration of epitope peptide fragment sample

Using VP3 polyclonal antibody as detection reagent, positive hole OD is selected by square matrix titration using indirect ELISA The antigen diluent degree of value 1.0 or so, negative control OD value 0.1 or so finally determines that best antigen diluent degree is 0.174ng/ μ L (1:1280 dilution), the result is shown in tables 6.

The determination of 6 epitope peptide fragment optimum dilution degree of table

Note: runic is denoted as corresponding P, N value of best antigen concentration.

(3) identification of epitope peptide fragment

Each epitope peptide fragment is diluted with 1:1280 and is coated with, and carry out indirect ELISA is compared with negative serum, the results are shown in Table 7. The P/N of VP3a and VP3b peptide fragment show VP3a (¹³¹FNTGRYQMSWYPIADGEQSL¹⁵⁰) and VP3b (²⁰⁰VNSSAPSNID²⁰⁹) energy Obviously in conjunction with VP3 polyclonal antibody, they are the B cell epitope of VP3, as shown in figure 14.

7 epitope screening qualification result of table

Note: runic is denoted as corresponding P, N value of satisfactory dilution.

Preparing polyclonal antibody there are certain requirements the structure and activity of recombinant protein, that is, need that there is stimulation body to generate The epitope of specific antibody, antigen protein (comformational epitope) in the form of primary structure (linear epitope) and higher structure show Epitope out can be identified by body, mostly anti-can also meet preparation after the recombinant protein dissolution renaturation of inclusion bodies expression Requirement because its epitope can sufficiently be shown (especially primary structure show linear epitope).In the present invention Western blot proves that VP3 recombinant protein can show the VP3 recombinant protein of preparation in conjunction with rabbit-anti DHAV-1 serum specificity Epitope is shown, and animal test results also turn out, after being immunized for several times, the fine jade of anti-VP3 recombinant protein rabbit anteserum Expand potency and is attained by 1:16 or more, and VP3 recombinant protein and DHAV-1 serum, polyclonal antibody and the equal energy of VP3 recombinant protein Specific binding.The rabbit-anti VP3 recombinant protein polyclonal antibody of this research preparation is identified correct, can be used as VP3B cell table The primary antibody of position identification, lays the foundation for follow-up test.

VP3 recombinant protein prepared by the present invention shows the energy for having in conjunction with DHAV-1 serum through Western blot verifying Power, however whether have that generate significant Neutralization antibody still unknown, in picornavirus, only fowl brain ridge at present The VP3 of marrow scorching viral (avian encephalomyelitis virus) is proved to be have immunogenicity, because passing through recombination Eukaryotic vector makees the specific antibody that certain potency can be detected after DNA gene vaccine mouse.The present invention is using laboratory Can DHAV-1 embryo-culture viruses --- CH60 plants of detection VP3 recombinant protein excite body to generate neutralizing antibody, and test measures VP3 The neutralization titer of recombinant protein polyclonal antibody is 2^-5.29(0.2mL) illustrates to be excited as VP3 recombinant protein anti-caused by body Body can neutralize DHAV-1, show that VP3 recombinant protein has the antibody that induction body generates neutralization activity, both illustrated VP3 albumen In contain B cell antigen epi-position, there are the potentiality of exploitation subunit vaccine, also illustrate that VP3 recombinant protein polyclonal antibody is available B cell antigen epi-position in identification VP3 albumen.

Indirect ELISA detection method of the embodiment 4 based on VP3 recombinant protein and its B cell antigen epi-position

1, the foundation of the indirect ELISA detection method based on VP3 recombinant protein

1.1, indirect ELISA detecting step:

(1) be coated with: antigen is diluted with 9.6 carbonate buffer solution of 0.05mol/L pH, 100 holes μ L/ addition enzyme mark hole, and 4 DEG C It is incubated overnight；

(2) board-washing: abandoning antigen liquid, with PBS (pH7.4) board-washing 4 times containing 0.05% polysorbas20,5min/ time, for the last time It is dried after board-washing, liquid in hole is patted dry on blotting paper (following board-washing step is identical)；

(3) it closes: confining liquid, 200 holes μ L/, 37 DEG C of incubation 90min being added into enzyme mark hole；

(4) add primary antibody: abandoning hole inner sealing liquid, board-washing simultaneously pats dry.Add serum dilution to be checked, 100 holes μ L/, 37 DEG C of incubations 90min；

(5) add ELIAS secondary antibody: abandoning serum dilution in hole, board-washing simultaneously pats dry.The rabbit-anti duck IgG dilution of HRP label is added Liquid, 100 holes μ L/, 37 DEG C of incubation 45min；

(6) it develops the color: abandoning liquid in hole, board-washing simultaneously pats dry.TMB working solution, 100 holes μ L/ is added, room temperature is protected from light effect After 10min, 2mol/L H is added in 50 holes μ L/₂SO₄Reaction is terminated, measures OD value under 450nm-630nm dual wavelength with microplate reader.

Judgment criteria bibliography: corresponding positive value (P) 1.0 or so (> 0.4), feminine gender value (N) < 0.4, and P/N is most Big person is as optimum reaction condition.

1.2, the optimization of indirect ELISA testing conditions:

(1) best antigen, serum dilution (chessboard method): VP3 recombinant protein makees 1:20,1:40,1:80,1:160,1: 320,1:640,1:1280 and 1:2560 series doubling dilution, 100 holes μ L/, each dilution add two holes (to be used separately as yin, yang Property hole), female plate and positive plates separate.Positive and negative serum makees 1:40,1:80,1:160,1:320,1:640 and 1:1280 system Column doubling dilution.

(2) it best ELIAS secondary antibody concentration: is coated with and is incubated for respectively with the best antigen of optimization, antibody concentration, by enzyme mark Antibody is made 1:500,1:1000,1:2000 and 1:4000 and is serially diluted.

(3) selection of best confining liquid: with best antigen, antibody and ELIAS secondary antibody concentration, being coated with respectively and be incubated for, closing Liquid selects 1%BSA, 5%BSA, 5% skimmed milk, 1% gelatin and 5% gelatin respectively.

It is 1.5mg/mL that VP3 recombinant protein, which is detected, as the initial concentration of antigen, according to square matrix method as a result, be shown in Table 8, Determine that antigen optimum dilution degree is 1:160 (requiring best antigen diluent degree is 9.375ng/ μ L), corresponding serum dilution For 1:160.Really definite opinion table 9, best confining liquid are 1% gelatin to best confining liquid.Best secondary antibody dilution is that 1:2000 is shown in figure 13.Note: runic is denoted as corresponding P, N and P/N value of best antigen, serum dilution in table 8.Runic is denoted as most preferably in table 9 Corresponding P, N and P/N value of confining liquid.

The determination of table 8 best antigen coat concentration, serum dilution

The optimization of 9 confining liquid of table

1.3, the determination of critical value

It takes 40 parts of DHAV-1 feminine gender duck bloods clear, is detected according to the indirect ELISA method of above-mentioned foundation.By negative sample The critical value of the sum of the average value (X (-)) of OD value and 3 times of standard variances (SD) as negative sample, i.e. the OD value of measuring samples It is determined as the positive when > (X (-)+3SD), otherwise is feminine gender.

40 parts of DHAV-1 negative serums are detected, the results are shown in Table 10, maximum value 0.27, minimum value 0.035, (average value) is that 0.122, SD (standard deviation) is 0.07, according to critical value calculation formula: X+3SD=0.122+3 × 0.07= 0.332。

Table 10 detects the OD value of 40 parts of negative serums

Note: runic mark is respectively OD peak and minimum.

1.4, sensitivity tests

By a DHAV-1 positive serum 1:80,1:160,1:320,1:640, l:1280 and l:2560 doubling dilution, if The vertical DHAV-1 positive and negative control, are then detected with the indirect ELISA method established, every part of serum sets 3 repetitions.

Continuous gradient dilutions are made by 1:80~1:2560 to a DHAV-1 positive serum, are repeated three times.As a result most Big 1:1280 times of diluted positive serum can be detected, as shown in figure 15.

1.5, specific test

The swollen head septicemia of DHAV-1, DHAV-3, duck plague virus, duck virus, duck is diluted respectively with best serum-concentration Virus, riemerella anatipestifer, duck source Escherichia coli, the positive duck blood of Salmonella anatis are clear, are setting up DHAV-1 negative serum (just Normal duck blood is clear) control, 3 repetitions of every part of serum, the cross reactivity of the method for inspection.

By DHAV-1 and DPV antigen, 10:1 is mixed by volume with DHAV-1 positive serum respectively, separately in the same way will DHAV-1 antigen is mixed with DHAV-3 positive serum.After acting on lh at 37 DEG C, it is diluted to best serum-concentration, it is indirect with foundation ELISA method detection.Meanwhile control is not blocked with the diluted DHAV-1 positive serum of optimum dilution degree, every part of serum sets 3 It repeats, blocking rate: PI (%)=100 × [1- (OD value/control serum OD value of test serum)] is calculated as follows.

As shown in figure 16, specific cross test result finds that only the OD value of DHAV-1 and DHAV-3 positive serum is greater than Critical value, mean OD value is respectively 1.347 and 1.232, and the positive serum of other cause of diseases is respectively less than critical value (0.332), Show that the method that this research is established can become while detect the universal method that DHAV-1 and DHAV-3 infects, without sick with other Cross reaction occurs for the serum of poison.

DHAV-1 positive serum is neutralized with DHAV-1 antigen and DPV antigen respectively, the same DHAV-1 of DHAV-3 positive serum Antigen neutralizes, and testing result as shown in figure 17 shows that DHAV-1 positive serum can be by DHAV-1 specific inhibition, and cannot be by DPV Antigen blocks, and calculates its blocking rate and reaches 82%, testing result as shown in figure 18 shows that DHAV-3 positive serum can be by DHAV-1 It blocks, blocking rate is 56%.

1.6, repetitive test

The positive and negative serum for taking difference OD value known to 6 parts (antibody level is different), is diluted by optium concentration, is added to same The VP3 recombinant protein of batch preparation is made in the ELISA Plate hole of antigen coat, and every part sets 4 repetitions, calculates being averaged for every part of serum OD valueWith standard deviation (SD), and then the coefficient of variation (CV) of every part of serum is calculated, formula is

The VP3 recombinant protein for taking different batches to prepare makees the ELISA Plate of antigen coat, detects above-mentioned 6 parts of serum, every part sets 4 A repetition, calculate CV value, evaluate sample batch between repeatability.

Make the ELISA Plate of antigen coat with the VP3 recombinant protein of different batches of preparations, while detecting different serum samples (3 respectively The positive sample of part, 3 parts of negative samples), every part of sample repeats detection 4 times, calculates its coefficient of variation CV%.

It is shown in Table 11 as the result is shown, variation within batch coefficient is up to 8.64%, minimum 0.73%, average out to 5.36.Between batch Coefficient of variation maximum 7.68%, minimum 3.35%, average out to 5.53%.There is no any portion in 6 parts of tested serum CV% is more than 10%, shows that this method has preferable repeatability.

11 repeatability analysis of table

1.7, VP3I-ELISA application and between DHAV-1I-ELISA coincidence rate assessment

Indirect ELISA method (DHAV-1I-ELISA) reference literature [Zhao X L, Phillips the R M, Li of totivirus G D,et al.Studies on the detection of antibody to duck hepatitis virus by Enzyme-linked immunosorbent assay [J] .Avian diseases, 1991,35 (4): 778-782.], it improves It is as follows: (1) to be proliferated DHAV-1 and purified；(2) make confining liquid with 5% skimmed milk, and act on 1.5h at 37 DEG C；(3) two Anti- action time is adjusted to 45min；(4) it is substituted with commercialization TMB developing solution；(5)OD_450nm-OD_630nmLower reading.

The indirect ELISA method (VP3I-ELISA) and DHAV-1 established with VP3 recombinant protein make the indirect ELISA of antigen (DHAV-1I-ELISA) the method clinical duck blood that the doubtful DHAV-1 of 60 parts of detection infects simultaneously respectively is clear, compares two methods Testing result, and the coincidence rate of two methods is calculated as follows: (the sum of common anode and common cathode sample number)/sample to be tested Sum.

It the results are shown in Table 12, make the indirect ELISA of antigen with DHAV-1 and the VP3I-ELISA method of foundation is examined simultaneously respectively The duck serum sample of 60 parts of doubtful DHAV-1 infection is surveyed, positive rate is respectively 86.7% (52/60) and 83.3% (50/60).Have The negative serum of a DHAV-1I-ELISA detection, VP3I-ELISA tests positive.There are 3 parts of DHAV-1I-ELISA to be positive Serum, VP3I-ELISA, which detected, to be negative.The symbol of VP3I-ELISA and DHAV-1I-ELISA are calculated according to formula Conjunction rate is 93.3% [(49+7)/60].

The coincidence rate of table 12VP3I-ELISA and DHAV-1I-ELISA method

Note: runic mark is respectively the common anode of two methods detection, negative sample number.

2, the indirect ELISA detection of VP3 protein B cell epitope

Antigen, the hole 200ng/ coated elisa plate are made with the VP3 recombinant protein of the mono- peptide of VP3a, the mono- peptide of VP3b and purifying respectively； The DHAV-1 duck blood final proof (being beforehand with ELISA detection as antigen with DHAV-1) of different antibodies level known to 10 parts is taken to make 1:40 Dilution, while setting negative duck serum control；Make secondary antibody with the rabbit-anti duck IgG (1:1000) that HRP is marked, it is thin referring to B in embodiment 1 The ELISA detecting step of the step of extracellular antigen Epitope Identification is detected.

The clear testing result of the DHAV-1 duck blood of 10 parts of different antibodies levels is shown in the same VP3 of Figure 19, VP3a and VP3b epitope peptide Recombinant protein can equally distinguish different DHAV-1 antibody levels, and the height of the antibody level reflected for testing result Regular three is consistent.But the indirect ELISA detected value that VP3 recombinant protein makees antigen is significantly higher than VP3a and VP3b epitope peptide Make the indirect ELISA detected value of antigen respectively, and is not significantly different between VP3a epitope peptide and the value of VP3b epitope peptide.

VP3 is one of main structural proteins of picornavirus, positioned at the outside of virion capsid, is exempted from induction body Epidemic disease answer party face has unique advantage, thus has the potentiality for developing diagnostic reagent and subunit vaccine.However at present to DHAV The research of structural proteins is also seldom, and immunological investigation is carried out in a deep going way dependent on each species specific antibody, for DHAV-1's The research of VP3 is also the same.The present invention carries out prokaryotic expression to the VP3 of DHAV-1, obtains VP3 recombinant protein, is used for for preparation Go deep into the antibody (such as Epitope Identification) of DHAV immunological investigation and establishes the indirect ELISA based on VP3 recombinant protein Detection method lays the foundation.The formulation immune programme of adaptation to local conditions is clinically needed to the control of DHAV, and this needs a kind of spirit Quick, easy, efficient method carries out antibody surveillance to special group.Due to present invention demonstrates that having B cell on VP3 albumen Epitope, therefore make antigen with the VP3 recombinant protein of prokaryotic expression establishes a kind of indirect ELISA method and preparation 1 type duck first of detection The ELISA kit of hepatovirus, detection duck blood it is clear in DHAV-1 antibody.By carrying out index evaluation to the method established, And Preliminary Applications have good clinical application potentiality in the detection of clinical sample；The present invention utilizes bioinformatics soft in turn Part comprehensive analysis, and 131aa~150aa and 200aa~209aa energy and rabbit-anti are shown by indirect ELISA method detection The how anti-generation specific reaction of VP3 recombinant protein.In order to assess potential value of the VP3 protein B cell epitope in checkout and diagnosis, Make antigen respectively with the B cell epitope peptide and VP3 recombinant protein that identify, has detected 10 parts of different antibodies water with indirect ELISA Flat DHAV-1 duck serum sample, as a result, it has been found that epitope peptide can recognize that the duck blood of DHAV-1 is clear as VP3 recombinant protein, and VP3 recombinant protein can be used to detect DHAV-1 antibody, this shows that epitope peptide has detection DHAV-1 anti-as VP3 recombinant protein The potentiality of body.It, can although epitope peptide can also identify that DHAV-1 serum, reactionogenicity are strong not as good as VP3 recombinant protein in this research It can be because not detected to epitope peptide separately using the testing conditions of the VP3 recombinant protein after optimized in the present embodiment The condition of DHAV-1 antibody optimizes；It could also be possible that epitope peptide molecular weight is too small, when coating, fails to be adsorbed onto well On elisa plate；It is also possible to being contained on VP3 than B cell epitopes more in this research, this needs further research confirmation. This, which also enlightens us, need to consider the technologies hands such as multi-epitope overlapping or coupling carrier to the research of DHAV epitope diagnostic reagent and vaccine Section.

Above-mentioned detailed description is illustrating for the possible embodiments invented, and the embodiment is not to limit this hair Bright the scope of the patents, it is all without departing from equivalence enforcement or change of the invention, it should all be contained in the scope of the patents of the invention.

In addition, those skilled in the art can also the claims in the present invention scope of disclosure and spirit in do other forms and Various modifications, addition and replacement in details.Certainly, these spirit is done according to the present invention various modifications, addition and replacements It, all should be comprising within scope of the present invention Deng variation.

Claims

1. a kind of B cell epitope of 1 type duck hepatitis A virus VP3 albumen, which is characterized in that its amino acid sequence such as SEQ ID NO: Shown in 4 or shown in SEQ ID NO:5.

2. the identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope of one kind described in claim 1, which is characterized in that packet Include following steps:

Step 1: it obtains VP3 target fragment: determining 1 type duck hepatitis A virus VP3 truncation gene restriction enzyme site and specific primer, Upstream primer is 5'-GGATCCGCAATGGACAATCAGGGAAAGAGA-3 ', downstream primer 5'-CTCGAGAACTGGCTAATCATCCCCTA-3'；Be proliferated DHAV-1 Strain, and extract the RNA of DHAV-1 Strain, using with The RNA of 1 type duck hepatitis A virus of machine primer pair carries out PCR amplification after carrying out cDNA synthesis for template with specific primer, obtains VP3 Target fragment；

Step 2: the VP3 target fragment T-A building recombinant expression plasmid pGEX-VP3: is cloned into pMD19-T (Simple) In carrier, building recombination T plasmid pMD19-VP3 is distinguishing double digestion empty plasmid pGEX-4T-1 and identification just with BamH I and Xho I True positive recombinant plasmid pMD19-VP3, recycling, connection, identification, obtains recombinant expression plasmid pGEX-VP3；

Step 3: preparation VP3 recombinant protein: the recombinant expression plasmid pGEX-VP3 is converted to e. coli bl21 DE3 In, recombinant protein inducing expression is carried out, after, by bacterium solution purifying, identification, 1 type duck hepatitis A virus VP3 recombinant protein is obtained, The VP3 recombinant protein amino acid sequence is as shown in SEQ ID NO:1；

Step 4: preparation VP3 recombinant protein polyclonal antibody detects serum titer and identifies the specificity of polyclonal antibody；

Step 7: B cell antigen epi-position is identified on VP3 albumen: prediction VP3 protein B cell antigen epitope, epitope synthesis Peptide carries out indirect ELISA with VP3 recombinant protein polyclonal antibody and negative serum.

3. a kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope according to claim 2, feature exist In the VP3 recombinant protein Anti-TNF-α preparation step includes: to prepare anti-blood with the immune male rabbit of the VP3 recombinant protein of purifying Clearly, with the serum titer of agar gel diffusion test detection last time 7d after immune, VP3 recombinant protein polyclonal antibody is obtained.

4. a kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope according to claim 3, feature exist In, the immune program include: it is first exempt to be emulsified with the VP3 recombinant protein solution of 0.5mg and isometric Freund's complete adjuvant be After Water-In-Oil state, the nape of the neck is intradermal, rabbit is subcutaneously injected；Head exempts from the latter 11 days VP3 recombinant protein solution with 1mg and isometric Incomplete Freund's adjuvant emulsifies after Water-In-Oil state, to carry out two in the nape of the neck subcutaneous injection of rabbit and exempt from；Two exempt from rear 22 days use After the VP3 recombinant protein of 1mg and the emulsification of isometric incomplete Freund's adjuvant are Water-In-Oil state, the nape of the neck be subcutaneously injected rabbit into Row three is exempted from；Head exempts from 34 and 47 days afterwards, carries out four with the VP3 recombinant protein auricular vein of 0.5mg injection rabbit and exempts to exempt from five.

5. a kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope according to claim 2, feature exist In median lethal dose ELD of the measurement DHAV-1 virus to chicken embryo₅₀Step includes: to make the virus liquid of DHAV-1 low virulent strain Continuous 10 times dilute and are inoculated with 9 age in days chick embryo allantoic cavities, shine egg observation daily to the chicken embryo after inoculation, count each group chicken embryo death Situation calculates the ELD of virus liquid using Reed-Muench method₅₀。

6. a kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope according to claim 2, feature exist In the neutralization titer step of the measurement VP3 recombinant protein polyclonal antibody includes: with surveying ELD₅₀DHAV-1 low virulent strain Virus liquid, by fixed virus-diluted blood heat-clearing method measurement VP3 recombinant protein polyclonal antibody neutralization titer, with Reed- Muench method calculates its neutralization titer.

7. a kind of identification method of 1 type duck hepatitis A virus VP3 protein B cell epitope according to claim 2, feature exist Including: in, the measurement B cell antigen epi-position authentication step first respectively can to the flexibility of VP3 protein amino acid sequence, surface And property and hydrophily are analyzed；Further forecasting sequence antigenicity, prediction obtains the B cell epitope of VP3 albumen, artificial synthesized Then epitope peptide fragment sample makees antigen with the VP3 recombinant protein of purifying, VP3 recombinant protein polyclonal antibody as detection reagent, Using indirect ELISA by square matrix titration, the antigen packet of positive hole OD value 1.0 or so, negative control OD value 0.1 or so is selected By concentration as best antigen diluent degree, each epitope peptide is finally diluted with best antigen concentration, with the rabbit-anti of doubling dilution VP3 recombinant protein polyclonal antibody makees primary antibody, and the control of negative rabbit anteserum is arranged in parallel, and HRP- goat anti-rabbit igg makees secondary antibody, according to ELISA method is connect to identify artificial synthesized epitope peptide fragment.

8. a kind of B cell epitope of 1 type duck hepatitis A virus VP3 albumen described in claim 1 detects 1 type duck hepatitis A in preparation Purposes in the 1 type duck hepatitis A virus antibody assay kit of kit or preparation of poison infection.

9. a kind of purposes of the B cell epitope of 1 type duck hepatitis A virus VP3 albumen described in claim 1, which is characterized in that institute The B cell epitope for stating 1 type duck hepatitis A virus VP3 albumen is applied in preparation 1 type duck hepatitis A virus recombinant vaccine of prevention.