CN105273065B - 1 type duck hepatitis A virus VP2 recombinant protein of one kind, ELISA kit and preparation method thereof - Google Patents
1 type duck hepatitis A virus VP2 recombinant protein of one kind, ELISA kit and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to 1 type duck hepatitis A virus VP2 recombinant protein of one kind, ELISA kit and preparation method thereof, the amino acid sequence such as SEQ ID NO of the 1 type duck hepatitis A virus VP2 recombinant protein:Shown in 1.The preparation method, includes the following steps：Obtain VP2 target fragment；Construct recombinant expression plasmid pProEx-HTb-VP2；Prepare VP2 recombinant protein.It successfully obtains VP2 recombinant protein to be expressed in non-solubility inclusion body, and there is good reactionogenicity with rabbit-anti DHAV-1 serum, illustrate that prokaryotic expression has successfully been obtained in the VP2 albumen of DHAV-1.The ELISA kit of 1 type duck hepatitis A virus antibody of detection is established with the VP2 recombinant protein of expression, provides test data and basic material for the detection and the further correlative study for carrying out DHAV-1 of DHAV-1 antibody.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of 1 type duck hepatitis A virus VP2 recombinant protein, ELISA
Kit and preparation method thereof.
Background technique
Duck hepatitis A virus (Duck Hepatitis A Virus, DHAV) can cause duckling to break out duck virus hepatitis,
Have the characteristics that morbidity is anxious, the course of disease is short, propagation is rapid, case fatality rate is high, be clinically mainly shown as appetite stimulator, nervous symptoms,
Die by visitation of God and liver enlargement bleeding, also known as wryneck disease.The each duck culturing countries and regions in the world are almost spread at present, give duck culturing industry
Bring biggish economic loss.DHAV can be divided into tri- genotype of DHAV-1, DHAV-2 and DHAV-3.DHAV-1 is to cause duck
A kind of most important cause of disease of virus hepatitis is distributed the most extensive and pathogenic strong, infection rate height, may be up to duckling lethality
90% or more.The each duck culturing countries and regions in the world are almost spread at present, bring biggish economic loss to duck culturing industry.
According to the epidemic of disease, clinical symptom characteristic and the lesion of analysing for having diagnostic significance, can generally do
Tentative diagnosis out, but make a definite diagnosis that it is still necessary to by laboratory testing.The laboratory method for detecting DHAV is numerous, respectively there is advantage and disadvantage, in
It is most classic method with test, although result is accurate, when operating cost, is not suitable for clinical detection.With DHAV genome
The announcement successively of complete sequence, a series of new skill of molecular biology such as isothermal amplification technology (LAMP) that RT-PCR technology, ring mediate
Art arises, and provides quick, efficient method for the detection of the virus.And it is used for the ELISA method of the disease antibody test
It is easy to operate, it is easily mastered, does not need the instrument and equipment of high-end precision, it is easy to spread.It is examined at present for duck hepatitis A virus antibody
The ELISA method of survey is indirect ELISA, but existing indirect ELISA method still remains that time-consuming, spends big, purifying protein
It measures small, is unable to large-scale production.
Picornavirus is the smallest one kind in RNA virus, and distribution is extremely wide, can be classified as enterovirus genus
(enterovirus), aphthovirus genus (Aphthovirus), cardiovirus (Cardiovirus), Liposcelis entomophila
(Hepatovirus), secondary intestines orphan Tobamovirus (Parechovirus), equine rhinoviruses category (Erbovirus), ridge Tobamovirus
(Kobuvirus), prompt Shen Tobamovirus (Teschovirus), fowl hepatitis virus category (Avihepatovirus) etc..Picornavirus
It is rounded, virion diameter be 20~40nm, no cyst membrane, viral structural proteins (capsid protein) generally comprise VP1, VP2,
VP3 and VP4.Wherein VP4 is embedded in inside particles, and the inside in protein coat is connected with geneome RNA.VP1,VP2,VP3
It is exposed to virus surface, forms a subunit.VP2 albumen is located at capsid surface, contains epitope and some antigen tables
The inducible body in position generates neutralizing antibody, and there are phase interactions between capsid protein VP2 and pro apoptotic protein/proapoptotic protein Siva
With Coxsackie virus B3 VP2 albumen may be which can specifically bind on proapoptotic protein Siva, to influence Apoptosis
Pathology process caused by induction, the diffusion and virus of virus, but it is relatively seldom to the research of DHAV capsid protein.Biology letter
Breath credit analysis and supposition show that the capsid protein of DHAV may be VP1, VP3 and VP0, rather than as other picornavirus
For VP1, VP2, VP3 and VP4, therefore DHAV is VP0 or VP2 and VP4 on earth, they whether be located at be exposed to virus surface and
How antigenicity has not been reported at present.
Can it can be seen that further investigate to VP2 albumen, the VP2 albumen based on duck hepatitis A virus be established a kind of heavy
The method of histone so that the purity of protein that purifying obtains is high, and establishes indirect ELISA method, makes its sensibility, repeatability
And specificity is good, easy to operate, be produced on a large scale the technical problem urgently to be resolved as those skilled in the art.
Summary of the invention
In order to solve the above-mentioned technical problem the present invention, provides a kind of 1 type duck hepatitis A virus VP2 recombinant protein, ELISA reagent
Box and preparation method thereof.
Technical solution of the present invention includes the following contents：
A kind of 1 type duck hepatitis A virus VP2 recombinant protein, the amino acid sequence of the 1 type duck hepatitis A virus VP2 recombinant protein
Column such as SEQ ID NO:Shown in 1.
A kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein, includes the following steps：
Step 1：Obtain VP2 target fragment：Determine that 1 type duck hepatitis A virus VP2 truncation gene restriction enzyme site and specificity draw
Object, the upstream primer are 5'-GAATTCACTCCTGTTCTTATGAAGTAGGAGC-3', sequence such as SEQ ID NO:Shown in 2,
Downstream primer is 5'-CTCGAGCCTGATTGTCAAATGGTC-3', sequence such as SEQ ID NO:Shown in 3, it is proliferated 1 type duck hepatitis A
Strain extracts the RNA of 1 type duck hepatitis A virus strain, carries out RT-PCR amplification using specific primer, obtains VP2 target fragment；
Step 2：Construct recombinant expression plasmid pProEx-HTb-VP2：The VP2 target fragment is cloned into pMD19-T
In Simple carrier, ligase is added, obtains connection product I, the connection product I is converted and is cultivated into competent cell,
Positive colony body is filtered out, recombinant plasmid pMD19-T Simple-VP2 is obtained after identified, by the recombinant plasmid pMD19-T
The bacterial strain of Simple-VP2 and expression plasmid pProEx-HTb extract Plasmid DNA after expanding culture, use EcoR I and Xho I respectively
The VP2 target fragment that recycling obtains is connected with carrier segments pProEx-HTb, obtains connection product by double digestion, purification and recovery
The connection product II is converted and is cultivated into competent cell, filters out positive colony, recombinantly expressed after identified by II
Plasmid pProEx-HTb-VP2；
Step 3：Prepare VP2 recombinant protein：Recombinant expression plasmid pProEx-HTb-VP2 is converted to e. coli bl21
In, carry out recombinant protein inducing expression, after purifying, identification, obtain 1 type duck hepatitis A virus VP2 recombinant protein.
In the step 2, the ratio between amount of substance of VP2 target fragment and the pMD19-T Simple carrier is 6:1,
Take connection product I described in 5 μ L that 50 μ L competent cell E.coli DH5 α are added, piping and druming mixes, 42 DEG C of thermal shocks after ice bath 30min
60~90s, ice bath 1min after LB liquid medium 450 μ L, 37 DEG C of shaking table 1.5h is added, are trained through Amp resistance LB solid and liquid
Support base screening positive clone.
In the step 2, the amount of the substance for recycling obtained VP2 target fragment and carrier segments pProEx-HTb
The ratio between be 6:1, take connection product II described in 5 μ L that 50 μ L competent cell E.coli DH5 α are added, piping and druming mixes, ice bath 30min
42 DEG C of 60~90s of thermal shock afterwards, ice bath 1min, it is solid through Amp resistance LB after LB liquid medium 450 μ L, 37 DEG C of shaking table 1.5h is added
Body and fluid nutrient medium screening positive clone.
In the step 2, the recombinant plasmid pMD19-T Simple-VP2 and recombinant expression plasmid pProEx-HTb-
The identification method of VP2 includes bacterium solution PCR identification, double digestion identification and sequencing identification, and it is correct to choose the bacterium solution PCR identification
Positive bacterium solution extracts Plasmid DNA, carries out double digestion identification with EcoR I and Xho I to the Plasmid DNA of extraction, chooses through bacterium
Liquid PCR and the correct bacterial strain of double digestion identification carry out the sequencing identification.
Preferably, digestion condition is added 10 × Loading buffer after being 37 DEG C of water-bath 2h and terminates reaction, 1% agarose
Gel electrophoresis.
Preferably, the recombinant protein expression induction step includes：IPTG concentration be 0.2~1.0mM/L induce 8~10h,
Temperature is 37 DEG C.
Preferably, purification process is to cut glue purification method in the step 3.
In above-mentioned preparation 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.
It is a kind of for detecting the ELISA kit of 1 type duck hepatitis A virus antibody, including elisa plate, PBST buffer, envelope
Liquid, ELIAS secondary antibody, developing solution and terminate liquid are closed, the ELISA kit further includes 1 type duck hepatitis A described in claim 1
Virus VP 2 recombinant protein.
Preferably, the confining liquid is the PBS buffer solution containing 5% skimmed milk power；The ELIAS secondary antibody is HRP label
Rabbit or goat-anti duck IgG dilution.
A method of above-mentioned ELISA kit is prepared, is included the following steps：
Step 1：Coating：1 type duck hepatitis A virus VP2 recombinant protein primordial covering enzyme mark elisa plate, 100 holes μ L/, 4 DEG C of mistakes
Night, next day PBST buffer board-washing 3~5 times, each 3min are patted dry；
Step 2：Closing：It is added containing 5% skimmed milk power, 150 hole μ L/ in 37 DEG C of closing 1h, is washed according to the method for step 1
Plate pats dry；
Step 3：Serum to be checked is incubated for：Serum to be checked is added, is patted dry in 37 DEG C of 2h according to step 1 board-washing；
Step 4：ELIAS secondary antibody is incubated for：The rabbit of the HRP label of addition working concentration or goat-anti duck IgG dilution, 37 DEG C
2h is patted dry according to step 1 board-washing.
Step 5：Colour developing：100 hole μ L/ of TMB developing solution is added, is protected from light colour developing 10min；
Step 6：It terminates：Terminate liquid 2mol/L H is added2SO4, 50 holes μ L/；
Step 7：Reading：OD is read with double wave long form with microplate reader450nm-OD630nmValue.
Beneficial effects of the present invention include：The present invention provides a kind of 1 type duck hepatitis A virus VP2 recombinant protein, ELISA reagent
Box and preparation method thereof successfully constructs pProEx-HTb-VP2 recombined pronucleus expression plasmid, successfully obtains VP2 recombinant protein table
There is good reactionogenicity up in non-solubility inclusion body, and with rabbit-anti DHAV-1 serum, illustrate the VP2 egg of DHAV-1
It is white that prokaryotic expression has successfully been obtained.Prokaryotic expression is carried out to the VP2 of DHAV-1 and obtains recombinant protein, and with the recombinant protein of expression
The indirect ELISA detection method for establishing detection DHAV-1 antibody establishes the ELISA examination of 1 type duck hepatitis A virus antibody of detection
Agent box provides test data and basic material for the detection and the further correlative study for carrying out DHAV-1 of DHAV-1 antibody.
Detailed description of the invention
Fig. 1 show VP2 gene RT-PCR amplification figure of the present invention.
Fig. 2 show positive colony bacterium solution PCR mirror of the present invention containing recombinant plasmid pMD19-T simple-VP2 thallus
Fixed figure.
Fig. 3 show positive colony EcoR I described in Fig. 2 and Xho I double digestion qualification result figure.
Fig. 4 show probable positive clone bacterium solution of the present invention containing recombinant expression plasmid pProEx-HTb-VP2 thallus
PCR qualification figure.
Fig. 5 show positive colony EcoR I described in Fig. 4 and Xho I double digestion qualification result figure.
Fig. 6 show VP2 recombinant protein Western blot qualification figure of the present invention.
Fig. 7 show VP2 recombinant protein purification qualification figure of the present invention.
Fig. 8 show expression-form figure of the VP2 recombinant protein of the present invention in host strain.
Fig. 9 show the optimization qualification figure of 2 difference IPTG concentration of embodiment induction VP2 recombinant protein expression.
Figure 10 show the optimization qualification figure of 2 different time of embodiment induction VP2 recombinant protein expression.
Figure 11 show the optimization qualification figure of different temperatures induction VP2 recombinant protein expression in embodiment 2.
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.
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。
Embodiment 1
A kind of 1 type duck hepatitis A virus VP2 recombinant protein, the amino acid sequence of the 1 type duck hepatitis A virus VP2 recombinant protein
Column such as SEQ ID NO:Shown in 1.
A kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein, includes the following steps：
Step 1：Obtain VP2 target fragment：Determine that 1 type duck hepatitis A virus VP2 truncation gene restriction enzyme site and specificity draw
Object, the upstream primer are 5'-GAATTCACTCCTGTTCTTATGAAGTAGGAGC-3'(SEQ ID NO:2), downstream primer
For 5'-CTCGAGCCTGATTGTCAAATGGTC-3'(SEQ ID NO:3) the DHAV-1 Strain stoste that, laboratory is saved
With 5 times of PBS dilutions of sterilizing, after the dual anti-rear 37 DEG C of incubation 1h for adding 1/100 volume, 8000r/min is centrifuged 5min, takes supernatant
It is inoculated with 9~11 ages in days health, the duck embryos without DHAV-1 maternal antibody, dead embryo in for 24 hours is discarded, collects 24~72h death embryo
Allantoic fluid and idiosome extract viral RNA according to Trizol kit specification.According to the PrimeScript of TaKaRaTM RT-
PCR kit carries out viral RNA template and Priming, carries out RT-PCR amplification immediately, obtains VP2 target fragment；Expand body
System is shown in Table 1, the RT-PCR program first step (RT)：(30 DEG C of 10min, 42 DEG C of 15min, 95 DEG C of 5min, 4 DEG C of 5min) × 1cycle；
Second step (PCR)：94 DEG C of 5min, (95 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 40sec) × 30cycle, 72 DEG C of 10min.
1 VP2 gene RT-PCR amplification system of table
Step 2：Construct recombinant expression plasmid pProEx-HTb-VP2：
1, preparation and reorganization plasmid pMD19-T Simple-VP2：
The VP2 target fragment of glue recycling and 1 μ L pMD19-T simple carrier, 5 μ L ligase solution I are mixed
It is even, make the amount of VP2 target fragment substance：Amount=6 of pMD19-T simple carrier mass:1, add ultrapure water to complement to 10 μ L,
16 DEG C of connections are overnight.Connection product I is obtained, connection product I is proceeded as follows：Take 5 μ L connection products that 50 μ L competence are added
Cell E.coli DH5 α, piping and druming mix, and LB liquid medium is added in 42 DEG C of 60~90s of thermal shock, ice bath 1min after ice bath 30min
After 450 μ L, 37 DEG C of shaking table 1.5h, through Amp resistance LB solid and fluid nutrient medium screening positive clone.
Positive colony body identification to filtering out, includes the following steps：
(1), bacterium solution PCR is identified：The positive colony that above-mentioned Amp resistance LB solid and fluid nutrient medium are screened, carries out bacterium
Liquid PCR identification, shown in PCR system and program reference table (PRT) 1 and corresponding program；
(2), double digestion is identified：It chooses above-mentioned PCR and identifies that positive bacterium solution extracts the explanation of Plasmid DNA according to Axygen in a small amount
Book extracts plasmid, carries out double digestion identification with EcoR I and Xho I to the Plasmid DNA of extraction, reaction system is shown in Table 2.
2 EcoR I and Xho I double enzyme digestion reaction system of table
| Reagent | EcoR I and Xho I double digestion |
| 10xH buffer | 2μL |
| pMD19-T simple-VP2 | 12μL |
| EcoR I(15U/μL) | 1μL |
| Xho I(12U/μL) | 1μL |
| ddH2O | 4μL |
| Total volume | 20μL |
Digestion condition：10 × Loading buffer is added after 37 DEG C of water-bath 2h and terminates reaction, 1% Ago-Gel electricity
Swimming, is observed through gel imaging imaging；
(3), sequencing identification：It chooses and carries out the sequencing through bacterium solution PCR and the correct bacterial strain of double digestion identification and identify.It will
Correct plasmid pMD19-T simple-VP2 bacterium solution is sequenced by 1:50 LB liquid mediums of the inoculation containing Amp, 37 DEG C of shaking table vibrations
It swings and 2~2.5h is pre-chilled in 4 DEG C of refrigerators after overnight incubation.It being carried out under sterile working, 4000r/min is centrifuged 5min, supernatant is abandoned,
Every pipe thallus is pressed and is centrifuged preceding bacterium solution volume 1:Isometric 30% glycerine physiological saline and LB/Amp liquid is added in 20 ratio
Culture medium is blown and beaten uniformly with the pipette tips of sterilizing, is sub-packed in strain tube, and every pipe packing bacterium solution amount is no more than the 1/2 of strain tube, envelope
Chewing-gum is sealed and is marked, and -70 DEG C save backup.
Qualification result：As shown in Figure 1,1：VP2 gene RT-PCR amplified fragments, M:2000 DNA marker, with extraction
DHAV-I viral nucleic acid is template, and RT-PCR is amplified and expected VP2 (target sequence 573bp, containing PCR product after restriction enzyme site
Total 585bp) segment that is consistent of gene size, after the RT-PCR VP2 gene expanded is connected to pMD19-T simple carrier, sieve
The positive colony of choosing carries out bacterium solution PCR identification as shown in Fig. 2, in Fig. 2, and 1~2:VP2 gene bacterium solution PCR identification, M:2000 DNA
marker；Positive colony with EcoR I and Xho I double digestion qualification result as shown in figure 3, in Fig. 3,1~2:pMD19-T
EcoRI and XhoI double digestion, the M of simple-VP2:15000 DNA marker.By Fig. 1~3 and sequencing result and known array
Unanimously, show successfully to construct pMD19-T simple-VP2 cloning vector.
Preparation DH5 α competent cell is proceeded as follows with reference to related document：
(1) DH5 α strain is taken to be inoculated on LB solid medium, one single colonie of picking connects after 37 DEG C of inversion overnight incubations
Kind into the LB liquid medium of 5mL, stay overnight by 37 DEG C of shaken cultivations.
(2) 1 is pressed:100 ratio is seeded in the LB liquid medium for being preheated to 37 DEG C, violent 2~3h of shaken cultivation, until
OD600nmAbout 0.4~0.5.
(3) 4 DEG C of refrigerators of bacterium solution are taken out, 2~2.5h is pre-chilled.
(4) inoculum is dispensed into the 50mL centrifuge tube of pre-cooling, 4 DEG C are centrifuged 10min with 4000r/min.
(5) supernatant is abandoned, centrifuge tube residual liquid is got rid of to the greatest extent, is added in 1/10 ratio of bacterium solution volume before being centrifuged into centrifuge tube
Enter the 0.1mol/LCaCl of pre-cooling2Suspend precipitating, and ice bath 30min, 4000r/min are centrifuged 10min.
(6) abandon supernatant, by centrifuge tube inversion 1min flow to end residual liquid, in be centrifuged before bacterium solution volume 1/10 ratio to
The 0.1mol/L CaCl containing 15% glycerol of pre-cooling is added in centrifuge tube2Sufficiently piping and druming, which suspends, precipitates.
(7) suspension is dispensed into EP pipe, -70 DEG C save backup.
2, preparation and reorganization expression plasmid pProEx-HTb-VP2：
(1) recycling of target gene and expression vector：The simple-VP2 of pMD19-T containing recombinant plasmid and expression plasmid
The bacterial strain of pProEx-HTb is stayed overnight after distinguishing streak inoculation in the LB solid medium containing Amp through 37 DEG C of shaking tables of fluid nutrient medium
Expand culture, gained bacterium solution uses Axygen kit to extract Plasmid DNA, then uses EcoR I and Xho I double digestion respectively；It is double
Digestion system and condition referring to above-mentioned table 2 and corresponding digestion condition, digestion products through agarose gel electrophoresis, by target gene and
The size of expression vector cuts the segment on gel, is purified back according to Axygen DNA purification kit step
It brings drill to an end work.
(2) connection of target gene and expression vector：The target gene fragment VP2 and carrier segments that recycling is obtained
PProEx-HTb is placed in linked system by agarose gel electrophoresis estimated concentration：The amount and carrier of VP2 target fragment substance
The ratio between amount of pProEx-HTb substance is 6:1, linked system is added, is attached, reaction system is as shown in table 3, is connected
Product II.
3 coupled reaction system of table
It is placed in PCR reaction tube, pipettor gently blows and beats mixing, and low speed brief centrifugation is stayed overnight in 16 DEG C of constant temperature connections.
The connection product II is converted into competent cell, under aseptic condition, connection product II is grasped as follows
Make：Take 7.5 μ L connection product II that 50 μ L competent cell E.coli DH5 α are added, piping and druming mixes, 42 DEG C of heat after ice bath 30min
60~90s, ice bath 1min are hit, after LB liquid medium 450 μ L, 37 DEG C of shaking table 1.5h is added, through Amp resistance LB solid and liquid
Screening of Media positive colony.
(3) screening and identification of recombinant expression plasmid：
The single colonie grown on random picking conversion plate is inoculated into the LB liquid medium containing Amp, 37 DEG C of shaking table vibrations
After swinging culture 16h, bacterium solution PCR and double digestion screening and identification select two methods and identify that correct bacterial strain is sequenced, will survey
The correct plasmid of sequence is named as pProEx-HTb-VP2, specific identification method and above-mentioned recombinant plasmid pMD19-T simple-VP2
Identification method is identical.
Qualification result：By the pMD19-T simple-VP2 after EcoRI and XhoI double digestion, pProEx-HTb two kind digestion
Product carries out electrophoresis respectively, is attached after glue recycling, and conversion to DH5 α obtains the thallus containing recombinant expression plasmid, the positive gram
Grand progress bacterium solution PCR identification, as shown in figure 4, M in figure:2000 DNA marker, 1~4:Different suspicious bacterium colony pProEx-
HTb-VP2 bacterium solution PCR, as a result 1 is feminine gender, and 2~4 be the positive；Positive colony EcoRI and XhoI double digestion result as shown in figure 5,
M in figure:15000 DNA marker, 1~2：EcoRI the and XhoI double digestion of pProEx-HTb-VP2 is identified.Through being sequenced, purpose
Gene VP2 sequence is correct.Therefore, recombined pronucleus expression plasmid pProEx-HTb-VP2 is constructed successfully.
Step 3：Prepare VP2 recombinant protein：
1, recombinant plasmid pProEx-HTb-VP2 is converted into expression bacterium BL21
The recombinant plasmid pProEx-HTb-VP2 of extraction is converted into competence host strain BL21, to being transformed into BL21
PProEx-HTb-VP2 carry out bacterium solution PCR identification.
2, inducing expression of the recombinant protein VP2 in host strain
Recombinant expression plasmid pProEx-HTb-VP2 is converted into expression bacterium BL21 and after identifying correctly, carries out recombinant protein
The condition of inducing expression, VP2 recombinant protein inducing expression is：IPTG concentration is 0.2~1.0mM/L induction, 8~10h, temperature is
37℃。
3, the Western blot identification of recombinant protein VP2：
Identify that recombinant protein, primary antibody are rabbit-anti DHAV-1 serum with Western blot method, secondary antibody is to be marked with HRP
Goat anti-rabbit igg, concrete operation step are as follows：The sample of the VP2 containing recombinant protein after inducing expression is subjected to SDS-PAGE electrophoresis, point
It is 12% from gum concentration, removes glue after electrophoresis, in transferring film buffer.Conventional method transfer.The good pvdf membrane of trace is taken out, by it
Infiltration 10sec in 100% methanol, is washed with water film three times, and film, which is placed on drying, film on filter paper, becomes translucent from transparent.One
Anti- incubation：1 is pressed with the confining liquid containing 0.05%Tween-20:The rabbit-anti DHAV-1 serum of 100 dilution proportions, 37 DEG C of incubation 1h,
Primary antibody is abandoned, washes film three times with PBS, about 10min/ times.Secondary antibody is incubated for：With the confining liquid 1 containing 0.05%Tween-20:3000 dilutions
The goat anti-rabbit igg of HRP label is incubated for 30min in 37 DEG C of incubators, abandons secondary antibody, wash film three times with PBS, and about 10min/ times.DAB
Colour developing：DAB developing solution is added and is protected from light about 5min, should not develop the color too long, otherwise background colour blackening.
4, the processing of recombinant protein VP2 great expression bacterium sample：
By the pProEx-HTb-VP2/BL2 bacterium solution being incubated overnight by 1：50 are added to the fluid nutrient medium of LB/Amp containing 1L
In conical flask, 37 DEG C of water-bath shaken cultivation about 2h~2.5h, until bacterial growth reaches logarithmic growth phase, i.e. OD600nmValue 0.6 is left
It is right.Best IPTG concentration, Best Times and optimum temperature obtained in optimization according to inducing expression condition continue after cultivating in 4
8000r/min is centrifuged 10min in DEG C centrifuge, collects thallus.Thallus is pressed with 20mM Tris-Cl (pH8.0) with original bacteria liquid volume
1:5 ratios are added, suspension thalline, and thallus is intermittently crushed in Ultrasonic Cell Disruptor, and 30 seconds ultrasounds of every septum secundum are primary, until thallus becomes clear
It is bright, after 4 DEG C of broken bacterium solution, 12000r/min be centrifuged 10min, recombinant protein VP2 is due to being in pProEx-HTb-VP2/BL2
The expression of inclusion body non-solubility, therefore supernatant will be abandoned after cracking bacterium solution centrifugation, precipitating is pressed and original bacteria liquid volume 1:50 ratios add
Enter 8M urea, piping and druming promotes its dissolution to take finally, 8000r/min is centrifuged 10min in 4 DEG C of centrifuges again repeatedly for the dissolution that suspends
Supernatant abandons non-solubility impurity in precipitating removal sample；The recombinant protein VP2 sample of above-mentioned acquisition is pressed 4:1 ratio addition contains
5 × SDS loading buffer of beta -mercaptoethanol, boils 10min, and 8000r/min is centrifuged 5min, 4 DEG C of preservations, for cutting glue purification
Albumen.
5, recombinant protein VP2 cuts glue purification：
With the large size SDS-PAGE electrophoresis tank of Bio-Rad, by specification installs electrophoresis tank and glass plate；To glass
Configured separation gel and concentration glue are added in plate, does not have to insert comb, remaining space is used to add protein sample after solidification.First use
70mA current electrophoresis about 2.5h uses 100mA current electrophoresis about 4.5h instead until sample enters separation gel；KCl dyeing：It cuts
It is soaked in after bromophenol blue on removing photoresist in the KCl of the 0.125mol/L of pre-cooling, stands 10min colour developing (principle:It is stranded in gel
Protein combine a large amount of SDS, SDS combines the KDS to be formed and be easier to precipitating compared with SDS with potassium ion, when low temperature largely not
Molten KDS is precipitated, and forms pale precipitation), purpose band is cut according to the size position of albumen；The processing of bag filter：Containing
2%NaHCO3Bag filter 10min is boiled in 1mM EDTA mixed liquor, uses ddH2O is thoroughly cleaned, then is boiled in 1mMEDTA
10min uses ddH2O cleaning is immersed in 4 DEG C of preservations in 20% ethyl alcohol；Gel will be cut to be fitted into processed bag filter, added
It is fastened after PBS with rope, is put into 100V electrophoresis 2h in Horizontal electrophoresis tank, destination protein VP2 is made to swim out from gel into PBS,
Counterelectrophoresis 10min again collects the VP2 for dissolving in PBS；The destination protein VP2 50mL super filter tube for dissolving in PBS is concentrated, 4 DEG C,
8000r/min be centrifuged 10min, sampling carry out SDS-PAGE electroresis appraisal, remaining -70 DEG C freeze it is spare.
Qualification result：Western blot identifies the result of VP2 recombinant protein as indicated with 6, in Fig. 6, M：Pre-dyed albumen
Marker,1：The sample of the recombinant protein containing VP2, VP2 recombinant protein can be identified by DHAV-1 serum, show that VP2 recombinant protein has
There is good reactionogenicity.As shown in fig. 7, cutting glue purification mode obtains purity and concentration all higher destination protein VP2, Fig. 7
In, M:Albumen Marker, 1：VP2 recombinant protein after cutting glue purification, 2：Unpurified recombinant protein VP2 is induced to forgive through IPTG
Body sample.
The expression-form of 2 VP2 recombinant protein of embodiment is analyzed and inducing expression condition optimizing
1, the analysis of expression-form：
(1) taking identification, correctly expression bacterium streak inoculation is chosen single colonie and is trained through LB liquid in the LB solid medium containing Amp
It supports 37 DEG C of rejuvenation of base to stay overnight, takes 2mL bacterium solution to be inoculated with 100ml LB/Amp, 37 DEG C of water-baths vibrate 2.5~3h, until OD600nmAbout
0.6。
(2) be added IPTG to final concentration be 0.4mmol/L, 37 DEG C of water-bath inducing expression 4h.
(3) bacterium solution is centrifuged 10min in 4 DEG C, 8000r/min, abandons supernatant.
(4) the 20mM Tris-HCl suspension thalline of 10mL pH8.0 is added, under condition of ice bath, ultrasonication 30sec/ times,
Every minor tick 30sec for several times, until bacterium solution is limpid, is centrifuged 10min through 4 DEG C, 12000r/min, it is heavy to collect supernatant respectively
It forms sediment；Precipitating is againWith2mL 8M urea blows and beats dissolution repeatedly, and 4 DEG C, 8000r/min centrifugation 10min, dissolution must be precipitated by collecting supernatant
Object.
(5) it takes supernatant to precipitate each 80 μ L of dissolved matter, 5 × SDS loading buffer of the 20 μ L containing beta -mercaptoethanol, water is added
10min is boiled in bath, and 8000r/min is centrifuged 5min, the observation of SDS-PAGE electrophoresis.
(6) if in supernatant visible purpose band, for soluble protein, if in precipitating dissolved matter, with inclusion body
Form exists.
Recombinant plasmid pProEx-HTb-VP2 and corresponding empty plasmid are transformed into BL21 respectively and expand culture, IPTG is added
It induces, after the supernatant inclusion body sample treatment obtained after ice-bath ultrasonic is broken, SDS-PAGE electrophoresis result is as shown in figure 8, Fig. 8
In, M is albumen Marker, swimming lane 1~3 is followed successively by：PProEx-HTb-VP2 turn the inclusion body after BL21 ultrasonication,
PProEx-HTb-VP2 turns the supernatant pProEx-HTb zero load after BL21 ultrasonication and turns BL21 sample.Induction contains pProEx-
The BL21 expression bacterium supernatant of HTb-VP2 does not occur obvious purpose VP2 expression protein band, and there are VP2 to express egg in inclusion body
Informal voucher band, size are about 24KDa, i.e., expression vector pProEx-HTb-VP2 is mainly forgiven in host strain BL21 with non-solubility
Body form expresses recombinant VP 2 albumen.
2, the optimization of expression condition
(1) optimization of IPTG concentration
The fresh bacterium solution 1mL of expressive host bacterium BL21 of the pProEx-HTb-VP2 containing recombinant plasmid is inoculated in 50mL LB/
In Amp fluid nutrient medium, 37 DEG C of shaking table shaken cultivation about 2.5h to OD600nmValue is 0.6 or so, and IPTG, which is added, makes its final concentration
For：0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L and 1.2mmol/L induce table
Up to 4h, after handling sample, SDS-PAGE electrophoresis, Coomassie brilliant blue dye, decoloration observation.
(2) optimization of induction time
The expressive host bacterium BL21 1mL of the pProEx-HTb-VP2 containing recombinant plasmid is inoculated in the training of 50mL LB/Amp liquid
It supports in base, 37 DEG C of shaking table shaken cultivations to OD600nmAbout 0.6 or so, the optium concentration that IPTG is obtained to above-mentioned test is added, then divide
Sample You Dao not be handled, SDS-PAGE electrophoresis after 2h, 4h, 6h, 8h, 10h and 12h.
(3) optimization of inducing temperature
The expressive host bacterium BL21 1mL of the pProEx-HTb-VP2 containing recombinant plasmid is inoculated in the training of 50mL LB/Amp liquid
It supports in base, 37 DEG C of shaking table shaken cultivations to OD600nmAbout 0.6 or so, addition IPTG obtains best to final concentration of above-mentioned test
IPTG concentration and best induction time, then Fiber differentiation under the conditions of 20 DEG C, 25 DEG C, 30 DEG C and 37 DEG C, at sample
After reason, SDS-PAGE electrophoresis.
As shown in Fig. 9,10 and 11, after different IPTG concentration, different inducing expression times and different temperatures optimization, obtain
PProEx-HTb-VP2 containing recombinant expression plasmid using BL21 as host strain express VP2 recombinant protein optimum condition of the expression be：
IPTG concentration is 0.2mM/L induction 8h, temperature is 37 DEG C of (wherein 8~10h of 0.2~1.0mM/L of IPTG concentration and induction time
When, the variation of destination protein expression quantity is little), in Fig. 9：M be albumen Marker, 1~7 be IPTG induced concentration 0,0.2,0.4,
0.6,0.8,1.0 and 1.2mmol/L；In Figure 10：M is albumen Marker, swimming lane 1~6 is followed successively by：Induction time 2,4,6,8,10
And 12h；In Figure 11：M is albumen Marker, swimming lane 1~4 is followed successively by：37 DEG C, 30 DEG C, 25 DEG C and 20 DEG C of inducing temperature forgive
Body, 5~8 are followed successively by the supernatant of 37 DEG C of inducing temperature, 30 DEG C 25 DEG C and 20 DEG C.
Embodiment 3
It is a kind of for detecting the ELISA kit of 1 type duck hepatitis A virus antibody, including elisa plate, PBST buffer, envelope
Liquid, ELIAS secondary antibody, developing solution and terminate liquid are closed, the ELISA kit further includes 1 type duck hepatitis A described in claim 1
Virus VP 2 recombinant protein.
The confining liquid is the PBS buffer solution containing 5% skimmed milk power；The ELIAS secondary antibody be HRP label rabbit or
Goat-anti duck IgG dilution.
A method of ELISA kit is prepared, is included the following steps：
Step 1：Coating：1 type duck hepatitis A virus VP2 recombinant protein primordial covering enzyme mark elisa plate, 100 holes μ L/, 4 DEG C of mistakes
Night, next day PBST buffer board-washing 3~5 times, each 3min are patted dry；
Step 2：Closing：150 hole μ L/ of PBST buffer containing 5% skimmed milk power is added in 37 DEG C of closing 1h, according to step
Rapid one method board-washing, pats dry；
Step 3：Serum to be checked is incubated for：Serum to be checked is added, is patted dry in 37 DEG C of 2h according to step 1 board-washing；
Step 4：ELIAS secondary antibody is incubated for：The rabbit of the HRP label of addition working concentration or goat-anti duck IgG dilution, 37 DEG C
2h is patted dry according to step 1 board-washing.
Step 5：Colour developing：100 hole μ L/ of TMB developing solution is added, is protected from light colour developing 10min；
Step 6：It terminates：Terminate liquid 2mol/L H is added2SO4, 50 holes μ L/；
Step 7：Reading：OD is read with double wave long form with microplate reader450nm-OD630nmValue.
The foundation and application of ELISA method of the embodiment 4 based on VP2 recombinant protein
1, the determination of the best peridium concentration of antigen and yin and yang attribute serum optimum dilution degree：
It is titrated, the VP2 recombinant protein coating buffer (pH9.6) of purifying is diluted and is added in ELISA Plate using Founder, pressed
Two repetitions of each dilution, are added to the coated ELISA Plate of VP2 recombinant protein after DHAV-1 yin and yang attribute serum is diluted respectively
In, by ELIAS secondary antibody HRP- rabbit-anti duck IgG first with 1:2000 times of dilutions carry out indirect ELISA detection referring to embodiment 3, finally
Best envelope antigen dilution and best serum dilution are determined with the maximum dilution of P/N value.
The VP2 albumen initial concentration that purifying is measured with nucleic acid-protein instrument is 2.170mg/ml, each antigen diluent degree setting 2
A repetition, is averaged.The results are shown in Table 4, selects P/N value the maximum for optimum condition, i.e. the best packet of VP2 recombinant protein
It is 1 by concentration:800 dilution (2.713 μ g/ml), serum 1:40 dilutions.
4 VP2 recombinant protein antigen peridium concentration of table and serum dilution selection
2, the determination of ELIAS secondary antibody best effort concentration
By the VP2 recombinant protein of purifying with optimal antigen diluent degree come coated elisa plate, by DHAV-1 yin and yang attribute serum
It is diluted by optimum dilution degree, ELIAS secondary antibody HRP- rabbit-anti duck IgG is diluted to various concentration, is carried out indirectly referring to embodiment 3
ELISA detection, using the maximum extension rate of P/N value as the optimal working concentration of ELIAS secondary antibody.
As shown in table 5, three repetitions of each concentration, are averaged, and select P/N value the maximum for best secondary antibody dilution,
The best ELIAS secondary antibody dilution of ELISA i.e. based on VP2 albumen is 1:2000.
Table 5 is coated with the optimization of recombinant protein VP2 ELIAS secondary antibody working concentration
| Enzyme labelled antibody dilution | 1:400 | 1:500 | 1:800 | 1:1000 | 1:1600 | 1:2000 | 1:3200 |
| Positive OD value | 1.509 | 1.483 | 1.390 | 1.319 | 1.121 | 1.069 | 0.741 |
| Negative OD value | 0.370 | 0.230 | 0.157 | 0.125 | 0.086 | 0.078 | 0.057 |
| P/N value | 4.077 | 6.435 | 8.841 | 10.595 | 12.965 | 13.720 | 13.098 |
3, most preferably it is coated with condition：
By the best antigen diluent degree coated elisa plate of above-mentioned VP2 recombinant protein, 37 DEG C 4 DEG C of 1h overnight, 37 DEG C of 3h, 4 DEG C of mistakes
These three coating condition setting positive and negative serum controls, each 6 repetitions, the optimal extension rate of ELIAS secondary antibody are dilute overnight with 4 DEG C
Secondary antibody is released, indirect ELISA is carried out referring to the operation of embodiment 3 and detects sequence, when with P/N value maximum, determines best coating condition.
As shown in table 6,6 repetitions of every kind of coating condition, are averaged, and select P/N value the maximum as best coating item
Part, i.e., the ELISA based on VP2 albumen are most preferably coated with condition and stay overnight for 4 DEG C.
6 VP2 recombinant protein of table is coated with condition optimizing
| Coating condition | 37 DEG C 4 DEG C of 1h overnight | 37 DEG C 4 DEG C of 3h overnight | 4 DEG C overnight |
| Positive mean value | 1.355 | 1.342 | 1.342 |
| Negative mean value | 0.229 | 0.250 | 0.196 |
| P/N value | 5.920 | 5.366 | 6.857 |
4, best confining liquid determines：
With the best antigen diluent multiple groped above, it is most preferably coated with condition, VP2 recombinant protein coated elisa plate is used
Eight kinds of confining liquids are closed below：5% fetal calf serum, 10% fetal calf serum, 1% gelatin, 5% gelatin, 1% skimmed milk power, 5%
3 repetitions are done in skimmed milk power, 1%BSA and 5%BSA, each hole, with optimal antibody extension rate dilute serum and enzyme mark two
It is anti-, OD is finally measured in microplate reader450nm-OD630nmValue.With the maximum confining liquid of P/N value for best confining liquid.
As shown in table 7, each 3 repetitions of eight kinds of confining liquids, are averaged, and select P/N value the maximum as best confining liquid,
The best confining liquid of ELISA i.e. based on VP2 recombinant protein is 5% skimmed milk power.
The selection of the best confining liquid of table 7
| Confining liquid | 5% fetal calf serum | 10% fetal calf serum | 1% skimmed milk power | 5% skimmed milk power |
| Positive mean value | 1.412 | 1.372 | 1.313 | 1.266 |
| Negative mean value | 0.079 | 0.066 | 0.061 | 0.050 |
| P/N value | 17.863 | 20.895 | 21.570 | 25.420 |
| 1%BSA | 5%BSA | 1% gelatin | 5% gelatin | |
| Positive mean value | 1.133 | 1.084 | 0.756 | 0.004 |
| Negative mean value | 0.104 | 0.078 | 0.128 | 0.093 |
| P/N value | 10.918 | 13.899 | 5.884 | 0.040 |
5, best sealing condition determines：
According to the best confining liquid selected, tetra- off-period gradients of 30min, 60min, 90min and 120min are set, often
A gradient sets 3 repetitions, carries out best off-period and gropes.
As shown in table 8,3 repetitions of each off-period, are averaged, and select P/N value the maximum as best closing
Time, i.e., the best off-period of the ELISA based on VP2 recombinant protein are 37 DEG C of 1h.
The coating recombinant protein VP2 off-period of table 8 selects
| Off-period | 0.5h | 1h | 1.5h | 2h |
| Positive value | 1.299 | 1.101 | 1.037 | 1.199 |
| Feminine gender value | 0.187 | 0.106 | 0.109 | 0.117 |
| P/N value | 6.961 | 10.424 | 9.539 | 10.287 |
6, serum and ELIAS secondary antibody incubation time determine：
It is operated by the coating condition groped above, sealing condition, ELIAS secondary antibody optimum dilution degree, setting 30min,
Tetra- incubation time gradients of 60min, 90min and 120min, each gradient set 3 repetitions, carry out best positive and negative serum and enzyme
Mark secondary antibody incubation time is groped.
As shown in Table 9 and Table 10,3 repetitions of each time point, are averaged, and select P/N value the maximum, that is, are based on VP2
The best serum incubation time and best secondary antibody incubation time of the ELISA of recombinant protein is 2h.
Table 9 is coated with the optimization of recombinant protein VP2 serum incubation time
| Serum incubation time | 0.5h | 1h | 1.5h | 2h |
| Positive value | 1.408 | 1.385 | 1.398 | 1.375 |
| Feminine gender value | 0.315 | 0.256 | 0.271 | 0.187 |
| P/N value | 4.473 | 5.406 | 5.167 | 7.369 |
Table 10 is coated with the optimization of recombinant protein VP2 ELIAS secondary antibody incubation time
7, developing time determines：
If six time gradients of 5min, 10min, 15min, 20min, 25min and 30min, it is protected from light in 37 DEG C and bottom is added
Object developing solution, each developing time positive and negative serum are respectively arranged 6 repetitions, are averaged, finally measure OD450nm-OD630nm
Value, with positive value >=1.0 and P/N value maximum substrate-function time for best developing time.
As shown in table 11,3 repetitions of each time point, are averaged, and select P/N value the maximum, i.e., recombinate egg based on VP2
The best developing time of white ELISA is 10min.
Table 11 is coated with the best developing time selection of recombinant protein VP2
| Developing time (min) | 5min | 10min | 15min | 20min | 25min | 30min |
| Positive mean value | 0.876 | 1.089 | 1.226 | 1.253 | 1.276 | 1.266 |
| Negative mean value | 0.119 | 0.142 | 0.173 | 0.176 | 0.185 | 0.193 |
| P/N value | 7.347 | 7.670 | 7.080 | 7.134 | 6.903 | 6.568 |
8, the determination of yin and yang attribute critical value：
60 parts of negative serum OD are detected with the optimum condition of the VP2 recombinant protein of above-mentioned optimization respectively450nm-OD630nmValue,
Cut-off value=mean value+3 × variance is calculated, as positive threshold value.
As shown in table 12,60 parts of negative serum OD are measured450nm-OD630nmValue, positive threshold value=average value+3 × SD conduct
Positive threshold value, obtaining VP2 positive threshold value is 0.207+3 × 0.051=0.359.
Table 12 is coated with 60 parts of negative serum OD of recombinant protein VP2450nm-OD630nmValue
9, repetitive test：
The coated ELISA Plate of the VP2 recombinant protein purified respectively with same batch and different batches detects 6 parts of serum
OD450nm-OD630nm6 repetitions are arranged in value, every part of serum, detect in its plate, the coefficient of variation between plate, analyze the side ELISA of foundation
The repeatability of method.
(1) repetitive test in plate
The VP2 recombinant protein coefficient of variation is between 1.45%~3.47%, less than 10%, as shown in table 13, illustrates to establish
ELISA method have preferable plate in repeatability.
Table 13 is coated with coefficient of variation measurement in recombinant protein VP2 plate
| Positive serum | Mean value | SD | The coefficient of variation in plate |
| 1 | 1.398 | 0.048 | 3.47% |
| 2 | 1.375 | 0.037 | 2.68% |
| 3 | 1.329 | 0.035 | 2.66% |
| 4 | 1.382 | 0.030 | 2.14% |
| 5 | 1.387 | 0.022 | 1.60% |
| 6 | 1.445 | 0.021 | 1.45% |
(2) repetitive test between plate
The VP2 coefficient of variation, less than 10%, is shown in Table 14 between 0.90%~1.84%, illustrates the side ELISA established
Method has the repeatability between preferable plate.
The coefficient of variation measures between table 14 is coated with recombinant protein VP2 plate
10, specific test：
(1) specific cross is tested
With the ELISA method detection Salmonella anatis, E. coli isolated from ducks, duck swollen head septicaemia virus, avian flu of foundation
The positive serum of Mo Shi bacillus in malicious (H5), duck plague virus and duck, is arranged DHAV-1 positive serum, negative serum control, such as table
Shown in 15, each serum is arranged 3 repetitions and is averaged, six kinds of other cause of disease positive serums OD450nm-OD630nmValue is respectively less than sun
Property threshold value 0.359 and the ratio of positive serum and negative serum be respectively less than 2.1, show establish ELISA method specificity very well.
15 VP2 recombinant protein ELISA method specific detection of table
(2) specific inhibition is tested
By antigen 10:The volume ratio of 1 yin or positive serum is after 37 DEG C of neutralization 1h, respectively with the ELISA method inspection established
The serum for neutralizing front and back is surveyed, 6 repetitions of every kind of serum calculate blocking rate.As shown in table 16, the positive, negative serum by DHAV-1
It is reacted with VP2 recombinant protein, the serum of front and back will be blocked to detect, 6 repetitions are respectively set, are averaged.Obtain VP2 sun
Property serum blocking rate be 88.9%, negative serum blocking-up rate 8.7%.Show that VP2 recombinant protein can be with DHAV-1 positive serum
Specificity neutralizes.
16 VP2 recombinant protein blocking test of table
| VP2 | Before blocking | After blocking | Blocking rate |
| Positive value | 1.087 | 0.121 | 88.9% |
| Feminine gender value | 0.122 | 0.110 | 8.7% |
| P/N value | 8.910 | 1.100 | / |
11, ELISA method sensitivity Detection：
DHAV-1 positive serum known to taking 8 parts, with 2 times of doubling dilutions, the ELISA method for foundation is detected, can be examined
Measuring positive maximum serum dilution is sensitivity.
As shown in table 17,8 parts of duck hepatitis A virus positive serums are taken, from 1:100 start to carry out 2 doubling dilutions, can detect
Positive maximum extension rate is as sensitivity out.Show that the VP2 recombinant protein ELISA method of foundation is sensitive with the data of table 17
Degree is at least 1:6400.
17 VP2 recombinant protein ELISA method sensitivity technique of table
12, based on VP2 recombinant protein ELISA detection method application and with the ELISA method coincidence rate based on DHAV-1
Compare：
ELISA method based on DHAV-1：It is coated with overnight for 4 DEG C after 37 DEG C of 3h with the DHAV-1 of 8 μ g/mL, is added 1:160
Diluted tested serum is incubated for 1h, with 5% gelatin, 37 DEG C of closing 0.5h, is added 1:The rabbit-anti duck IgG of 2000 diluted HRP labels
It is incubated for 40min, yin-yang critical value is 0.210.
48 parts of serum to be checked, detection are detected with the ELISA detection method based on VP2 albumen of foundation shown in embodiment 3
As a result it is compared with the testing result of the ELISA method based on DHAV-1, calculates coincidence rate.
48 parts of serum to be checked are detected jointly using VP2 recombinant protein, DHAV-1 as the ELISA method that antigen is established, and compare two
The coincidence rate of kind method is as shown in table 18 and 19, the results show that ELISA method of the invention and DHAV-1 are the ELISA of antigen
The positive coincidence rate of method detection is 93.9%, and feminine gender detection coincidence rate is 88.2%, and total coincidence rate is 91.1%.Explanation is based on
The alternative totivirus method of ELISA detection method that VP2 recombinant protein is established detects blood serum sample.
The ELISA method of 18 VP2 recombinant protein of table and DHAV-1 detect 48 parts of serum OD450nm-OD630nmValue
The coincidence rate of indirect ELISA method of the table 19 based on VP2 recombinant protein and DHAV-1
The present invention is selected according to analysis and the VP2 gene order speculated, design primer according to primer and restriction enzyme site
PProEx-HTb expression vector.Secondly, when the selection for the restriction endonuclease sites being inserted into primer it has to be ensured that amplification
The restriction enzyme site is not contained in VP2 gene, while also to orient insertion in carrier, by screening before VP2 nucleotide sequence
Introduce EcoR I (GAATTC) and XhoI (CTCGAG) the two restriction enzyme sites respectively afterwards.The present invention successfully constructs pProEx-
HTb-VP2, sequencing result speculate that VP2 gene order is consistent in GenBank DHAV-1H plants with analysis, missing, insertion do not occur
Deng mutation, the correct expression of VP2 recombinant protein ensure that.The prokaryotic expression of foreign gene mainly with inclusion body and can dissolve two kinds
Form, VP2 albumen are to turn host strain BL21 by pProEx-HTb-VP2 plasmid to be expressed, soluble analysis show VP2 with
Inclusion bodies exist.The albumen of inclusion bodies is the correct non-natural denatured protein of primary structure.Using gel extraction
Method purify destination protein VP2, the higher VP2 of concentration and purity is then concentrated to get by super filter tube.It is subcloned plasmid
PProEx-HTb tag size is 3KDa, and being subcloned insertion VP2 segment in recombinant plasmid is 573bp, and coding polypeptide size is about
The restructuring destination protein VP2 size of 21.2KDa, the prokaryotic expression plasmid pProEx-HTb-VP2 expression of building are about 24KDa.This
The recombinant protein and DHAV-1 serum of invention have preferable reactionogenicity, are based on VP2 albumen indirect ELISA detection side to be subsequent
The foundation of method provides theoretical and data basis and material.Using VP2 recombinant protein as envelope antigen, inspection is successfully established
The indirect ELISA method of duck hepatitis A virus antibody is surveyed, this method repeatability with higher and specificity, are duck first as the result is shown
The diagnosis of duck hepatitis caused by hepatovirus is provided with reference to method.
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 (10)
1. a kind of 1 type duck hepatitis A virus VP2 recombinant protein, which is characterized in that the 1 type duck hepatitis A virus VP2 recombinant protein
Amino acid sequence such as SEQ ID NO:Shown in 1.
2. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein, which is characterized in that include the following steps：
Step 1：Obtain VP2 target fragment：Determine 1 type duck hepatitis A virus VP2 truncation gene restriction enzyme site and specific primer,
The upstream primer is 5'-GAATTCACTCCTGTTCTTATGAAGTAGGAGC-3', downstream primer 5'-CTCGAGCCTGATTGTCAAATGGTC-3' is proliferated 1 type duck hepatitis A virus strain, extracts the RNA of 1 type duck hepatitis A virus strain, utilizes
Specific primer carries out RT-PCR amplification, obtains VP2 target fragment；
Step 2：Construct recombinant expression plasmid pProEx-HTb-VP2：The VP2 target fragment is cloned into pMD19-T
In Simple carrier, ligase is added, obtains connection product I, the connection product I is converted and is cultivated into competent cell,
Positive colony body is filtered out, recombinant plasmid pMD19-T Simple-VP2 is obtained after identified, by the recombinant plasmid pMD19-T
The bacterial strain of Simple-VP2 and expression plasmid pProEx-HTb extract Plasmid DNA after expanding culture, use EcoR I and Xho I respectively
The VP2 target fragment that recycling obtains is connected with carrier segments pProEx-HTb, obtains connection product by double digestion, purification and recovery
The connection product II is converted and is cultivated into competent cell, filters out positive colony, recombinantly expressed after identified by II
Plasmid pProEx-HTb-VP2；
Step 3：Prepare VP2 recombinant protein：Recombinant expression plasmid pProEx-HTb-VP2 is converted into e. coli bl21, into
Row recombinant protein inducing expression, after purifying, identification, obtain 1 type duck hepatitis A virus VP2 recombinant protein.
3. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein according to claim 2, which is characterized in that institute
It states in step 2, the ratio between amount of substance of VP2 target fragment and the pMD19-T Simple carrier is 6:1, it takes and connects described in 5 μ L
50 μ L competent cell E.coli DH5 α are added in the object I that practices midwifery, and piping and druming mixes, 42 DEG C of 60~90s of thermal shock after ice bath 30min, ice bath
1min after LB liquid medium 450 μ L, 37 DEG C of shaking table 1.5h is added, is screened positive through Amp resistance LB solid and fluid nutrient medium
Clone.
4. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein according to claim 2, which is characterized in that institute
It states in step 2, obtained the ratio between the amount of substance of VP2 target fragment and carrier segments pProEx-HTb that recycles is 6:1,
Take connection product II described in 5 μ L that 50 μ L competent cell E.coli DH5 α are added, piping and druming mixes, 42 DEG C of thermal shocks after ice bath 30min
60~90s, ice bath 1min after LB liquid medium 450 μ L, 37 DEG C of shaking table 1.5h is added, are trained through Amp resistance LB solid and liquid
Support base screening positive clone.
5. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein according to claim 2, which is characterized in that institute
It states in step 2, the identification side of the recombinant plasmid pMD19-T Simple-VP2 and recombinant expression plasmid pProEx-HTb-VP2
Method includes bacterium solution PCR identification, double digestion identification and sequencing identification, and choosing the bacterium solution PCR identification, correctly positive bacterium solution mentions
Plasmid DNA is taken, double digestion identification is carried out with EcoR I and Xho I to the Plasmid DNA of extraction, is chosen through bacterium solution PCR and double
The correct bacterial strain of digestion identification carries out the sequencing identification.
6. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein according to claim 2, which is characterized in that institute
Stating recombinant protein inducing expression condition is：IPTG concentration is 0.2~1.0mM/L induction, 8~10h, temperature is 37 DEG C.
7. a kind of preparation method of 1 type duck hepatitis A virus VP2 recombinant protein according to claim 2, which is characterized in that institute
Stating purification process in step 3 is to cut glue purification method.
8. a kind of for detecting the ELISA kit of 1 type duck hepatitis A virus antibody, which is characterized in that including elisa plate, PBST
Buffer, confining liquid, ELIAS secondary antibody, developing solution and terminate liquid, the ELISA kit further include described in claim 11
Type duck hepatitis A virus VP2 recombinant protein.
9. according to claim 8 a kind of for detecting the ELISA kit of 1 type duck hepatitis A virus antibody, feature exists
In the confining liquid is the PBS buffer solution containing 5% skimmed milk power；The ELIAS secondary antibody is the rabbit or goat-anti duck of HRP label
IgG dilution.
10. a kind of method for preparing the described in any item ELISA kits of claim 8~9, which is characterized in that including following
Step：
Step 1：Coating：1 type duck hepatitis A virus VP2 recombinant protein is coated with elisa plate, and 100 holes μ L/, 4 DEG C overnight, next day PBST
Buffer board-washing 3~5 times, each 3min is patted dry；
Step 2：Closing：It is added and is clapped in 37 DEG C of closing 1h according to the method board-washing of step 1 containing 5% skimmed milk power, 150 hole μ L/
It is dry；
Step 3：Serum to be checked is incubated for：Serum to be checked is added, is patted dry in 37 DEG C of 2h according to step 1 board-washing；
Step 4：ELIAS secondary antibody is incubated for：The rabbit or goat-anti duck IgG dilution, 37 DEG C of 2h that the HRP label of working concentration is added are pressed
According to step 1 board-washing, pat dry.
Step 5：Colour developing：100 hole μ L/ of TMB developing solution is added, is protected from light colour developing 10min；
Step 6：It terminates：Terminate liquid 2mol/L H is added2SO4, 50 holes μ L/；
Step 7：Reading：OD is read with double wave long form with microplate reader450nm-OD630nmValue.
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| CN108445208B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | Universal DHAV polypeptide indirect ELISA antibody detection kit and application thereof |
| CN108845127A (en) * | 2018-04-13 | 2018-11-20 | 四川农业大学 | The indirect ELISA detection method of DHAV-3 antibody based on VP2 or VP4 recombinant protein antigen and application |
| CN110283836B (en) * | 2019-06-24 | 2021-07-06 | 四川农业大学 | Type 3 duck hepatitis A virus mutant gene ISA-T1142A and construction method |
| CN113817070B (en) * | 2021-08-12 | 2022-11-22 | 国药集团动物保健股份有限公司 | ELISA detection kit for type 1 duck hepatitis A virus specific fusion protein S1 antigen and type 1 duck hepatitis A virus antibody |
| CN113929754A (en) * | 2021-10-18 | 2022-01-14 | 西南民族大学 | Sheep derived cristae virus VP1 recombinant protein, ELISA detection kit, preparation method and application thereof |
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| WO2011041788A3 (en) * | 2009-10-02 | 2011-06-03 | The Trustees Of Columbia University In The City Of New York | Turkey viral hepatitis virus and uses thereof |
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