CN104293823B - The preparation method and application of soluble Type I DHV 3D albumen - Google Patents

Abstract

The invention discloses the preparation method and application of soluble Type I DHV 3D albumen, preparation method is as follows：9th～1376 nucleotide of sequence shown in SEQ ID NO.3 is connected at the polyclone enzyme enzyme site of pET 32 (a)+carrier, then with escherichia coli Rosetta, BL21 or BL21 (DE3) PLYS as Host Strains, after activating in the LB culture medium of Amp resistances, IPTG to final concentration of 0.2～1.0mmol/L is added, is 20～39 DEG C of 4～12h of abduction delivering in temperature；Obtained I types DHV 3D albumen is solubility, and there is immunogenicity, duck body can be stimulated to produce the antibody of anti-I type DHV 3D albumen, the reagent of the serum sample or the 3D protein antibodies of DHV containing anti-I type of detection I type duck viral hepatitiss is can act as, significant to the diagnosis of I type duck viral hepatitiss and the research of vaccine.

Description

The preparation method and application of soluble Type I DHV 3D albumen

Technical field

The invention belongs to biochemical field, and in particular to the preparation method of soluble Type I DHV 3D albumen, also It is related to the application of the albumen.

Background technology

China is the most country of duck culturing quantity in the world, and the number of animals raised of duck accounts for the 70% of the world, and duck culturing industry is in China Occupy critical role in agricultural economy.Therefore duck source microorganism is carried out studying and there are important society and economic implications.Duck liver Scorching virus (Duck Hepatitis Virus, DHV) can cause duck viral hepatitiss (the Duck Viral of duckling Hepatitis, DVH).DVH is a kind of height lethal infectious diseases, so that urgency of falling ill, the course of disease be short, a height of spy of M ＆ M Point.DHV point is 3 each independent serotypes without serological relation：I types, II types and type III.Wherein, serum I type duck Hepatitis viruss (DHV-I), also known as DHAV (Duck hepatitis A virus, DHAV), are that current China is popular Main DHV, cause huge economic loss to China's duck culturing industry, threaten the sound development of duck culturing industry.And Three kinds of serotypes belong to Picornaviridae fowl hepatovirus, and virion is spherical in shape or spherical, and nucleocapsid is in 20 face bodies pair Claim, a diameter of 20-40nm, without breeding in cyst membrane, endochylema, single-stranded positive RNA, long 7000- of the genomic nucleic acids for non-segmented negative 8500nt, 5 ' untranslated regions (5 ' UTR) terminal covalent combine VPg, 3 ' untranslated regions (3 ' UTR) end tail containing polyA, and two non-turn over It is unique open reading frame (ORF) to translate between area, after ORF gives expression to polyprotein in host cell, is compiled by virus itself The proteolytic cleavage of code is segmented into maturation protein.1/3 coding structure albumen P1 of polyprotein N sections, is assembled into the capsid of virus；Remaining Coding P2 and P3 non-structural proteins, the structure such as the associated protein being primarily involved in needed for virus replication and host defense system and work( The regulation of energy, makes viral nucleic acid and albumen satisfactory duplication, translation in host cell.P3 coding nonstructural protein 3A, 3B, 3C and 3D, 3D albumen is the important non-structural protein of DHV, is the key enzyme of virus replication, therefore, to I type DHVs 3D carry out research there is most important theories and practical significance.

The basis that 3D is studied and is applied first has to obtain 3D albumen.The acquisition of target protein can adopt genetic engineering The method of vivoexpression, is presently expressed by the conventional eucaryon of exogenous gene and prokaryotic system, and eukaryotic system commonly uses insecticide, animal, yeast With cells of mamma animals etc., prokaryotic system is then mainly widely used using escherichia coli and at present, but be not each gene all Effective expression can be carried out wherein, and escherichia coli expression easily forms insoluble inclusion body, inclusion body is contained due to foreign protein Amount is relatively low, make it easier to isolate and purify the features such as can avoid proteasome degradation, be insoluble in water.The Crack cause of inclusion body is multiple Various, wherein topmost reason is probably that albumen aggregate velocity is too fast, is correctly folded without time enough；In addition, When recombiant protein lacks post translational modification, intermediate can be also made to accumulate in a large number and form inclusion body.Target egg in inclusion body Although its peptide chain white is complete, but the expressing protein of unnatural forms, no biological activity, the dissolving of inclusion body is very Difficulty, needs to interrupt intramolecular and intermolecular non-covalent bond, ionic bond etc. with strong denaturant high concentration urea, SDS etc., is Activated albumen is obtained, is needed then to carry out protein renaturation, and the renaturation after inclusion body protein dissolving is not only time-consuming, laborious, And renaturation yield is low.Therefore, carry out vivoexpression to obtain diagnosis of the activated 3D albumen to I type DHVs to 3D genes Significant with the research of recombinant vaccine.

Content of the invention

In view of this, an object of the present invention is the preparation method for providing soluble Type I DHV 3D albumen； The second object of the present invention is that providing soluble Type I DHV 3D albumen is preparing the DHV infection of detection I types Application of the Sanguis Anas domestica clearly or in the ELISA kit of the DHV 3D antibody of type containing I；The third object of the present invention is that offer can Application of the dissolubility I type DHV 3D albumen in 3D protein immunization serums are prepared；The fourth object of the present invention is to provide The immune serum prepared using soluble Type I DHV 3D albumen is preparing detection I types DHV or I type duck hepatitis Application in the reagent of viral 3D albumen.

For achieving the above object, the present invention provides following technical scheme：

1st, the preparation method of soluble Type I DHV 3D albumen, comprises the steps：By shown in SEQ ID NO.3 9th～1376 nucleotide of sequence is connected at the polyclone enzyme enzyme site of pET-32 (a)+carrier, then with escherichia coli Rosetta, BL21 or BL21 (DE3) PLYS is Host Strains, after activating in the LB culture medium of Amp resistances, in IPTG final concentrations It is abduction delivering 4～12 hours under the conditions of 20～39 DEG C for 0.2～1.0mmol/L, temperature.

Preferably, the Host Strains are e. coli bl21 (DE3) PLYS.

Preferably, abduction delivering 6 hours under the conditions of the final concentration of 0.8mmol/L of IPTG, temperature are for 25 DEG C.

Preferably, the activation is concretely comprised the following steps：Expression strain is inoculated in the LB culture medium of Amp resistances, 37 DEG C, incubated overnight under the conditions of 120r/min, be then 1 by culture fluid by volume with the LB culture medium of Amp resistances：100 expand Cultivate to OD₆₀₀For 0.6.

It is furthermore preferred that also include purification step after abduction delivering, specific as follows：Bacterium solution is collected, in 12000r/min conditions Collects thalline after lower centrifugation 10min, the thalline of collection are suspended with the Tris-HCl that 20mmol/L, pH are 8.0, then under ice bath Ultrasound, the thalline after crushing are centrifuged 10min under the conditions of 4 DEG C, 12000r/min, collect supernatant, supernatant Ni²⁺- NTA agar Sugared gel column purification, dialysis, the soluble Type I DHV 3D albumen for obtaining purification with 0.45 μm of ultrafiltration through membranes.

Most preferably, under the ice bath, ultrasound is ultrasonication 6 times under condition of ice bath, 30sec/ time, every time between Every 30sec.

2nd, methods described is obtained soluble Type I DHV 3D albumen and is preparing detection I type DHV infected ducks Application in the ELISA reagents of serum or the DHV 3D antibody of type containing I.

Preferably, the soluble Type I DHV 3D albumen is as I type duck viral hepatitiss antibody or I type ducks Application in the trapping agent of hepatitis viruss 3D protein antibodies.

3rd, methods described is obtained soluble Type I DHV 3D albumen and is preparing I type DHV 3D protein immunizations Application in serum.

4th, the I type DHV 3D protein immunization serums prepared using soluble Type I DHV 3D albumen are in system Application in the reagent of standby detection I types DHV or I type DHV 3D albumen.

The beneficial effects of the present invention is：The invention discloses the preparation method of soluble Type I DHV 3D albumen, The 3D albumen that the method is obtained is solubility, overcomes the defect that prior art easily forms inclusion body, the egg for therefore obtaining It is natural in vain, it is not necessary to carry out degeneration, renaturation using denaturant and obtain activated albumen.And by obtained solubility I Hyper-immune serum is obtained in that after type DHV 3D protein immunization rabbits, is shown that the recombiant protein has immunogenicity, can be pierced Sharp rabbit produces the polyclonal antibody of anti-3D albumen；Also with the 3D albumen for obtaining as I types can be captured after antigen coat agent DHV 3D protein antibodies, therefore, it is possible to the ELISA reagents as diagnosis I type duck viral hepatitiss, to I type duck virals The diagnosis of hepatitis and the research of recombinant vaccine are significant.

Description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below：

3D gene PCR amplified production electrophoresis result (Ms of the Fig. 1 for I type DHV strain DHAV-H：DL 2000Marker, 1：3D gene PCR amplified productions).

Fig. 2 is PCR identifications and enzyme action qualification result in the 3D genophore building processs of I type DHV strain DHAV-H (A：The identification of pJET-1.2+/DHAV-H-3D plasmid PCRs, M：DL2000Marker, 1：PCR primer；B：pJET-1.2+/DHAV- The identification of H-3D plasmid enzyme restrictions, M：DL15000Marker, 1：I/Xho of Kpn, I double digestion bands, 2：I single endonuclease digestion bands of Xho；C： The PCR identifications of pET-32 (a) +/DHAV-H-3D plasmids, M：DL2000Marker, 1：PCR primer；D：pET32a+/DHAV-H- The enzyme action identification of 3D plasmids, 1：I/Xho of Kpn, I double digestion results, 2：I single endonuclease digestion bands of Xho).

Fig. 3 for solubility 3D protein expression condition optimization (M be low molecular weight protein standard substance；A：The sieve of expression bacterium Choosing, 1 be pET-32 (a)+empty carrier expression product, 2-4 be respectively pET-32 (a) +/DHAV-H-3D recombinant plasmid transformeds The expression product of Rosetta, BL21 and tri- kinds of different expressive host bacterium of BL21 (DE3) PLYS；B：The optimization of abduction delivering time, 1-5 is followed successively by BL21 (DE3) the PLYS Host Strains expression products of induction 12h, 10h, 8h, 6h and 4h；C：Abduction delivering temperature Optimize, 1-6 is respectively 39 DEG C, 37 DEG C, 35 DEG C, 30 DEG C, 25 DEG C and 20 DEG C of BL21 (DE3) PLYS Host Strains expression products；D： IPTG concentration optimizations, 1-5 are respectively 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mmol/L The expression product of IPTG inductions.

Fig. 4 is that solubility 3D albumen Western-blot is detected and (immunoblotting inspections of the A for 3D albumen of purifying protein electrophoresis Result is surveyed, M is protein Marker, and 1 is 3D Western blots；B is the 3D albumen that SDS-PAGE detects purification, and M is low-molecular-weight Protein Marker, 1 is the 3D albumen of purification).

Fig. 5 is that rabbit-anti solubility 3D albumen hyper-immune serum fine jade expands bioactivity.

Immunohistochemical Method detection DHAV and 3D distributions in immune duck liver of the Fig. 6 for rabbit-anti 3D albumen hyper-immune serum (A：SABC detects distribution of the solubility 3D protein immunization duck 3D albumen in liver；B：The weak poison of SABC detection DHAV Seedling and distribution of the solubility 3D albumen co-immunization duck 3D albumen in liver；C：SABC detects DHAV Attenuate vaccine immune ducks Distribution of the 3D albumen in liver；D：SABC detection control duck liver organization (10 × 60)；Arrow shows specific stain).

Specific embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted concrete in embodiment The experimental technique of condition, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. write) Described in condition, or according to the condition proposed by manufacturer.

Embodiment 1, the 3D genes of clone I type DHV strain DHAV-H

(genome sequence is shown in GenBank to I types DHV strain H (DHAV-H)：JQ301467), E.coli DH5 α bacterium Plant and pET-32 (a)+carrier is preserved and provided by Sichuan Agricultural University's poultry disease prevention and control research center.Various molecular biology examinations Agent is purchased from biological reagent company.

Genome sequence (GenBank according to DHAV-H：JQ301467) the primer of design amplification 3D genes, concrete primer As follows：

P1：5’-ggggtaccGatcaagggaaagtagtgagcaag-3 ' (SEQ ID NO.1), underscore represents Kpn I restriction enzyme site；

P2：5’-acgcctcgagTcagatcatcatgcaagctgt-3 ' (SEQ ID NO.2), underscore represents Xho I Restriction enzyme site；Then the primer of design is synthesized by precious biological engineering (Dalian) company limited.

The DHAV-H virus liquids that deposits of going bail for make 5 times of dilutions with the PBS that sterilizes, and add dual anti-(penicillin, the chain of 1/100 volume The final concentration of mycin be respectively 100IU/mL and 100 μ g/mL) after 37 DEG C incubate 1h, be then inoculated with 9 ages in days physically well develop, nothing The duck embryos of maternal antibody, discard dead embryo in 24h, collect the allantoic fluid and idiosome of 24～72h death embryos, according to Trizol reagents Box description extracts viral RNA.

By the reverse transcription of viral RNA synthesis cDNA for extracting, performing PCR amplification, reverse transcription and PCR are entered as template with cDNA then Amplification system is as shown in table 1.

1 reverse transcription of table and PCR reaction systems

Reverse transcription program is：30 DEG C of 10min, 42 DEG C of 15min, 95 DEG C of 5min, 4 DEG C of 5min, circulation primary；PCR programs are： 94 DEG C of denaturations 5min；94 DEG C of degeneration 40sec, 56 DEG C of annealing 40sec, extend 30sec after 72 DEG C, 30 circulate, last 72 DEG C Extend 10min afterwards.Pcr amplification product is entered row agarose gel electrophoresis, as a result as shown in Figure 1.As a result show, amplification is grown The fragment (not including that restriction enzyme site and protectiveness base are 1368bp) of degree about 1386bp, its nucleotide sequence such as SEQ ID Shown in NO.3, identical with expected clip size, therefore according to the glue reclaim test kit of TIANGEN Biotech (Beijing) Co., Ltd. Description reclaims purpose band, reclaims the fragment for obtaining and is named as DHAV-H-3D.

Embodiment 2, the expression vector of construction expression I type DHV strain H 3D albumen

The DHAV-H-3D for reclaiming is connected with pJET1.2 carriers, coupled reaction is with reference to pJET1.2 Cloning Kit explanations Book is carried out, and reaction system is as shown in table 2.

Table 2, connection DHAV-H-3D and pJET1.2 carriers

By brief centrifugation being carried out after the mixing of 2 system of table, then connect 15min under the conditions of 20 DEG C.Connection product converts DH5 α competent cells, and the LB solids with Amp resistances and fluid medium screening, and will screening bacterium colony with SEQ ID NO.1 and Sequence shown in SEQ ID NO.2 enters performing PCR detection for primer, and amplified production enters row agarose gel electrophoresis, as a result such as A in Fig. 2 Shown.As a result show, screening obtains positive colony.

In order to further detect positive colony, the bacterial strain of positive colony is extracted plasmid, then through Kpn I and Xho I couple Enzyme action and the identification of I single endonuclease digestions of Xho, enzyme action system are as shown in table 3.

Table 3, I enzyme action identification reaction system of Kpn I and Xho

Brief centrifugation after the mixing of 3 system of table is pressed, then in 37 DEG C of condition water-bath 3h, reaction terminates for digestion products to carry out fine jade Sepharose electrophoresis detection, as a result as shown in B in Fig. 2.As a result show, DHAV-H-3D fragments are correctly connected into pJET1.2 carriers In, and it is named as pJET-1.2+/DHAV-H-3D.Invitrogen companies are sent to be sequenced pJET-1.2+/DHAV-H-3D, screening Unmutated sequence is used for construction of expression vector.

Taking correct pJET-1.2+/DHAV-H-3D and pET-32 (a)+carrier of sequencing is carried out with Kpn I and Xho I respectively Double digestion, reclaims 3D genes and carrier framework, and then system as shown in table 4 is attached.

Table 4,3D genes and carrier framework linked system

Brief centrifugation after the mixing of 4 system of table is pressed, is then overnight connected under the conditions of 16 DEG C, is obtained pET-32 (a) +/DHAV-H- 3D plasmids.Connection product converts DH5 α Host Strains, is screened with Amp resistances LB solid and fluid medium, then enters performing PCR mirror Fixed, as a result as shown in C in Fig. 2.Then the bacterial strain that PCR is accredited as the positive is used for extracting plasmid, and by plasmid with I Hes of Kpn Xho I carries out double digestion and Xho I and carries out single endonuclease digestion, and digestion products enter row agarose gel electrophoresis, as a result as shown in D in Fig. 2. Correctly connected from C in Fig. 2 and D, 3D gene and carrier framework.

Embodiment 3, the expression of I type DHV strain H solubility 3D albumen

Expressive host bacterium escherichia coli (Escherichia coli) Rosetta, e. coli bl21 and escherichia coli BL21 (DE3) PLYS strains are preserved by Sichuan Agricultural University's poultry disease prevention and control research center；The DHV strain H 3D eggs of type containing I White expression vector pET-32 (a) +/DHAV-H-3D is built by embodiment 2；Various molecular biology reagents are purchased from biological examination Agent company.

The pET-32 (a) that embodiment 2 is obtained +/DHAV-H-3D plasmids convert BL21, Rosetta and BL21 respectively (DE3) PLYS expression bacterium, transformed bacteria is 37 DEG C in the LB fluid mediums of Amp resistances, incubated overnight under the conditions of 120r/min, secondary Day it is 1 by volume by the LB fluid mediums of overnight culture and fresh Amp resistances：100 amplification culture about 3 hours, bacterium solution OD₆₀₀Up to addition IPTG when 0.6 to final concentration of 0.2mmol/L, and 12h is induced at 37 DEG C, then collect bacterium solution, by bacterium solution Under the conditions of 12000r/min, centrifugation 10min, thalline presses 1 with 20mmol/L Tris-HCl (pH 8.0):10 (V/V) suspend. Simultaneously using pET-32 (a)+carrier conversion respective table up to bacterium as negative control.

In order to screen the expression bacterium for giving expression to solubility 3D albumen, then by suspension bacteria liquid obtained above in condition of ice bath Lower ultrasonication 6 times, 30sec/ time, every time between be spaced 30sec, then will broken after bacterium solution in 4 DEG C, 12000r/min bars 10min is centrifuged under part, precipitation (for insoluble inclusion body expressing protein) is abandoned, supernatant (for soluble express protein) is collected.Draw 80 μ L of supernatant, add 5 × SDS loadings buffer of the 20 μ L containing beta -mercaptoethanol, boil after 10min under the conditions of 12000r/min Room temperature is centrifuged 5min, then carries out SDS-PAGE electrophoretic examinationss, as a result as shown in A in Fig. 3.As a result show, by matter of recombinating After grain pET-32 (a) +/DHAV-H-3D is transformed into three kinds of expression bacterium, purpose band is occurred in that at about 68kD, and negative control This band is not contained, and converts the expressing quantity maximum of BL21 (DE3) PLYS bacterial strains, the expression of BL21 and taken second place, so Optimum expression bacterium is filtered out for BL21 (DE3) PLYS according to expression.

The optimization of I type DHV strain H solubility 3D protein expression conditions：

(1) optimization of IPTG concentration：Will be containing BL21 (DE3) the PLYS bacterium of pET-32 (a) +/DHAV-H-3D recombiant plasmid The bacterium solution of strain is 1 by volume:100 are inoculated in 5 LB fluid medium test tubes containing Amp resistances, and in 37 DEG C of vibration trainings Support to OD₆₀₀=0.6 or so, add IPTG to final concentration be respectively 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mmol/L, then inducing culture 12h under the conditions of 37 DEG C, then detects solubility 3D egg with SDS-PAGE White expression, as a result as shown in B in Fig. 3.As a result show, solubility 3D albumen under the conditions of the final concentration of 0.8mmol/L of IPTG Expression highest, i.e. the final concentration of 0.8mmol/L of IPTG be optimum concentration.

(2) abduction delivering temperature optimization：Will be containing BL21 (DE3) PLYS of pET-32 (a) +/DHAV-H-3D recombiant plasmid The bacterium solution of bacterial strain is 1 by volume:100 are inoculated in 6 LB fluid medium test tubes containing Amp resistances, and vibrate in 37 DEG C Cultivate to OD₆₀₀=0.6 or so, add IPTG to final concentration of 0.8mmol/L, then respectively at temperature be 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, induce 12h under the conditions of 37 DEG C and 39 DEG C, then detect the expression of solubility 3D albumen with SDS-PAGE, as a result such as In Fig. 3 shown in C.As a result show, the expression highest of temperature solubility 3D albumen under the conditions of 25 DEG C, i.e. inducing temperature are 25 DEG C For suitableeest expression temperature.

(3) abduction delivering is time-optimized：Will be containing BL21 (DE3) PLYS of pET-32 (a) +/DHAV-H-3D recombiant plasmid The bacterium solution of bacterial strain is 1 by volume:100 are inoculated in 5 LB fluid medium test tubes containing Amp resistances, and vibrate in 37 DEG C Cultivate to OD600=0.6 or so, add IPTG to final concentration of 0.8mmol/L, be then to induce under the conditions of 25 DEG C in temperature 4h, 6h, 8h, 10h and 12h, then detect the expression of solubility 3D albumen, as a result as shown in D in Fig. 3 with SDS-PAGE.Knot Fruit shows, the expression of temperature solubility 3D albumen after abduction delivering 6h is reaching highest, i.e. abduction delivering time for 6h.

Through above-mentioned optimization, it can be seen that BL21 (DE3) PLYS containing pET-32 (a) +/DHAV-H-3D recombiant plasmid Bacterium optimal expression condition is 0.8mmol/L IPTG, 25 DEG C of induction 6h.Great expression is carried out to 3D albumen according to this condition subsequently.

I type DHV strain H solubility 3D albumen Western-blot is detected, is comprised the following steps that：Press optimal expression Condition express solubility 3D albumen, then carry out SDS-PAGE, subsequently by electrophoresis after gel be transferred on pvdf membrane, 80V turn Pvdf membrane is taken out after finishing by print 90min, transfer, with 1%BSA in 37 DEG C of shake incubation 1h, then with 1:100 dilutions anti- The IgG of DHAV is taken out after 37 DEG C of shake incubation 1h, is washed 3 times with TBS, and each 2min, subsequently with 1:The HRP marks of 3000 dilutions Two anti-igg of note treat purpose band in 37 DEG C of shake incubation 1h to develop the color according to DAB colour reagent boxes description after TBS washings With distilled water flushing color development stopping when high-visible, as a result as shown in A in Fig. 4, finally pvdf membrane is dried and is kept in dark place.

The purification of I type DHV strain H solubility 3D albumen：To contain recombiant plasmid pET-32 (a) +/DHAV-H-3D's BL21 (DE3) PLYS is expressed under optimal condition, then collects thalline, after being suspended with 20mmol/L Tris-HCl (pH 8.0) Ultrasonication 6 times under condition of ice bath, 30sec/ time, every time between be spaced 30sec, then will broken after bacterium solution 4 DEG C, 10min is centrifuged under the conditions of 12000r/min, supernatant (for soluble express protein) is collected；Then by the supernatant that collects in Bio- The Ni2+-NTA agarose gel column purifications (refined solution and purification process are carried out by kit specification) that rad companies provide, will The purifying protein liquid of collection dialysed after with 0.45 μm of ultrafiltration through membranes, be finally placed in 4 DEG C, -20 DEG C, -70 DEG C or lyophilizing protected Deposit.Solubility 3D albumen after purification is carried out SDS-PAGE electrophoresis, as a result as shown in B in Fig. 4.As a result the 3D eggs of expression are shown In vain through Ni²⁺After-NTA affinity purifications, the higher albumen of purity, concentration is obtained, dense through nucleic acid-protein analysis-e/or determining albumen Spend for 1.5mg/mL.

Embodiment 4, using solubility 3D albumen with ELISA method detect I type duck viral hepatitiss antibody

ELISA method detects comprising the following steps that for I type duck viral hepatitiss antibody：

(1) antigen coat Sptting plate：The solubility 3D albumen for adding 3 purification of embodiment to obtain, 100 μ L/ holes are coated, secondary Day PBST board-washing 5 times, each 3min is patted dry；

(2) closed with the PBST containing 1%BSA, 1h is closed under the conditions of 37 DEG C, ibid board-washing；

(3) add Sanguis Anas domestica to be checked clear, 1.5h is incubated under the conditions of 37 DEG C, ibid board-washing；

(4) enzyme labelled antibody (HRP labelling goat-anti duck IgG) of working concentration is added, and 0.5h is processed under the conditions of 37 DEG C, ibid Board-washing；

(5) TMB nitrite ions, lucifuge is added to react 15min；

(6) terminate liquid (2mol/L H are added₂SO₄), 50 μ L/ holes；

(7) microplate reader reads OD with double wave long form₄₅₀-OD₆₃₀Value；

(8) it is optimum reaction condition to select P/N values the maximum.

In order to obtain optimal Detection results, condition optimizing as shown in table 5：

Table 5, indirect ELISA condition optimizing

The reaction condition optimization of ELISA method

(1) optimal enzyme labelled antibody concentration

Enzyme labelled antibody (the HRP labelling goat-anti duck IgG of KPL companies, concentration are 100 μ g/mL) is made 1 respectively:100、1: 200、1:400、1:800、1:1600 concentration dilutions, each concentration arrange two repetitions, are incubated in 37 DEG C, while arranging negative and positive Property serum, then detect OD₄₅₀-OD₆₃₀Value, and average, as a result as shown in table 6.Select P/N values the maximum anti-for optimal two Dilution factor.

Table 6, enzyme labelled antibody working concentration optimize

As shown in Table 6, enzyme labelled antibody makees 1:Best results during 800 dilution.

(2) optimal antigen coat concentration and serum dilution

The solubility 3D albumen that 3 purification of embodiment is obtained is made 1 respectively:50、1:100、1:200、1:400、1:800 and 1: 1600 dilutions, the male/female property serum of duck viral hepatitiss make 1 respectively:100、1:200、1:400、1:800 dilutions, each concentration set 2 repetitions are put, OD is detected₄₅₀-OD₆₃₀Average after value, as a result as shown in table 7.It is optimum condition to select P/N values the maximum.

Table 7, antigen coat concentration and serum dilution are selected

As shown in Table 7, solubility 3D albumen is made 1：1:1600, serum makees 1：400 dilution effects are optimal.

(3) optimal developing time

Developing time is respectively set to 5min, 10min, 15min, 20min, 25min and 30min, each developing time Positive, negative serum is respectively provided with 4 repetitions, in 37 DEG C of lucifuge colour developings, then detects OD₄₅₀-OD₆₃₀Value, reads meansigma methodss, knot Fruit is as shown in table 8.It is optimal developing time to select P/N values the maximum.

Table 8, optimal developing time are selected

As a result show, best results when developing time is 15min.

(4) be most preferably coated condition

After 1h is processed at 37 DEG C respectively at 4 DEG C overnight, overnight and direct at 4 DEG C overnight three kinds at 4 DEG C after 37 DEG C of process 4h Under the conditions of coated, while arranging positive and negative serum, OD is read in each 7 repetitions₄₅₀-OD₆₃₀Meansigma methodss, as a result such as 9 institute of table Show.P/N values the maximum is selected for optimal coated condition.

Table 9, coated condition optimizing

As shown in Table 9, coated under the conditions of 4 DEG C is overnight optimal coated condition.

(5) optimal confining liquid, off-period

Respectively with 1%BSA, 5%BSA, 1% defatted milk powder, 5% defatted milk powder, 1% calf serum, 5% calf serum, 1% gelatin and 5% gelatin are confining liquid, and each confining liquid arranges 3 repetitions, and closes under the conditions of 37 DEG C, reads OD₄₅₀- OD₆₃₀Meansigma methodss, as a result as shown in table 10.Then off-period is set for 30min, 60min, 90min and 120min, is read OD₄₅₀-OD₆₃₀Meansigma methodss, as a result as shown in table 11.P/N values the maximum is selected as optimal confining liquid, off-period.

Table 10, optimal confining liquid are selected

Table 11, off-period is selected

From table 10 and table 11, optimal confining liquid is 1%BSA, and optimal off-period is 0.5h.

According to ELISA kit and detection method that above-mentioned optimal conditions obtain detection I type duck viral hepatitiss antibody：

ELISA kit is：The coated Sptting plate of soluble Type I DHV 3D albumen, enzyme labelled antibody, nitrite ion and Terminate liquid, wherein Sptting plate are with the soluble Type I DHV 3D albumen of 1 μ g/mL of concentration by the addition Sptting plate per 100 μ L of hole In, 4 DEG C are overnight coated, next day PBST board-washing 5 times, pat dry, its best results；Enzyme labelled antibody be HRP labelling goat-anti duck IgG, institute Nitrite ion is stated for TMB, the terminate liquid is the H that concentration is 2mol/L₂SO₄.

ELISA detection method is：Overnight it is coated with 4 DEG C of the solubility 3D protein liquid of 1 μ g/mL, using 1%BSA as closing 37 DEG C of closing 0.5h of liquid, an antiserum dilution factor 1:400 are incubated 1.5h in 37 DEG C, and the goat-anti duck two of HRP labellings is anti-with 1:800 is dilute Releasing and 0.5h being incubated in 37 DEG C, develop the color 15min.

According to above-mentioned ELISA reaction condition optimizations result, the specific detection of ELISA kit is carried out：

(1) specific cross experiment

With ELISA kit detection Salmonella anatis (Sal), E. coli isolated from ducks (E.coli), the swollen head deteriorated blood of duck set up Mo Shi bacillus (RA) positive serum in syndrome virus (DSHDV), bird flu viruss H5 (AIV), duck plague virus (DPV) and duck, is arranged Duck hepatitis are positive, negative serum control, and each serum arranges 3 repetitions, reads OD₄₅₀-OD₆₃₀Meansigma methodss, as a result such as 12 institute of table Show.

Table 12, solubility 3D protein ELISA method specific detection

As a result show, six kinds of positive serums to be checked are respectively less than 2.1 with the ratio of negative serum, show the ELISA side for setting up Method specificity is fine.

(2) specific inhibition experiment

The positive serum infected with I types DHV with solubility 3D albumen is 10 by volume：1 mixing, in 37 DEG C Optimal serum dilution is diluted to after neutralization 1h, with the ELISA method detection that sets up, blocking rate is calculated, as a result as shown in table 13.

Table 13, solubility 3D albumen blocking experiment

As a result show, positive serum blocking-up rate is 72.8%, negative serum blocking-up rate is 16.4%.Show that 3D albumen can be with Duck hepatitis positive serum specific recognition is simultaneously neutralized.

(3) coefficient of variation

Different batches coating enzyme mark strip is pressed with the solubility 3D albumen of a collection of purification, the OD of 6 parts of serum is then detected₄₅₀- OD₆₃₀Value, every part of serum arrange 6 repetitions, detect that its coefficient of variation makees the coefficient of variation, coefficient of variation CV=standard in plate, between plate Difference/meansigma methodss (SD/X × 100%), as a result as shown in table 14 and table 15.

In table 14, plate, the coefficient of variation is determined

Make a variation between table 15, plate coefficient determination

As a result show, in plate the coefficient of variation in 1.9%-6.6%, respectively less than 10%, between plate, the coefficient of variation is in 1.4%- 6.7%, respectively less than 10%.Show the ELISA method based on solubility 3D Protein Detection I type duck viral hepatitiss antibody that sets up Favorable reproducibility.

(4) positive threshold value determines

With solubility 3D albumen optimum dilution degree coating enzyme mark strip, reacted with optimum reaction condition, detected 60 parts of the moon Property serum OD₄₅₀-OD₆₃₀Value, calculates cut-off values=average+3 × variance (V_cut-off=X+3 × SD), as a result such as 16 institute of table Show.

16,60 parts of negative serum OD of table₄₅₀-OD₆₃₀Value

+ 3 × SD of average is taken as positive threshold value, is as a result shown, positive threshold value is 0.155+3 × 0.028=0.238.

(5) sensitivity Detection

8 parts of I type duck viral hepatitiss positive serums are taken, respectively from 1:100 start, with 2 times of doubling dilutions, with set up ELISA method detection, so that the greatest dilution of the positive can be detected as sensitivity, as a result as shown in table 17.

Table 17, solubility 3D protein ELISA method sensitivity technique

As a result show, the sensitivity of ELISA detection I type duck viral hepatitiss serum is 1:3200.

(6) solubility 3D albumen ELISA detection clinical serum sample and with the ELISA method based on DHAV-1 totiviruss Coincidence rate compares

ELISA method with solubility 3D albumen, DHAV-1 totiviruss as antigen detects 60 parts of identical clinic blood respectively Final proof product are in OD₄₅₀-OD₆₃₀Value, then compares the coincidence rate of two methods, as a result as shown in table 18 and 19.Table 18, respectively with can ELISA method detection 60 parts of serum OD of Parallel testing of dissolubility 3D albumen and DHAV-1 totiviruss₄₅₀-OD₆₃₀Value

A meets for the positive, and b meets for feminine gender, and c is not meet

Table 19, based on solubility 3D albumen and the coincidence rate of the indirect ELISA method of DHAV-1 totiviruss

As a result show, the positive coincidence rate of two methods detection is 83.3%, negative detection coincidence rate is 85.7%, total symbol Conjunction rate is 84.5%, illustrates that the ELISA method that sets up has good accuracy.

In sum, I type duck viral hepatitiss are detected by ELISA method using solubility 3D albumen obtained in the present invention Serum sample has high specificity, favorable reproducibility, sensitivity high, the symbol with the ELISA method detection for making antigen coat with totiviruss Conjunction rate is high, can be used for the detection of clinical sample.

Embodiment 5, the application for preparing hyper-immune serum and its hyper-immune serum using solubility 3D albumen

(1) solubility 3D albumen hyper-immune serum is prepared, antibody titer is determined with agarose diffusion test

The solubility 3D albumen obtained using 3 purification of embodiment, according to the method immunity rabbit of table 20.

Table 20, rabbit-anti 3D albumen hyper-immune serum prepare immune programme for children

Immunity completes rear neck artery and takes blood system from serum, determines antibody titer with agarose diffusion test, as a result such as Fig. 5 institutes Show.As a result show, the expansion fine jade potency of hyper-immune serum is 1:16.Then gained serum subpackage is stored in -20 DEG C or lyophilizing is preserved.

(2) the Immunohistochemical Method detection distributions of the DHAV and 3D in immune duck liver of rabbit-anti 3D albumen hyper-immune serum

By DHAV Attenuate vaccines, solubility 3D albumen individually and 1 age in days duck of co-immunization, after immunity, 11d is with 10000ELD₅₀ Survival duck is put to death by the strong poison of duck hepatitis to immune duckling counteracting toxic substances on the 21st.The dead duck of collection and execution duck liver are dirty, are fixed on rapidly In 4% paraformaldehyde, take out after fixed 48h, wash 20min.Detected by following procedure：

A. according to common pathology section flow process, serial dehydration, waxdip, embedding, section, dewaxing and water are carried out by dirty for duck liver Change, with being washed 3 times with PBST after the Ethanol Treatment that 5 volume fractions are 50% after aquation, each 5min；

B. antigen retrieval：Repaired using enzyme or hot restorative procedure reparation, enzyme restorative procedure is 50 μ L of every microscope slide Deca Mass fraction is 0.05% trypsin solution, and 37 DEG C of digestion 20min in wet box, digestion finish rear PBST and wash 3 times；Hot restorative procedure It is that section is positioned in the citrate buffer of 0.01mol/L pH 6.0, the middle-grade reparation 30min of microwave oven, after reparation certainly Room temperature is so cooled to, and PBST is washed 3 times；

C. close：Closing endogenous peroxydase and closing nonspecific binding site, close Endogenous peroxide The method of enzyme is Deca 3%H₂O₂- methanol solution, every 100 μ L of microscope slide, 37 DEG C of incubation 30min in wet box, after incubation is finished PBST is washed 3 times；Closing nonspecific binding site is 100 μ L 10%BSA of every microscope slide Deca, 37 DEG C of incubations in wet box 30min, PBST are washed 3 times；

D. anti-incubation of plus：1 is pressed with the rabbit-anti solubility 3D protein I gG of purification:200 dilutions, every microscope slide Deca 100 μ L, put overnight incubation in 4 DEG C of wet box, and next day PBST is washed three times；

E. plus HRP goat anti-rabbit iggs incubation：1 is selected with reference to kit specification:2000 dilutions, are incubated in 37 DEG C of wet box 45min, incubation finish rear PBST and wash three times；

F.DAB develops the color：Nitrite ion is prepared according to kit specification, is developed the color under low light level dark situation, every microscope slide Deca 50 μ L, develop the color about 40sec, when observing that tissue changes colour immediately with distilled water wash, color development stopping；

G. redye and mounting：Dyeing 15min or so in haematoxylin dye liquor, basis of microscopic observation is coloured, and contrast is substantially Can terminate dyeing, take out washing and return indigo plant.Then differentiation, dehydration, the transparent rear mounting of dimethylbenzene, observed and recorded result (figure after drying 6).

Through Immunohistochemical detection, visible positive specific stain in hepatocyte cell matter.Wherein DHAV Attenuate vaccines with Positive specific stain during solubility 3D albumen co-immunization duck liver is dirty is most (B), during solubility 3D protein immunization duck liver is dirty Positive specific stain take second place (A), and the positive specific stain in DHAV Attenuate vaccine immune duck livers is minimum (C), control Group has no specific stain (D).It can thus be seen that the Immunohistochemical Method of rabbit-anti 3D albumen hyper-immune serum can be used for tissue sample The positioning of middle 3D and distribution detection, it can also be used to the detection that infection group and viral infection group is knitted.

Finally illustrate, preferred embodiment above is only unrestricted in order to technical scheme to be described, although logical Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be In form and various changes are made to which in details, without departing from claims of the present invention limited range.

Claims (7)

1. the preparation method of soluble Type I DHV 3D albumen, it is characterised in that comprise the steps：By SEQ ID 9th ~ 1376 nucleotide of sequence shown in NO.3 is connected at the polyclone enzyme enzyme site of pET-32 (a)+carrier, then with big Enterobacteria BL21 (DE3) PLYS is Host Strains, after activating in the LB culture medium of Amp resistances, in IPTG final concentration of 0.2 ~ 1.0 Mmol/L, temperature are abduction delivering 4 ~ 12 hours under the conditions of 20 ~ 39 DEG C, collect bacterium solution, are centrifuged 10 under the conditions of 12000 r/min Collects thalline after min, the thalline of collection are suspended with the Tris-HCl that 20 mmol/L, pH are 8.0, then ultrasound under ice bath, will Thalline after broken is centrifuged 10min under the conditions of 4 DEG C, 12000r/min, collects supernatant, supernatant Ni²⁺- NTA agarose gel Column purification, dialysis, the soluble Type I DHV 3D albumen for obtaining purification with 0.45 μm of ultrafiltration through membranes.

2. preparation method according to claim 1, it is characterised in that：In final concentration of 0.8 mmol/L of IPTG, temperature it is Abduction delivering 6 hours under the conditions of 25 DEG C.

3. preparation method according to claim 1, it is characterised in that the activation is concretely comprised the following steps：By expression strain It is inoculated in the LB culture medium of Amp resistances, then culture fluid is resisted by incubated overnight under the conditions of 37 DEG C, 120 r/min with Amp Property LB culture medium by volume be 1：100 amplification culture are to OD₆₀₀For 0.6.

4. method according to claim 1, it is characterised in that：Under the ice bath, ultrasound is ultrasonication under condition of ice bath 6 times, 30sec/ time, every time between be spaced 30sec.

5. any one of claim 1-4 methods described is obtained soluble Type I DHV 3D albumen and is preparing detection I type duck liver Application in the ELISA kit of scorching virus infected duck serum or the DHV 3D antibody of type containing I.

6. application according to claim 5, it is characterised in that：The soluble Type I DHV 3D albumen is in conduct Application in the trapping agent of anti-I type DHV 3D protein antibodies or anti-I type duck viral hepatitiss antibody.

7. any one of claim 1-4 methods described is obtained soluble Type I DHV 3D albumen and is preparing I types duck hepatitis disease Application in malicious 3D protein immunization serums.