CN103837680B - The preparation method of the total antibody ELISA detection kit of one boar HEV - Google Patents
The preparation method of the total antibody ELISA detection kit of one boar HEV Download PDFInfo
- Publication number
- CN103837680B CN103837680B CN201410084515.4A CN201410084515A CN103837680B CN 103837680 B CN103837680 B CN 103837680B CN 201410084515 A CN201410084515 A CN 201410084515A CN 103837680 B CN103837680 B CN 103837680B
- Authority
- CN
- China
- Prior art keywords
- antigen
- preparation
- solution
- hev
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the preparation method of the total antibody ELISA detection kit of a boar HEV, the preparation method of the total antibody ELISA detection kit of this pig HEV comprises the following steps: preparation HEV envelope antigen; Preparation HEV labelled antigen; The preparation of HEV labelled antigen-HRP label; Adopt carbonate buffer solution with certain proportion by S antigen diluent, 100 μ l/ holes join ELISA Plate, are packaged in and are added with in the aluminum foil bag of drying agent, wrap complete.The present invention establishes one and rapidly and efficiently detects the total antibody ELISA detection method of pig HEV, the present invention is according to dual-antigen sandwich method principle, envelope antigen and Serum Antibody produce specific binding, conjugated antigen antibody complex and labelled antigen produce antigen-antibody reaction again, two antigens are in conjunction with different antigen binding sites, highly sensitive, specificity is good, reproducible, for the large-scale inquiry of hepatitis E virus epidemiology provides relevant art guarantee, also provide theoretical foundation for better the popular of prevention and corntrol pig HEV epidemic disease.
Description
Technical field
The invention belongs to the detection technique field of hepatitis E virus, particularly relate to the preparation method of the total antibody ELISA detection kit of a boar HEV.
Background technology
Hepatitis E (Hepatitis E) is a kind of infectious diseases common to human beings and animals through transmission caused by hepatitis E virus (Hepatitis E virus, HEV).This disease easily develops into fulminant hepatitis in teenager, and pregnant woman can be caused to miscarry, and case fatality rate is up to 21%.HEV is widely distributed, and harm is serious.
Countries in the world have the report that pig source HEV is relevant in succession, found that both have high homology with mankind HEV sequence alignment, find again that the RNA virus extracted can infect chimpanzee and rhesus macaque subsequently.Subsequently, experimental result in succession shows that pig hepatitis E is a kind of zoonosis, pig is as the Major Natural host of hepatitis E virus, HEV average rate reaches as high as 83.4%, many swinerys of carrying Hepatitis E virus do not show morbidity state (Meng X J et al., 1997).Pig is the important meat sources of the mankind, simultaneously as the main donor of human organ transplantation, effectively controls Hepatitis E and just must cut off pig and human route of transmission.This has just shown the meaning of the checkout and diagnosis of pig hepatitis E virus especially.
Due to the biological characteristics of Hepadna Virus itself, also do not have at present ripe Cell culture invitro system can virus of proliferation particle in a large number, environmental resistance is weak to external world to add virus itself, does not also develop gratifying pig hepatitis E diagnostic kit and vaccine so far.
HEV is RNA virus, and be mainly the detection of HEVRNA and adopt PCR method to carry out, but be not quite similar due to various places the primer, testing result is unsatisfactory; And life period is shorter in blood due to viral RNA, viral level is low, and RNA is easy to the factors such as degraded, makes to detect HEV RNA and there is certain difficulty.Therefore, at present the diagnosis of HEV is diagnosed mainly through detecting anti-HEV IgG and anti-HEV IgM in serum.
Immunological detection method specificity is high, susceptibility is strong, and the easy advantage such as fast, has become the method that HEV Serologic detection is the most frequently used, now along with the development of biology techniques, gene engineering method can be adopted to express associated protein to set up a series of Serology test.The present invention adopts dual-antigen sandwich method to set up the total antibody ELISA detection kit of pig hepatitis E virus, be intended to detect the total antibody of hepatitis E virus in Swine serum quick and precisely efficiently, thus set up a kind of sensitivity is good, specificity is high immunological detection method for detecting and assess the HEV antibody horizontal of swinery, for the clinical diagnosis of swinery hepatitis E virus and prevention and control significant.
At present, on market, the detection of hepatitis E virus mainly adopts serology ELISA detection method, this product is mainly used in the detection of Serum Antibodies, also there is no Related product for the detection of pig HEV antibody, pig is as the Major Natural host of hepatitis E virus, owing to lacking relevant feasible testing product, temporarily there is no the examining report of swinery HEV antibody horizontal yet.Pig source HEV and mankind HEV sequence have high homology, and its RNA virus can also infect other animal such as chimpanzee and rhesus macaque, in order to make up this technological gap, develop the total antibody ELISA detection method of pig source HEV, overcoming available reagent box can only the deficiency of specific detection people hepatitis E virus, and this product is first and is specifically designed to the immunoassay product series detecting the total antibody of animal Hepatitis E.
Summary of the invention
The invention reside in and provide the boar HEV preparation method of total antibody ELISA detection kit, the kit being intended to solve existing hepatitis E virus can only specific detection people hepatitis E virus, and can not detect the problem of the hepatitis E virus in pig source.
The present invention is achieved in that the preparation method of the total antibody ELISA detection kit of a boar HEV, and the preparation method of the total antibody ELISA detection kit of this pig HEV comprises the following steps:
The first step, the preparation of HEV envelope antigen:
First two fragments of pcr amplification pig source swCH189 strain HEV ORF2 Gene Partial, upstream and downstream primer is respectively with BamH I and Not I restriction enzyme site;
FS is S fragment upstream primer, and with BamH I restriction enzyme site, RS is fragment downstream primer, with Not I restriction enzyme site; FP12 is P12 fragment upstream primer, and with BamH I restriction enzyme site, RP12 is fragment downstream primer, with Not I restriction enzyme site;
Reclaim after PCR fragment amplification, adopt BamH I and Not I double digestion, be connected to pMD18-T carrier, product conversion bacillus coli DH 5 alpha will be connected, picking transformant, little upgrading grain, qualification is cut, qualification positive colony called after T-S and T-P12 respectively with BamH I and Not I enzyme;
By pET32a(+) digest with BamH I/Not I respectively with the cloned plasmids of qualification, carry out agarose electrophoresis, glue connects after reclaiming object fragment, structure can at the recombinant plasmid of expression in escherichia coli, name pET-S and pET-P12, will connect product conversion bacillus coli DH 5 alpha, picking transformant, little upgrading grain, carries out enzyme with BamH I and Not I restriction enzyme and cuts qualification;
Positive recombinant plasmid pET-S and pET-P12 transforms Rossetta (DE3) competent cell, select monoclonal bacterium colony and cultivate 10h in ampicillin sodium LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to OD600=0.8 with the ratio of 1:100, the final concentration added is the IPTG of 0.5mmol/L, 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, analysis determines that expression product is in colibacillary existence form, found that recombinant protein exists with the form of inclusion body.
Destination protein carries out SDS-PAGE, and band is consistent with expection size, called after S protein, and the S protein of purifying is envelope antigen;
Second step, the preparation of HEV labelled antigen:
Positive colony pET-P12 is cultivated 10h in Amp+LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to OD600=0.8 with the ratio of 1:100, add the IPTG that final concentration is 1mmol/L, 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, determine that expression product is present in precipitation with the form of inclusion body, carry out the purifying of albumen, the protein designations that purifying obtains is P12 albumen, for labelled protein,
3rd step, the preparation of HEV labelled antigen-HRP label, specifically comprises:
Step one, the coupling of HEV labelled antigen and HRP adopts NaIO
4oxidizing process, first by purifying protein P12 4 DEG C of dialysed overnight in PBS solution;
Step 2, ultraviolet spectrophotometer measures purifying protein concentration;
Step 3, analytical balance weighs HRP10mg and is dissolved in 2ml ultrapure water and prepares the HRP solution that final concentration is 5mg/ml, and analytical balance weighs NaIO
4, be dissolved in ultrapure water and prepare the NaIO that final concentration is 20mg/ml
4solution for standby;
Step 4, slowly drips 225 μ l NaIO
4solution is in 2ml HRP solution, and under room temperature, lucifuge leaves standstill 20 minutes;
Step 5, adds 20 μ l ethylene glycol in HRP mixed solution, left at room temperature 30 minutes afterwards;
Step 6, the labelled protein P12 getting 1ml2.1mg/ml slowly dropwise joins in the HRP solution activated;
Step 7, then dialyses P12 albumen-HRP bond 2.5 hours respectively at 4 DEG C of lucifuges in the solution of 50mM CB pH9.6;
Step 8, it is soluble in water that analytical balance weighs NaBH4, prepares the NaBH that final concentration is 4mg/ml
4solution;
Step 9, takes out bag filter, adds the NaBH of 162 μ l
4solution;
Step 10, shakes 2 hours gently under room temperature on shaking table;
Step 11, label is 4 DEG C of dialysed overnight in PBS solution;
Step 12, dialysis terminates rear 1:1 ratio and adds glycerine, and-20 degree are preserved, this label hereinafter referred to as HRP-P12;
4th step, adopt carbonate buffer solution with certain proportion by S antigen diluent, 100 μ l/ holes join ELISA Plate, 4 DEG C of bags are spent the night, adopt PBST (10mM PB next day, 150mM NaCl, 0.05%Tween-20, pH7.4) cleansing solution washes plate three times, pat dry for the last time, 150 μ l/ holes add containing 30% NBCS, 8% sucrose, 5% bovine serum albumin(BSA), the ρ Η 7.4 of 150mM NaCl, 10mM PB confining liquid, close 12 hours for 4 DEG C, get rid of liquid in hole next day, pat dry, put room temperature 20-25 DEG C, humidity 20%-25%, there is the interior air dried overnight of the drying shed of ventilation equipment, be packaged in and be added with in the aluminum foil bag of drying agent, wrap complete.
Further, protein purification in a first step adopts Invitrogene clonetech protein purification post, and concrete steps are as follows:
Step one, prepare 1L and express somatic cells, get the centrifugal 2min of 5ml bacterium liquid 12000r/min, supernatant discarded, the PBS adding 1mmol/LPH7.4 fully suspends, 4 DEG C of centrifugal 15min of 6000r/min, retains precipitation, centrifugal 3 times so repeatedly;
Step 2, is deposited in-70 DEG C and places 5min, takes out afterwards and dissolves; Multigelation circulation like this 3 ~ 5 times, to realize maximum cracking;
Step 3, the 1 × FastBreakTM cell Lysis Reagent solution adding 100 μ l, in precipitation, fully suspends;
Step 4, adds the DNase I of 1 μ l, 200r/min shaking 30min under room temperature;
Step 5, adds the Ni-Particles of 30 μ l or adds the NaCl solid of 0.03g;
Step 6, after adopting pipettor repeatedly to inhale to blow mixing 10 times, room temperature stationary incubation 30min;
Step 7, puts into 30s on carbon magnetic frame by EP pipe, sees what nickel post was adsorbed near magnetic frame, is sucked out by remaining liquid with pipettor;
Step 8, adds the Bind/Wash Buffer of 150 μ l, repeatedly inhales and blows mixing 10 times, room temperature stationary incubation 10min, puts into magnetic frame 30s afterwards, is sucked out by remaining liquid, adopt same liquid repeatedly to wash twice, last liquid must blot only, does not have residual;
Step 9, adds the Elution Buffer of 100 μ l, retains the liquid also difference label of different mycoprotein, the albumen of purifying and respective nickel post are carried out protein electrophoresis analysis result simultaneously after repeatedly acting on 30s after mixing piping and druming on magnetic frame.
Further, the concrete using method of the total antibody ELISA detection kit of this pig HEV is:
In ELISA Plate, every hole adds 100 μ l sample diluting liquids, sample diluting liquid is PH7.4PB, 150mMNaCl, 5% bovine serum albumin(BSA), 0.5% isinglass, 2%Tween-20,0.1%Triton X-100,0.1% thimerosal, add 50 μ l testing samples, select health pig negative serum as negative with reference to sample, select HEV antibody positive Swine serum to contrast as positive reference sample, 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry for the last time, I00 μ l/ hole adds containing 20% NBCS, with the HRP-P12 damping fluid of certain proportion dilution, and 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry;
Every hole adds containing the developer A of 0.5% hydrogen peroxide urea, 4.76% sodium acetate trihydrate, 0.9% glacial acetic acid and each 50 μ l of developer B containing 0.32%TMB, 5mM citric acid, 0.5mMEDTA-2Na, 5% methyl alcohol, 2% dimethyl formamide, 37 DEG C of lucifuges develop the color 15 minutes, every hole adds 50 μ l, containing the stop buffer cessation reaction of 2M sulfuric acid, behind microplate reader 450nm wavelength blank well school zero, read OD value.
Further, critical value COV calculates: COV=negative control mean OD value X1.5, and negative control OD value calculates by 0.065 lower than 0.065, calculates by actual OD value higher than 0.065, testing sample OD value >=COV is positive, and testing sample OD value <COV is negative.
Further, ELISA kit adopts dual-antigen sandwich method principle, and S antigen employing pH9.6CB is diluted to 4 μ g/ml and carries out wrapper sheet, and 4 DEG C of bags are spent the night afterwards; Enzyme mark confining liquid 0.5%BSA, 20mM PB, 150mM NaCl, 0.1% stabilizer T, 0.01%Proclin300 adds 150 μ l and closes; The dilution of positive sample; In P12-HRP marker enzyme labeling process, the mol ratio of HRP and P12 is 1:5, and adopt enzyme thin liquid 0.5%BSA afterwards, 20mM PB, 150mM NaCl, 5% NBCS, 0.01%Proclin300 dilutes the mark for ELISA kit after 2000 times.
The preparation method of the total antibody ELISA detection kit of pig HEV provided by the invention, according to pig source HEVORF2 protein structural information, carries out two antigen fragment prokaryotic expressions by pig source HEV ORF2, S domain protein and P12 domain protein.In ELISA kit, as envelope antigen after S protein purifying, carry out horseradish peroxidase mark after P12 protein purification as labelled antigen; Adopt pig source swCH189 strain HEV strain, according to the existing achievement in research of HEV viral protein structures, by adopting its capsid protein structure of related biological software analysis, and the capsid protein fragment that successful expression is different, through research and the exploration of different experiments method, filter out best experimental technique and antigen combination, establish a kind of ELISA detection method rapidly and efficiently detecting the total antibody of pig HEV; Of the present invention highly sensitive, specificity is good, reproducible, for the large-scale inquiry of hepatitis E virus epidemiology provides certain technical guarantee, also for better the popular of prevention and corntrol HEV epidemic disease provides technical support; Adopt dual-antigen sandwich method principle, envelope antigen and Serum Antibody produce specific binding, and conjugated antigen antibody complex and labelled antigen produce antigen-antibody reaction again, and two antigens are in conjunction with different antigen binding sites, and specificity is good, highly sensitive.
Accompanying drawing explanation
Fig. 1 is preparation method's process flow diagram of the total antibody ELISA detection kit of pig HEV that the embodiment of the present invention provides;
Fig. 2 is the pcr amplification schematic diagram of the S gene that the embodiment of the present invention provides;
In figure: M:DL2000marker; 1:S gene amplification fragment;
Fig. 3 is the pcr amplification schematic diagram of the P12 gene that the embodiment of the present invention provides;
In figure: M: λ-EcoT14 I digest marker; 1:P12 gene amplification fragment;
Fig. 4 is that the enzyme of the S gene that the embodiment of the present invention provides cuts qualification schematic diagram;
In figure: M:DL2000marker; 1:S gene enzyme cuts qualification fragment;
Fig. 5 is that the enzyme of the P12 gene that the embodiment of the present invention provides cuts qualification schematic diagram;
In figure: M:DL2000marker; 1:P12 gene enzyme cuts qualification fragment;
Fig. 6 is that the SDS-PAGE of the His-S albumen that the embodiment of the present invention provides analyzes schematic diagram;
Fig. 7 is the His-S protein purification schematic diagram that the embodiment of the present invention provides;
Fig. 8 is that the SDS-PAGE of the His-P12 albumen that the embodiment of the present invention provides analyzes schematic diagram;
Fig. 9 is the His-P12 protein purification schematic diagram that the embodiment of the present invention provides.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the preparation method of the total antibody ELISA detection kit of pig HEV of the embodiment of the present invention comprises the following steps:
S101: preparation HEV envelope antigen;
S102: preparation HEV labelled antigen;
The preparation of S103:HEV labelled antigen-HRP label;
S104: adopt carbonate buffer solution with certain proportion by S antigen diluent, 100 μ l/ holes join ELISA Plate, are packaged in and are added with in the aluminum foil bag of drying agent, wrap complete.
In conjunction with specific embodiments of the invention, the present invention is described further:
Embodiments of the invention comprise the following steps:
The first step, the preparation of HEV envelope antigen:
First pcr amplification pig source swCH189 strain HEV ORF2(Lanzhou veterinary institute infectious ward is preserved) two fragments of Gene Partial, upstream and downstream design of primers as shown in Table 1 and Table 2, respectively with BamH I and Not I restriction enzyme site;
Table 1:swCH189 strain S fragment design primer is:
FS is S fragment upstream primer, and with BamH I restriction enzyme site, RS is fragment downstream primer, with Not I restriction enzyme site;
Table 2:swCH189 strain P12 fragment is 328aa-End, and design primer is:
FP12 is P12 fragment upstream primer, and with BamH I restriction enzyme site, RP12 is fragment downstream primer, with Not I restriction enzyme site;
Reclaim after PCR fragment amplification, adopt BamH I and Not I double digestion, (Dalian is precious biological to be connected to pMD18-T carrier, article No. D103A), product conversion bacillus coli DH 5 alpha will be connected, picking transformant, little upgrading grain, cut qualification with BamH I and Not I enzyme, identify suitable positive colony called after T-S and T-P12 respectively; As shown in Figures 2 and 3;
By pET32a(+) (preservation of Lanzhou veterinary institute infectious ward) digest with BamH I/Not I respectively with the suitable cloned plasmids of qualification, carry out agarose electrophoresis, glue connects after reclaiming object fragment, structure can at the recombinant plasmid of expression in escherichia coli, name pET-S and pET-P12, (Dalian is precious biological will to connect product conversion bacillus coli DH 5 alpha, article No. 9027), picking transformant, little upgrading grain, with BamH I, (Dalian is precious biological, article No. 1605) and Not I restriction enzyme (the precious biology in Dalian, article No. 1166A) carry out enzyme and cut qualification, as shown in Figure 4 and Figure 5,
Positive recombinant plasmid pET-S and pET-P12 transforms Rossetta (DE3) competent cell, and (Beijing health is that century is biological, article No. CW0811A), select monoclonal bacterium colony in ampicillin sodium (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, article No. A0339) cultivate 10h in LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to about OD600=0.8 with the ratio of 1:100, the final concentration added is the IPTG (invitrogen of 0.5mmol/L, article No. AM9464), 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, analysis determines that expression product is in colibacillary existence form, found that recombinant protein mainly exists with the form of inclusion body,
Protein purification adopts Invitrogene clonetech protein purification post, and purification step is as follows:
(1) prepare 1L and express somatic cells, get the centrifugal 2min of 5ml bacterium liquid 12000r/min, supernatant discarded, the PBS adding 1mmol/LPH7.4 fully suspends, 4 DEG C of centrifugal 15min of 6000r/min, retains precipitation, centrifugal 3 times so repeatedly;
(2) be deposited in-70 DEG C and place 5min, take out afterwards and dissolve;
Multigelation circulation like this 3 ~ 5 times, to realize maximum cracking;
(3) 1 × FastBreakTM cell Lysis Reagent solution adding 100 μ l, in precipitation, makes it fully suspend;
(4) DNase I of 1 μ l is added, 200r/min shaking 30min under room temperature;
(5) adding the Ni-Particles of 30 μ l, experimentally need suitably to add into some, simultaneously in order to improve the binding ability of nickel post, also can add the NaCl solid of 0.03g;
(6) adopt pipettor repeatedly inhale blow mixing about 10 times after, room temperature stationary incubation 30min;
(7) add EP pipe and put into about 30s on carbon magnetic frame, can be clearly seen that what nickel post was adsorbed near magnetic frame, with pipettor, remaining liquid is sucked out;
(8) add the Bind/Wash Buffer of 150 μ l, repeatedly inhale and blow mixing about 10 times, room temperature stationary incubation 10min, put into magnetic frame 30s afterwards, remaining liquid is sucked out, adopt same liquid repeatedly to wash twice, last liquid must blot only, does not have residual;
(9) add the Elution Buffer of 100 μ l, retain the liquid also difference label of different mycoprotein after repeatedly acting on 30s after mixing piping and druming on magnetic frame, the albumen of purifying and respective nickel post are carried out protein electrophoresis analysis result simultaneously;
Destination protein carries out SDS-PAGE, and band is consistent with expection size, called after S protein, and S protein is envelope antigen of the present invention, as shown in Figure 6 and Figure 7;
Second step, the preparation of HEV labelled antigen:
Positive colony pET-P12 is cultivated 10h in Amp+LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to about OD600=0.8 with the ratio of 1:100, add the IPTG that final concentration is 1mmol/L, 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, determine that expression product is mainly present in precipitation with the form of inclusion body, following purification steps is the same with pET-S, the protein designations that purifying obtains is P12 albumen, for labelled protein of the present invention, as shown in Figure 8 and Figure 9,
3rd step, the preparation of HEV labelled antigen-HRP label, specifically comprises:
Step one, the coupling of HEV labelled antigen and HRP adopts NaIO
4oxidizing process, first by purifying protein P12 4 DEG C of dialysed overnight in PBS solution;
Step 2, ultraviolet spectrophotometer measures purifying protein concentration;
Step 3, analytical balance weighs HRP10mg and is dissolved in 2ml ultrapure water and prepares the HRP solution that final concentration is 5mg/ml, and analytical balance weighs NaIO
4, be dissolved in ultrapure water and prepare the NaIO that final concentration is 20mg/ml
4solution for standby;
Step 4, slowly drips 225 μ lNaIO
4solution is in 2ml HRP solution, and under room temperature, lucifuge leaves standstill 20 minutes;
Step 5, adds 20 μ l ethylene glycol in HRP mixed solution, left at room temperature 30 minutes afterwards;
Step 6, the labelled protein P12 getting 1ml2.1mg/ml slowly dropwise joins in the HRP solution activated;
Step 7, then dialyses P12 albumen-HRP bond 2.5 hours respectively at 4 DEG C of lucifuges in the solution of 50mM CB pH9.6;
Step 8, it is soluble in water that analytical balance weighs NaBH4, prepares the NaBH that final concentration is 4mg/ml
4solution;
Step 9, takes out bag filter, adds the NaBH of 162 μ l
4solution;
Step 10, shakes 2 hours gently under room temperature on shaking table;
Step 11, label is 4 DEG C of dialysed overnight in PBS solution;
Step 12, dialysis terminates rear 1:1 ratio and adds glycerine, and-20 degree are preserved, this label hereinafter referred to as HRP-P12;
4th step, adopt carbonate buffer solution (50mM, pH9.51) with certain proportion by S antigen diluent, 100 μ l/ holes join ELISA Plate (Shenzhen Jin Canhua Industrial Co., Ltd. irradiation ELISA Plate), 4 DEG C of bags are spent the night, adopt PBST (10mM PB next day, 150mM NaCl, 0.05%Tween-20, pH7.4) cleansing solution washes plate three times, pat dry for the last time, 150 μ l/ holes add containing 30% NBCS (Gibco, article No. 16010-159), 8% sucrose, 5% bovine serum albumin(BSA) (Sigma-Aldrich, article No. C-8645), the ρ Η 7.4 of 150mM NaCl, 10mM PB confining liquid, close 12 hours for 4 DEG C, get rid of liquid in hole next day, pat dry, put room temperature 20-25 DEG C, humidity 20%-25%, there is the interior air dried overnight of the drying shed of ventilation equipment, be packaged in and be added with in the aluminum foil bag of drying agent, wrap complete,
Concrete using method of the present invention is: in ELISA Plate, every hole adds 100 μ l sample diluting liquid (PH7.4PB, 150mM NaCl, 5% bovine serum albumin(BSA), 0.5% isinglass, 2%Tween-20,0.1%TritonX-100,0.1% thimerosal), add 50 μ l testing samples, negative with reference to sample (health pig negative serum), positive reference sample (HEV antibody positive Swine serum) contrast, 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry for the last time, I00 μ l/ hole adds containing 20% NBCS, with the HRP-P12 damping fluid of certain proportion dilution, and 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry;
Every hole adds containing 0.5% hydrogen peroxide urea (raw work, article No. UB1753), the developer A of 4.76% sodium acetate trihydrate, 0.9% glacial acetic acid and containing 0.32%TMB (raw work, article No. TB0514), each 50 μ l of developer B of 5mM citric acid, 0.5mM EDTA-2Na, 5% methyl alcohol, 2% dimethyl formamide, 37 DEG C of lucifuges develop the color 15 minutes, every hole adds 50 μ l, containing the stop buffer cessation reaction of 2M sulfuric acid, behind microplate reader 450nm wavelength (reference wavelength 630nm) blank well school zero, read OD value; Critical value calculates: (negative control OD value calculates by 0.065 lower than 0.065 COV=negative control mean OD value X1.5, calculate by actual OD value higher than 0.065), testing sample OD value >=COV is positive, and testing sample OD value <COV is negative.
Testing result of the present invention, select the 150 parts of pig HEV positive serums and 3000 parts of pig negative serums verified through RIBA2.0 (Ortho-Clinical Diagnostics), pig HEV dual-antigen sandwich method detection kit of the present invention is adopted to detect 300 parts of Swine serum, its sensitivity is 96.2%, specificity is 98%, accuracy rate is 97.2%
ELISA kit of the present invention adopts dual-antigen sandwich method principle, and S antigen employing pH9.6CB is diluted to 4 μ g/ml and carries out wrapper sheet, and 4 DEG C of bags are spent the night afterwards; Enzyme mark confining liquid (0.5%BSA, 20mM PB, 150mM NaCl, 0.1% stabilizer T, 0.01%Proclin300) adds 150 μ l and closes; The dilution of positive sample; In P12-HRP marker enzyme labeling process, the mol ratio of HRP and P12 is 1:5, and ((0.5%BSA, 20mM PB, 150mM NaCl, 5% NBCS, 0.01%Proclin300) dilutes the mark for ELISA kit after 2000 times to adopt enzyme thin liquid afterwards.
The present invention adopts S structure fragment and P12 structure fragment to express respectively, as envelope antigen and the labelled antigen of double antigens sandwich.Other invention may adopt different expression product to copy this product.Therefore, this product is the ELISA detection kit for adopting dual-antigen sandwich method to detect the total antibody of pig HEV.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a preparation method for the total antibody ELISA detection kit of pig hepatitis E virus, is characterized in that, the preparation method of the total antibody ELISA detection kit of this pig hepatitis E virus comprises the following steps:
The first step, the preparation of hepatitis E virus envelope antigen:
First two fragments of pcr amplification pig source swCH189 strain hepatitis E virus ORF2 Gene Partial, upstream and downstream primer is respectively with BamH I and Not I restriction enzyme site;
FS is S fragment upstream primer, and with BamH I restriction enzyme site, RS is fragment downstream primer, with Not I restriction enzyme site; FP12 is P12 fragment upstream primer, and with BamH I restriction enzyme site, RP12 is fragment downstream primer, with Not I restriction enzyme site;
The sequence of FS is 5'-CGCGGATCCGACACTGCACCCGTA-3', 132-146; The sequence of RS is 5'-ATTTGCGGCCGCTAGGTGTCAAGTT-3', 316-327; The sequence of FP12 is 5'-CGCGGATCCGGTAATACTAAT-3', 328-340; The sequence of RP12 is 5'-ATTTGCGGCCGCTATACTCCCGGGTTTT-3', 665-674;
Reclaim after PCR fragment amplification, adopt BamH I and Not I double digestion, be connected to pMD18-T carrier, product conversion bacillus coli DH 5 alpha will be connected, picking transformant, little upgrading grain, qualification is cut, qualification positive colony called after T-S and T-P12 respectively with BamH I and Not I enzyme;
PET32a (+) and the cloned plasmids of qualification are digested with BamH I/Not I respectively, carry out agarose electrophoresis, glue connects after reclaiming object fragment, structure can at the recombinant plasmid of expression in escherichia coli, name pET-S and pET-P12, will connect product conversion bacillus coli DH 5 alpha, picking transformant, little upgrading grain, carries out enzyme with BamH I and Not I restriction enzyme and cuts qualification;
Positive recombinant plasmid pET-S and pET-P12 transforms Rossetta (DE3) competent cell, select monoclonal bacterium colony and cultivate 10h in ampicillin sodium LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to OD600=0.8 with the ratio of 1:100, add the IPTG that final concentration is 0.5mmol/L, 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml 1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, analysis determines that expression product is in colibacillary existence form, found that recombinant protein exists with the form of inclusion body,
Destination protein carries out SDS-PAGE, and band is consistent with expection size, called after S protein, and the S protein after purifying is envelope antigen;
Second step, the preparation of hepatitis E virus labelled antigen:
Positive colony pET-P12 is cultivated 10h in Amp+LB nutrient solution, bacterium liquid joins 1L LB nutrient culture media 37 DEG C of shaken cultivation containing 100 μ g/ml ampicillin sodiums to OD600=0.8 with the ratio of 1:100, add the IPTG that final concentration is 1mmol/L, 25 DEG C of induction 5h, 4 DEG C of 6000r/min collect thalline in centrifugal 15 minutes, thalline adopts the PBS of 20ml 1%Triton X-100 to suspend, ultrasonic disruption, centrifugal 30 minutes of 4 DEG C of 12000r/min, get cleer and peaceful precipitation respectively and carry out SDS-PAGE, determine that expression product is present in precipitation with the form of inclusion body, carry out the purifying of albumen, the protein designations that purifying obtains is P12 albumen, for labelled protein,
3rd step, the preparation of hepatitis E virus labelled antigen-HRP label, specifically comprises:
Step one, the coupling of hepatitis E virus labelled antigen and HRP adopts NaIO
4oxidizing process, first by purifying protein P12 4 DEG C of dialysed overnight in PBS solution;
Step 2, ultraviolet spectrophotometer measures purifying protein concentration;
Step 3, analytical balance weighs HRP 10mg and is dissolved in 2ml ultrapure water and prepares the HRP solution that final concentration is 5mg/ml, and analytical balance weighs NaIO
4, be dissolved in ultrapure water and prepare the NaIO that final concentration is 20mg/ml
4solution for standby;
Step 4, slowly drips 225 μ l NaIO
4solution is in 2ml HRP solution, and under room temperature, lucifuge leaves standstill 20 minutes;
Step 5, adds 20 μ l ethylene glycol in HRP mixed solution, left at room temperature 30 minutes afterwards;
Step 6, the labelled protein P12 getting 1ml 2.1mg/ml slowly dropwise joins in the HRP solution activated;
Step 7, then dialyses P12 albumen-HRP bond 2.5 hours respectively at 4 DEG C of lucifuges in the solution of 50mM CB pH9.6;
Step 8, analytical balance weighs NaBH
4soluble in water, prepare the NaBH that final concentration is 4mg/ml
4solution;
Step 9, takes out bag filter, adds the NaBH of 162 μ l
4solution;
Step 10, shakes 2 hours gently under room temperature on shaking table;
Step 11, label is 4 DEG C of dialysed overnight in PBS solution;
Step 12, dialysis terminates rear 1:1 ratio and adds glycerine, and-20 degree are preserved, this label hereinafter referred to as HRP-P12;
4th step, adopt carbonate buffer solution with certain proportion by S antigen diluent, 100 μ l/ holes join ELISA Plate, 4 DEG C of bags are spent the night, content is adopted next day to be 10mM PB, 150mMNaCl, 0.05%Tween-20, the PBST cleansing solution of pH7.4 washes plate three times, pat dry for the last time, 150 μ l/ holes add containing 30% NBCS, 8% sucrose, 5% bovine serum albumin(BSA), the ρ Η 7.4 of 150mM NaCl, 10mM PB confining liquid, close 12 hours for 4 DEG C, get rid of liquid in hole next day, pat dry, put room temperature 20-25 DEG C, humidity 20%-25%, there is the interior air dried overnight of the drying shed of ventilation equipment, be packaged in and be added with in the aluminum foil bag of drying agent, wrap complete.
2. the preparation method of the total antibody ELISA detection kit of pig hepatitis E virus as claimed in claim 1, is characterized in that, protein purification in a first step adopts Invitrogeneclonetech protein purification post, and concrete steps are as follows:
Step one, prepare 1L and express somatic cells, get the centrifugal 2min of 5ml bacterium liquid 12000r/min, supernatant discarded, the PBS adding 1mmol/L pH7.4 fully suspends, 4 DEG C of centrifugal 15min of 6000r/min, retains precipitation, centrifugal 3 times so repeatedly;
Step 2, is deposited in-70 DEG C and places 5min, takes out afterwards and dissolves; Multigelation circulation like this 3 ~ 5 times, to realize maximum cracking;
Step 3, the 1 × FastBreakTM cell Lysis Reagent solution adding 100 μ l, in precipitation, fully suspends;
Step 4, adds the DNase I of 1 μ l, 200r/min shaking 30min under room temperature;
Step 5, adds the Ni-Particles of 30 μ l or adds the NaCl solid of 0.03g;
Step 6, after adopting pipettor repeatedly to inhale to blow mixing 10 times, room temperature stationary incubation 30min;
Step 7, puts into 30s on carbon magnetic frame by EP pipe, sees what nickel post was adsorbed near magnetic frame, is sucked out by remaining liquid with pipettor;
Step 8, adds the Bind/Wash Buffer of 150 μ l, repeatedly inhales and blows mixing 10 times, room temperature stationary incubation 10min, puts into magnetic frame 30s afterwards, is sucked out by remaining liquid, adopt same liquid repeatedly to wash twice, last liquid must blot only, does not have residual;
Step 9, adds the Elution Buffer of 100 μ l, retains the liquid also difference label of different mycoprotein, the albumen of purifying and respective nickel post are carried out protein electrophoresis analysis result simultaneously after repeatedly acting on 30s after mixing piping and druming on magnetic frame.
3. the preparation method of the total antibody ELISA detection kit of pig hepatitis E virus as claimed in claim 1, it is characterized in that, the concrete using method of the total antibody ELISA detection kit of this pig hepatitis E virus is:
In ELISA Plate, every hole adds 100 μ l sample diluting liquids, sample diluting liquid is pH7.4PB, 150mM NaCl, 5% bovine serum albumin(BSA), 0.5% isinglass, 2%Tween-20,0.1%Triton X-100,0.1% thimerosal, add 50 μ l testing samples, select health pig negative serum as negative with reference to sample, select the positive Swine serum of the hepatitis E virus antibody of preparation to contrast as positive reference sample, 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry for the last time, every hole add 100 μ l contain 20% NBCS with the HRP-P12 damping fluid of certain proportion dilution, 37 DEG C of incubations 30 minutes; Wash plate five times with PBST cleansing solution, pat dry;
Every hole adds containing the developer A of 0.5% hydrogen peroxide urea, 4.76% sodium acetate trihydrate, 0.9% glacial acetic acid and each 50 μ l of developer B containing 0.32%TMB, 5mM citric acid, 0.5m MEDTA-2Na, 5% methyl alcohol, 2% dimethyl formamide, 37 DEG C of lucifuges develop the color 15 minutes, every hole adds 50 μ l, containing the stop buffer cessation reaction of 2M sulfuric acid, behind microplate reader 450nm wavelength blank well school zero, read OD value.
4. the preparation method of the total antibody ELISA detection kit of pig hepatitis E virus as claimed in claim 3, it is characterized in that, critical value (Cut offValue (COV)) calculates: COV=negative control mean OD value X 1.5, negative control OD value calculates by 0.065 lower than 0.065, calculate by actual OD value higher than 0.065, testing sample OD value >=COV is positive, and testing sample OD value <COV is negative.
5. the preparation method of the total antibody ELISA detection kit of pig hepatitis E virus as claimed in claim 1, it is characterized in that, ELISA kit adopts dual-antigen sandwich method principle, and S antigen employing pH9.6CB is diluted to 4 μ g/ml and carries out wrapper sheet, and 4 DEG C of bags are spent the night afterwards; Enzyme mark confining liquid 0.5%BSA, 20mM PB, 150mMNaCl, 0.1% stabilizer T, 0.01%Proclin 300 adds 150 μ l and closes; The dilution of positive sample; In P12-HRP marker enzyme labeling process, the mol ratio of HRP and P12 is 1:5, and adopt enzyme thin liquid 0.5%BSA afterwards, 20mM PB, 150mMNaCl, 5% NBCS, 0.01%Proclin 300 dilutes the mark for ELISA kit after 2000 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410084515.4A CN103837680B (en) | 2014-03-07 | 2014-03-07 | The preparation method of the total antibody ELISA detection kit of one boar HEV |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410084515.4A CN103837680B (en) | 2014-03-07 | 2014-03-07 | The preparation method of the total antibody ELISA detection kit of one boar HEV |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103837680A CN103837680A (en) | 2014-06-04 |
| CN103837680B true CN103837680B (en) | 2015-08-26 |
Family
ID=50801392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410084515.4A Active CN103837680B (en) | 2014-03-07 | 2014-03-07 | The preparation method of the total antibody ELISA detection kit of one boar HEV |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103837680B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104792987B (en) * | 2015-04-29 | 2017-03-08 | 中国食品药品检定研究院 | For diagnosing method and the kit of hepatitis e virus infection |
| CN105044328A (en) * | 2015-05-28 | 2015-11-11 | 首都医科大学附属北京佑安医院 | Elispot pre-coated culture plate and preparation method thereof |
| CN105486863B (en) * | 2015-12-24 | 2018-03-02 | 中国农业科学院兰州兽医研究所 | One boar Sapporo virus total antibody ELISA detection kit and preparation method thereof |
| CN106168625B (en) * | 2016-08-03 | 2018-03-20 | 广州伯信生物科技有限公司 | A kind of RNA pulldown methods and kit |
| CN106771178B (en) * | 2016-11-18 | 2018-05-01 | 华南农业大学 | A kind of liquid-phase chip Multiple detection kit of PRRSV, SIV, pig HEV antibody |
| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0012100A1 (en) * | 1978-11-29 | 1980-06-11 | Siemens Aktiengesellschaft | Multi-core flat cable with round conductors |
| US20040234542A1 (en) * | 2001-05-07 | 2004-11-25 | Xiang-Jin Meng | Recombinant orf2 proteins of the swine hepatitis e virus and their use as a vaccine and as a diagnostic reagent for medical and veterinary applications |
| CN101034090B (en) * | 2006-03-08 | 2012-01-11 | 中国药品生物制品检定所 | Hepatitis E virus antibody and method and kit for detecting hepatitis E virus using same |
| KR101058486B1 (en) * | 2008-10-21 | 2011-08-22 | 대한민국(관리부서 : 농림수산식품부 농림수산검역검사본부) | The caffeine protein of the swine hepatitis E virus and the gene encoding the same |
| CN101620230A (en) * | 2009-08-11 | 2010-01-06 | 无锡益达生物技术有限公司 | Gold-labeled kit of beta-lactamase in dairy product and preparation method thereof |
| CN101710120A (en) * | 2009-12-14 | 2010-05-19 | 上海市农业科学院 | Application of stabilizer trehalose in pig hepatitis E diagnostic kit |
| CN101726595A (en) * | 2009-12-24 | 2010-06-09 | 上海市农业科学院 | Preparation method of diagnostic kit for pig hepatitis E virus ELISA |
-
2014
- 2014-03-07 CN CN201410084515.4A patent/CN103837680B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103837680A (en) | 2014-06-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103837680B (en) | The preparation method of the total antibody ELISA detection kit of one boar HEV | |
| CN103760365B (en) | A kind of BAS-ELISA kit detecting CSFV Erns and E2 antigen | |
| CN103588864B (en) | Classic swine fever virus (CSFV) C strain E2 truncated protein and its preparation method and use | |
| CN107942061A (en) | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody | |
| CN102731615B (en) | Detection reagent and detection method for PRRSV | |
| CN106771178B (en) | A kind of liquid-phase chip Multiple detection kit of PRRSV, SIV, pig HEV antibody | |
| CN104330572A (en) | Kit for indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection of IgG or IgA antibodies of PRRSV (Porcine reproductive and respiratory syndrome virus) of pigs | |
| CN103823057B (en) | Total antibody colloidal gold fast diagnose test paper bar of one boar HEV and preparation method thereof | |
| CN108802381A (en) | Bovine viral diarrhea virus differentiates test strip and preparation method thereof | |
| CN109187968B (en) | Bivalent gold-labeled test paper for detecting classical swine fever virus and porcine pseudorabies virus and preparation method thereof | |
| CN103616509B (en) | Detect E III-indirect ELISA antibody assay kit and the application of Latex agglutination test | |
| CN104215761A (en) | Kit for detecting anti-GP73 antibody in serum | |
| CN104166000A (en) | A method of indentifying brucella natural infection or immunifaction for livestock | |
| CN103499693A (en) | Competitive Alpha LISA (linked immuno sorbent assay) detection kit for classical swine fever virus (CSFV) antibody and detection method thereof | |
| CN104267181A (en) | African swine fever antigen and fluorescent nanocrystalline test strip prepared from African swine fever antigen | |
| CN104292311A (en) | Antigen for detecting porcine epidemic diarrhea virus neutralizing antibody, and preparation method and application thereof | |
| CN114152748A (en) | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof | |
| Lin et al. | Antibody responses of pigs to defined Erns fragments after infection with classical swine fever virus | |
| CN103834668B (en) | A kind of restructuring mycoplasma pneumoniae albumen and application thereof | |
| CN103197082A (en) | Rapid detection test strip for BVDV (bovine viral diarrhea virus) antibody and preparation method thereof | |
| CN113341138A (en) | Combined detection card for simultaneously detecting ASFV specific IgM and IgG antibodies and preparation method and application thereof | |
| CN105039406A (en) | Method for preparing vaccine vectors for swine from swine IgG1 Fc recombinant baculoviruses | |
| CN104569425A (en) | Antigen protein specifically bound with tyrosine phosphatase antibody | |
| CN103819565A (en) | HCV (hepatitis c virus) recombinant fusion antigen, and expressed gene and preparation method thereof | |
| CN106632618A (en) | Preparation method of swine hepatitis E virus ORF2 recombinant protein as well as ORF2 protein thereof and detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |