CN108546706A - A kind of preparation of yellow fever virus recombinant antigen and its kit production method - Google Patents
A kind of preparation of yellow fever virus recombinant antigen and its kit production method Download PDFInfo
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Abstract
The present invention relates to field of virus detection, disclose a kind of preparation method of yellow fever virus recombinant antigen, including predicting the higher virus antigen epitope segment design primer of score according to antigenicity, yellow fever virus RNA is extracted, PCR amplification, structure prokaryotic expression carrier and induced expression recombinant plasmid are then carried out；Also disclose a kind of production method of yellow fever virus recombinant antigen kit, yellow fever virus recombinant antigen kit includes PVC board, nitrocellulose filter, nitrocellulose filter is equipped with detection line and control line, yellow fever virus recombinant antigen is coated in detection line, the anti-human μ chain antibodies and substrate of IgM antibody and horseradish peroxidase-labeled are coated on control line.The high specificity for the yellow fever virus recombinant antigen that the present invention is prepared, affinity is high, will not intersect with other arthropod-borne viruses generation serum and react.
Description
Technical field
The present invention relates to field of virus detection, more particularly to a kind of yellow fever virus recombinant antigen preparation and its kit
Production method.
Background technology
Yellow fever virus (Yellow Fever Virus, YFV) belongs to the Flavivirus of flaviviridae, and viral genome is not
Segmented single-stranded positive RNA has several different genotypes, but only there are one serotype, antigenicity is conservative.Yellow fever virus
Grain is containing there are three types of structural proteins, large surface glycoprotein G, core coat protein C and surface protein M.Yellow fever is a kind of regional disease
Disease, torrid areas, the African incidence on the south the Sahara are higher in South America, mainly pass through Aedes aegypti (Aedes
Biting aegypti) is propagated between primate or people.
The detection of flavivirus infections relies primarily on nucleic acid and serological method.Serological method predominantly detects yellow fever virus
Specific antibody, cannot be only used for cause of disease monitoring, it may also be used for monitor the immunoreaction process of body, and evaluation vaccine
Effect of inoculation.The Serology test of presently used flavivirus infections, it is main or anti-using totivirus as detection
Former immunofluorescence technique, the yellow fever virus of in vitro culture is fixed on slide, and after being incubated jointly with sample serum, fluorescence is added
Plain labelled antibody carries out yin and yang attribute judgement by microscopically observation fluorescence power.This method is anti-using natural viral as detection
Original, it is as a result more accurate, but antigen slide preparation process is complicated, and there is also the infected wind of operator in antigen preparation process
Danger.
Moreover, because between different arboviruses, there are serious serological cross reactions, and at those, there are a variety of entomophilas
The region of viral prevalence, patient is because mixed infection leads to the presence of cross-reacting antibody in vivo so that immunology diagnosis and antidiastole
It is very difficult.Simultaneously as IgG antibody can exist for a long time in host, some is taken more than 10 months, or even throughout one's life
Band so that differentiate that infection in the past, recently or now is also very difficult, higher requirements are also raised to the quality of antigen.
Therefore, with the continuous development of Protocols in Molecular Biology, purity height is obtained, the genetic engineering recombination of high specificity resists
Original is important direction.Use kit made of Western-blot methods, generally conventional laboratory that can complete, no in addition
Need special installation, interpretation also simple and clear.And existing commercially available Western-blot reagents are import reagent, it is expensive.
Invention content
The present invention is directed to the shortcomings that cumbersome preparation procedure in the prior art, expensive reagents, provides a kind of yellow fever virus weight
The preparation of group antigen and its kit production method.
In order to solve the above-mentioned technical problem, the present invention is addressed by following technical proposals：
A kind of preparation method of yellow fever virus recombinant antigen, includes the following steps：
Step 1 chooses antigen fragment
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and is drawn in forward primer and reversely
The end of object 5 ' introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’；
Step 2, the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g
10min is centrifuged, sediment is collected, 5-10 times of volume Trizol liquid is added, after mixing, 5min is stood under the conditions of 4 DEG C, is then added
Chloroform sways 15s, take the first upper strata aqueous phase be added water-saturated phenol, sway 20s, take the second upper strata aqueous phase be added isopropanol, 4 DEG C
Under the conditions of place 10min, under the conditions of 4 DEG C, after 12000g centrifuges 10min, take and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water；
Step 3, PCR amplification
Yellow fever virus RNA reverse transcriptions obtain cDNA, then carry out PCR amplification：Take 2 μ L, 10 × PCR buffer solutions of cDNA, 5 μ
L, each 2 μ L of forward primer, reverse primer, 8 dNTP μ L, LATaq 2U, moisturizing to 50 μ L of total volume carry out PCR amplification, expand piece
Section it is purified, recycling after, -20 DEG C save backup；
Step 4, the structure of prokaryotic expression carrier
Restricted digestion and company are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III
It connects, obtains recombinant plasmid；
Step 5, the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, and the coli strain Top10F containing pET28a-17D exists
It is grown in LB culture solutions containing kanamycins, 1mM IPTG (isopropyl-beta D-thio galas is added during protein growth
Glucosides) carry out induced expression, induction time 3h；Carry out 12%SDS-PAGE (sodium dodecyl sulfate-polypropylene acrylamide gels
Electrophoresis) electrophoretic analysis.
Preferably, in step 2, the additive amount of chloroform is the 1/5 of Trizol liquid product, and the additive amount of water-saturated phenol is
The 1/3 of Trizol liquid product, the additive amount of isopropanol are the 1/2 of Trizol liquid product, and the first time additive amount of 75% ethyl alcohol is
1.5 times of Trizol liquid product, second of additive amount of 75% ethyl alcohol are 1.2 times of Trizol liquid product.
Preferably, the response parameter of PCR is in step 3：95 DEG C of pre-degeneration 5min, then 94 DEG C denaturation 30s, 57 DEG C
Anneal 40s, 72 DEG C of extension 90s, totally 32 cycles, 72 DEG C of extension 7min after last time recycles.
Preferably, further including the identification to recombinant plasmid in step 4：The L1-Blue senses of recombinant plasmid transformed Escherichia coli
It by state cell, is coated on the agar plate containing the ampicillins 100mg/mL, is containing X-gal (the chloro- 3- indoles-of the bromo- 4- of 5-
β-D- galactosides) and the agar plate of IPTG on screening recombinant plasmid and non-recombinant plasmid.
A kind of production method of yellow fever virus recombinant antigen kit, yellow fever virus recombinant antigen kit include PVC board,
Nitrocellulose filter, nitrocellulose filter are equipped with detection line and control line, and yellow fever virus recombinant antigen is coated in detection line, right
According to the anti-human μ chain antibodies and substrate for being coated with IgM antibody and horseradish peroxidase-labeled on line, include the following steps：
Step 1, the preparation of yellow fever virus recombinant antigen
(11), antigen fragment is chosen
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and is drawn in forward primer and reversely
The end of object 5 ' introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’；
(12), the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g
10min is centrifuged, sediment is collected, 5-10 times of volume Trizol liquid is added, after mixing, 5min is stood under the conditions of 4 DEG C, is then added
Chloroform sways 15s, take the first upper strata aqueous phase be added water-saturated phenol, sway 20s, take the second upper strata aqueous phase be added isopropanol, 4 DEG C
Under the conditions of place 10min, under the conditions of 4 DEG C, after 12000g centrifuges 10min, take and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water；
(13), PCR amplification
Yellow fever virus RNA reverse transcriptions obtain cDNA, then carry out PCR amplification：Take 2 μ L, 10 × PCR buffer solutions of cDNA, 5 μ
L, each 2 μ L of forward primer, reverse primer, 8 dNTP μ L, LATaq 2U, moisturizing to 50 μ L of total volume carry out PCR amplification, expand piece
Section it is purified, recycling after, -20 DEG C save backup；
(14), the structure of prokaryotic expression carrier
Restricted digestion and company are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III
It connects, obtains recombinant plasmid；
(15), the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, and the coli strain Top10F containing pET28a-17D exists
It is grown in LB culture solutions containing kanamycins, 1mM IPTG is added during protein growth and carry out induced expression, when induction
Between be 3h；Carry out 12%SDS-PAGE electrophoretic analysis；
Step 2, the protein that step 1 obtains is purified：The coli strain Top10F cultures of pET28a-17D
Liquid is seeded in the LB culture solutions containing kanamycins, 37 DEG C of cultures to OD450=0.6, IPTG shaken cultivations at 37 DEG C are added
5h, 12000g centrifuging and taking bacterial precipitation object, bacterial precipitation object, using affinity chromatography column purification, obtain yellow fever after PBS is washed
Malicious recombinant antigen protein；
Step 3, in the case where humidity is 45~65%, 1mg/mL sheep is prepared with the PBS solution that pH value is 8.2, a concentration of 10mM
Anti- mouse Anti-TNF-α liquid solution and 1mg/mL yellow fever virus recombinant antigen protein solution have been prepared being respectively charged into spray film instrument
10mL yellow fever virus recombinant antigen protein solution and 10mL people's IgM solution, start to spray film, nitrocellulose filter after spray moves into
It is taken out after dry 10h in 37 DEG C of thermostatic drying chambers, obtains the nitrocellulose filter that membrane aperture is 3 μm；
Step 4, the isoamyl alcohol solution that mass fraction is 1% is prepared, in the case where humidity is 75%, prepared by step 3
Nitrocellulose filter is soaked in the mixed solution of 100mL isoamyls alcohol solution and acetone, the body of isoamyl alcohol solution and acetone
Product is than being 5:1,40 DEG C are slowly heated to, 6min is kept, temperature is promoted to 65 DEG C later, keeps 5min, takes out cellulose nitrate
It is at least washed 3 times with deionized water after plain film, is then impregnated in the polyvinyl alcohol water solution that 60mL mass fractions are 5%
15min is preserved after drying at room temperature；
It being maintained under certain humidity, nitrocellulose filter is by absorbing vapor and acetone evaporated induces phase reversal, and third
Ketone induction time is maintained at 6min, and pore structure can tend to rule, then deionized water be used to remove remaining acetone and isoamyl
Alcohol consolidates nitrocellulose membrane structure.
Step 5, nitrocellulose filter is pasted onto PVC board upper surface, obtains detection diaphragm plate, on nitrocellulose filter successively
Equipped with detection line and control line, it is coated with yellow fever virus recombinant antigen at detection line, human IgM antibody is coated at control line, uses
Cutting machine is cut into the detection film item of strip by diaphragm plate is detected, with the monoclonal antibodies directed against human μ-chain of horseradish peroxidase-labeled and
Its substrate is assembled into yellow fever virus recombinant antigen kit jointly.
Preferably, in step (12), the additive amount of chloroform is the 1/5 of Trizol liquid product, the additive amount of water-saturated phenol
It is the 1/3 of Trizol liquid product, the additive amount of isopropanol is the 1/2 of Trizol liquid product, the first time additive amount of 75% ethyl alcohol
It it is 1.5 times of Trizol liquid product, second of additive amount of 75% ethyl alcohol is 1.2 times of Trizol liquid product.
Preferably, the response parameter of PCR is in step (13)：95 DEG C of pre-degeneration 5min, then 94 DEG C denaturation 30s, 57
DEG C annealing 40s, 72 DEG C extension 90s, totally 32 times cycle, last time recycle after 72 DEG C extension 7min.
Preferably, with mass/volume percentages, in step 5 substrate of horseradish peroxidase by 0.03% mistake
Hydrogen oxide and 0.07% diaminobenzidine composition.
Preferably, yellow fever virus recombinant antigen detection box further includes PVC board, further include being put into PVC board before step 5
20min is impregnated in treatment fluid, dry taking-up after 37 DEG C of thermostatic drying chambers, 10h is put into after immersion, treatment fluid is by a concentration of 1%
Bovine serum albumin, a concentration of 0.3% odium stearate, a concentration of 1% Tritonx-1000, a concentration of 0.1% 12
The filtrate of gained after alkyl dimethyl betaine, mixing filtering.
The present invention has significant technique effect as a result of above technical scheme：It need to keep certain wet when spraying film
Degree, humidity is too low, and static electricity gathered lotus is easy on nitrocellulose filter, and point film is susceptible to scatterplot, causes to occur when test hydrophobic
Spot；Humidity is excessively high, and capillarity is reinforced on nitrocellulose filter, and point film, which easily causes T lines, C lines broaden even spreads；To being made
Nitrocellulose filter post-processed, isoamyl alcohol can reduce the surface tension of solution and reduce electrostatic repulsion first, can be with
Nitrocellulose filter, is immersed in polyvinyl alcohol, can reduce albumen and exist by the significant wellability and stability for improving film later
Stability in solution will not lead to denaturation or the precipitation of protein, protein can be promoted in nitric acid since additive amount is less
Being adsorbed on cellulose membrane；Impregnation is carried out to sample pad, treatment fluid is penetrated into sample pad, then dried, and can avoid
Using complicated identification agent or the trouble of trace buffer solution sensitivity is improved to reduce sample difference.
The high specificity of yellow fever virus recombinant antigen that the preparation method of the present invention obtains, affinity is high, will not with it is other
Arthropod-borne virus occurs serum and intersects reaction.
Description of the drawings
Fig. 1 is that expression of the yellow fever virus recombinant plasmid in Escherichia coli uses 12%SDS-PAGE electrophoretic images.
Specific implementation mode
Present invention is further described in detail with embodiment below in conjunction with the accompanying drawings.
Embodiment 1
A kind of preparation method of yellow fever virus recombinant antigen, includes the following steps：
Step 1 chooses antigen fragment
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and is drawn in forward primer and reversely
The end of object 5 ' introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’。
Step 2, the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g
10min is centrifuged, sediment is collected, 5-10 times of volume Trizol liquid is added, after mixing, 5min is stood under the conditions of 4 DEG C, is then added
Chloroform sways 15s, take the first upper strata aqueous phase be added water-saturated phenol, sway 20s, take the second upper strata aqueous phase be added isopropanol, 4 DEG C
Under the conditions of place 10min, under the conditions of 4 DEG C, after 12000g centrifuges 10min, take and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water；
In step 2, the additive amount of chloroform is the 1/5 of Trizol liquid product, and the additive amount of water-saturated phenol is Trizol liquid
Long-pending 1/3, the additive amount of isopropanol are the 1/2 of Trizol liquid product, and the first time additive amount of 75% ethyl alcohol is Trizol liquid
Long-pending 1.5 times, second of additive amount of 75% ethyl alcohol are 1.2 times of Trizol liquid product.
Step 3, PCR amplification
Yellow fever virus RNA reverse transcriptions obtain cDNA, and reverse transcription step is：2 μ g, 10mmol/L dNTP of RNA, 1 μ L and
After 1 μ L of 500ng/ μ L OligodT heat 5min in 70 DEG C of water-baths, then 10 × Buffer, 2 μ L are added in ice bath 3min,
1 μ L of RNase inhibitor and reverse transcriptase (50unit/ μ L) 1 μ L react 60min after mixing under the conditions of 42 DEG C, then in 80 DEG C of items
5min is reacted under part, obtains cDNA；Then PCR amplification is carried out：Take 2 μ L, 10 × PCR buffer solutions of cDNA, 5 μ L, it is forward primer, anti-
To each 2 μ L of primer, 8 dNTP μ L, LA Taq 2U, moisturizing to 50 μ L of total volume carries out PCR amplification, and amplified fragments are purified, return
After receipts, -20 DEG C save backup；
The response parameter of PCR is in step 3：Then 95 DEG C of pre-degeneration 5min are denaturalized 30s, 57 DEG C of 40s that anneal, 72 for 94 DEG C
DEG C extend 90s, totally 32 times cycle, last time recycle after 72 DEG C extension 7min.
Step 4, the structure of prokaryotic expression carrier
Restricted digestion and company are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III
It connects, obtains recombinant plasmid；
It further include the identification to recombinant plasmid in step 4：Recombinant plasmid transformed Escherichia coli L1-Blue competent cells,
It is coated on the agar plate containing the ampicillins 100mg/mL, recombination is screened on the agar plate containing X-gal and IPTG
Plasmid and non-recombinant plasmid.
Step 5, the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, and the coli strain Top10F containing pET28a-17D exists
It is grown in LB culture solutions containing kanamycins, 1mM IPTG is added during protein growth and carry out induced expression, when induction
Between be 3h；12%SDS-PAGE electrophoretic analysis is carried out, as a result such as Fig. 1.
Yellow fever virus recombinant antigen detection box further includes PVC board, further includes that PVC board is put into treatment fluid before step 5
20min is impregnated, dry taking-up after 37 DEG C of thermostatic drying chambers, 10h is put into after immersion, treatment fluid is by a concentration of 1% cow's serum
Albumen, a concentration of 0.3% odium stearate, a concentration of 1% Tritonx-1000, a concentration of 0.1% dimethyl
The filtrate of gained after base glycine betaine, mixing filtering.
Embodiment 2
A kind of production method of yellow fever virus recombinant antigen kit, yellow fever virus recombinant antigen kit include PVC board,
Nitrocellulose filter, nitrocellulose filter are equipped with detection line and control line, and yellow fever virus recombinant antigen is coated in detection line, right
According to the anti-human μ chain antibodies and substrate for being coated with IgM antibody and horseradish peroxidase-labeled on line, include the following steps：
Step 1, the preparation of yellow fever virus recombinant antigen
(11), antigen fragment is chosen
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and is drawn in forward primer and reversely
The end of object 5 ' introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’；
(12), the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g
10min is centrifuged, sediment is collected, 5-10 times of volume Trizol liquid is added, after mixing, 5min is stood under the conditions of 4 DEG C, is then added
Chloroform sways 15s, take the first upper strata aqueous phase be added water-saturated phenol, sway 20s, take the second upper strata aqueous phase be added isopropanol, 4 DEG C
Under the conditions of place 10min, under the conditions of 4 DEG C, after 12000g centrifuges 10min, take and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water.
In step (12), the additive amount of chloroform is the 1/5 of Trizol liquid product, and the additive amount of water-saturated phenol is Trizol liquid
The 1/3 of volume, the additive amount of isopropanol are the 1/2 of Trizol liquid product, and the first time additive amount of 75% ethyl alcohol is Trizol liquid
1.5 times of volume, second of additive amount of 75% ethyl alcohol are 1.2 times of Trizol liquid product.
(13), PCR amplification
Reverse transcription is carried out to yellow fever virus RNA with conventional method, cDNA is obtained, then carries out PCR amplification：Take 2 μ of cDNA
5 μ L of L, 10 × PCR buffer solution, each 2 μ L of forward primer, reverse primer, 8 dNTP μ L, LATaq 2U, moisturizing to 50 μ L of total volume
PCR amplification is carried out, amplified fragments are purified, after recycling, and -20 DEG C save backup；
The response parameter of PCR is in step (13)：Then 95 DEG C of pre-degeneration 5min are denaturalized 30s, 57 DEG C of annealing 40s for 94 DEG C,
72 DEG C of extension 90s, totally 32 cycles, extend 7min for 72 DEG C after last time recycles.
(14), the structure of prokaryotic expression carrier
Restricted digestion and company are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III
It connects, obtains recombinant plasmid.
(15), the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, and the coli strain Top10F containing pET28a-17D exists
It is grown in LB culture solutions containing kanamycins, 1mM IPTG is added during protein growth and carry out induced expression, when induction
Between be 3h；Carry out 12%SDS-PAGE electrophoretic analysis.
Step 2, the protein that step 1 obtains is purified：The coli strain Top10F cultures of pET28a-17D
Liquid is seeded in the LB culture solutions containing kanamycins, 37 DEG C of cultures to OD450=0.6, IPTG shaken cultivations at 37 DEG C are added
5h, 12000g centrifuging and taking bacterial precipitation object, bacterial precipitation object, using affinity chromatography column purification, obtain yellow fever after PBS is washed
Malicious recombinant antigen protein；
Step 3, in the case where humidity is 45~65%, 1mg/mL sheep is prepared with the PBS solution that pH value is 8.2, a concentration of 10mM
Anti- mouse Anti-TNF-α liquid solution and 1mg/mL yellow fever virus recombinant antigen protein solution have been prepared being respectively charged into spray film instrument
10mL yellow fever virus recombinant antigen protein solution and 10mL people's IgM solution, start to spray film, nitrocellulose filter after spray moves into
It is taken out after dry 10h in 37 DEG C of thermostatic drying chambers, obtains the nitrocellulose filter that membrane aperture is 3 μm；
Step 4, the isoamyl alcohol solution that mass fraction is 1% is prepared, in the case where humidity is 75%, prepared by step 3
Nitrocellulose filter is soaked in the mixed solution of 100mL isoamyls alcohol solution and acetone, the body of isoamyl alcohol solution and acetone
Product is than being 5:1,40 DEG C are slowly heated to, 6min is kept, temperature is promoted to 65 DEG C later, keeps 5min, takes out cellulose nitrate
It is at least washed 3 times with deionized water after plain film, is then impregnated in the polyvinyl alcohol water solution that 60mL mass fractions are 5%
15min is preserved after drying at room temperature；
It being maintained under certain humidity, nitrocellulose filter is by absorbing vapor and acetone evaporated induces phase reversal, and third
Ketone induction time is maintained at 6min, and pore structure can tend to rule, then deionized water be used to remove remaining acetone and isoamyl
Alcohol consolidates nitrocellulose membrane structure.
Step 5, nitrocellulose filter is pasted onto PVC board upper surface, obtains detection diaphragm plate, on nitrocellulose filter successively
Equipped with detection line and control line, it is coated with yellow fever virus recombinant antigen at detection line, human IgM antibody is coated at control line, uses
Cutting machine is cut into the detection film item of strip by diaphragm plate is detected, with the monoclonal antibodies directed against human μ-chain of horseradish peroxidase-labeled and
Its substrate is assembled into yellow fever virus recombinant antigen kit jointly.
With mass/volume percentages, in step 5 substrate of horseradish peroxidase by 0.03% hydrogen peroxide and
0.07% diaminobenzidine composition.
Certain humidity need to be kept when spraying film, humidity is too low, and static electricity gathered lotus is easy on nitrocellulose filter, and point film is easy
There is scatterplot, leads to hydrophobic spot occur when test；Humidity is excessively high, and capillarity is reinforced on nitrocellulose filter, and point film is easy to draw
Play nature controlling line, detection line broadens and even spreads；Nitrocellulose filter obtained is post-processed, isoamyl alcohol can be reduced first
The surface tension and reduction electrostatic repulsion of solution, can significantly improve the wellability and stability of film, later by cellulose nitrate
Plain film is immersed in polyvinyl alcohol, can reduce the stability of albumen in the solution, since additive amount is less, will not lead to protein
Denaturation or precipitation, protein being adsorbed on nitrocellulose filter can be promoted；Impregnation, processing are carried out to sample pad
Liquid penetrates into sample pad, then dries, and the trouble using complicated identification agent or trace buffer solution is can avoid, to reduce sample
Difference improves sensitivity.
ELISA reactions are carried out to the kit that the present embodiment is prepared, evaluate sensitivity and the yellow fever of the kit
The binding specificity of malicious recombinant antigen, reaction condition are as follows：
1) sample carries out 1 using 0.01mol/L PBS (pH 7.2) -0.5%Tween-20:50 dilutions；
2) it closes, confining liquid is used as using 5% skim milk (preparation of 0.01mol/L PBS buffer solution), in 37 DEG C of incubations
45min；
3) Serum incubation：Detection film item needed for taking out is put into and incubates in slot, number.It is separately added into incubating slot
1.5mL diluted samples, 20min is incubated in 37 DEG C on shaking table；
4) it cleans：Tank liquid is sucked, 1.5mL 0.01mol/L PBS (pH 7.2) -0.5%Tween- is used on shaking table
20 cleaning detection film items 3 times, each 6min；
100 μ L (are diluted, 1 with -5% milk of 0.01mol/LPBS (pH 7.2) after washing:50) addition 3h, 37 DEG C, after washing,
The anti-ox IgM antibody dilution 1 of 100 μ L horseradish peroxidase-labeleds:1500 in -5% milk of 0.01mol/L PBS (pH 7.2)
It is added into 1h, 37 DEG C；
5) enzyme conjugate：1.5mL is added in incubating slot, and diluted enzyme conjugates (uses horseradish peroxidase mark
The anti-human IgM specific fragments μ chain monoclonal antibodies of note are detection antibody), 20min is incubated on shaking table in 37 DEG C.
6) it cleans：Tank liquid is sucked, detection film item 3 is cleaned with 1.5mL 0.01mol/L PBS buffer solution on shaking table
It is secondary, each 6min.
7) substrate incubates：1.5mL substrate solutions are separately added into incubating slot, in incubating 10min on 37 DEG C of shaking tables.
8) it terminates：Tank liquid is sucked, detects film item wash with distilled water 3 times, each 3min.
As a result judge：Detection film item is placed in result judgement template, judging result after air-drying.Any protein targets mark
Existing specific band can determine whether as the positive, can auxiliary diagnosis yellow fever.
Selection contains Syndebis, Xi Menlike, Ma Yaluo, Ross river, 1~2 type of Dengue and Japanese Type-B encephalitis blood
Clearly, yellow warm antibody rabbit anteserum, normal rabbit serum and normal mouse serum are used as and refer to sample, yellow fever virus recombinant antigen pH value
Different multiples are diluted for the PBS solution of 8.2, a concentration of 10mmol/L.As shown in table 1, yellow fever virus recombinant antigen of the invention
Kit and its yellow fever virus recombinant antigen protein of use there is good specificity, with above-mentioned reference sample without intersecting
Reaction；As shown in table 2, the inspection range for the kit that the present invention obtains is the 100~12800 of extension rate.
Table 1 dilutes the specific detection result of 200 times of yellow fever virus recombinant antigen
The testing result of 2 sensitivity of table
| Extension rate | Testing result | Extension rate | Testing result |
| 100 | It is positive | 3200 | It is positive |
| 200 | It is positive | 6400 | It is positive |
| 400 | It is positive | 12800 | It is negative |
| 800 | It is positive | 25600 | It is negative |
| 1600 | It is positive | 51200 | It is negative |
Therefore, the high specificity for the yellow fever virus recombinant antigen that preparation method of the invention obtains, affinity is high, Bu Huiyu
Other arthropod-borne viruses occur serum and intersect reaction, the kit high sensitivity that the present invention is prepared.
In short, the foregoing is merely presently preferred embodiments of the present invention, it is all according to impartial made by scope of the present invention patent
Variation and modification, should all belong to the covering scope of patent of the present invention.
Claims (9)
1. a kind of preparation method of yellow fever virus recombinant antigen, which is characterized in that include the following steps：
Step 1 chooses antigen fragment
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and in forward primer and reverse primer 5 '
End introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’；
Step 2, the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g centrifugations
10min collects sediment, and 5-10 times of volume Trizol liquid is added, and after mixing, 5min is stood under the conditions of 4 DEG C, chlorine is then added
It is imitative, 15s is swayed, takes the first upper strata aqueous phase that water-saturated phenol is added, sways 20s, takes the second upper strata aqueous phase that isopropanol, 4 DEG C of items are added
It places 10min under part, under the conditions of 4 DEG C, after 12000g centrifuges 10min, takes and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water；
Step 3, PCR amplification
Yellow fever virus RNA reverse transcriptions obtain cDNA, then carry out PCR amplification：5 μ L of cDNA2 μ L, 10 × PCR buffer solution are taken, it is positive
Each 2 μ L of primer, reverse primer, 8 dNTP μ L, LA Taq 2U, moisturizing to 50 μ L of total volume carry out PCR amplification, amplified fragments warp
After purifying, recycling, -20 DEG C save backup；
Step 4, the structure of prokaryotic expression carrier
Restricted digestion and connection are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III, obtained
To recombinant plasmid；
Step 5, the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, the coli strain Top10F containing pET28a-17D containing
It is grown in the LB culture solutions of kanamycins, 1mmol/L IPTG is added during protein growth and carry out induced expression, when induction
Between be 3h；Carry out 12%SDS-PAGE electrophoretic analysis.
2. a kind of preparation method of yellow fever virus recombinant antigen according to claim 1, it is characterised in that：In step 2,
The additive amount of chloroform is the 1/5 of Trizol liquid product, and the additive amount of water-saturated phenol is the 1/3 of Trizol liquid product, isopropanol
Additive amount is the 1/2 of Trizol liquid product, and the first time additive amount of 75% ethyl alcohol is 1.5 times of Trizol liquid product, 75% second
Second of additive amount of alcohol is 1.2 times of Trizol liquid product.
3. a kind of preparation method of yellow fever virus recombinant antigen according to claim 1, it is characterised in that：In step 3
The response parameter of PCR is：95 DEG C of pre-degeneration 5min, then 94 DEG C denaturation 30s, 57 DEG C annealing 40s, 72 DEG C extension 90s, totally 32 times
Cycle, 72 DEG C of extension 7min after last time recycles.
4. a kind of preparation method of yellow fever virus recombinant antigen according to claim 1, it is characterised in that：In step 4 also
It include the identification to recombinant plasmid：Recombinant plasmid transformed Escherichia coli L1-Blue competent cells, are coated on containing 100mg/mL
On the agar plate of ampicillin, recombinant plasmid and non-recombinant plasmid are screened on the agar plate containing X-gal and IPTG.
5. a kind of production method of yellow fever virus recombinant antigen kit, yellow fever virus recombinant antigen kit include PVC board, nitre
Acid cellulose film, nitrocellulose filter are equipped with detection line and control line, yellow fever virus recombinant antigen, control are coated in detection line
The anti-human μ chain antibodies and substrate of IgM antibody and horseradish peroxidase-labeled are coated on line, which is characterized in that including following
Step：
Step 1, the preparation of yellow fever virus recombinant antigen
(11), antigen fragment is chosen
The higher virus antigen epitope segment design primer of score is predicted according to antigenicity, and in forward primer and reverse primer 5 '
End introduces BamH I and Hind III digestions site, forward primer respectively：5’-
TAGGAATTCTCAGCTTTGACACTCAAGGGGACATCC-3 ', reverse primer：5'-
GACAAGCTTCTATTACTTTCCTATTGAGCTTCCCT-3’；
(12), the extraction of yellow fever virus RNA
Yellow fever virus 17D strain collects the supernatant of culture after BHK-21 cell culture 3d, under the conditions of 4 DEG C, 10000g centrifugations
10min collects sediment, and 5-10 times of volume Trizol liquid is added, and after mixing, 5min is stood under the conditions of 4 DEG C, chlorine is then added
It is imitative, 15s is swayed, takes the first upper strata aqueous phase that water-saturated phenol is added, sways 20s, takes the second upper strata aqueous phase that isopropanol, 4 DEG C of items are added
It places 10min under part, under the conditions of 4 DEG C, after 12000g centrifuges 10min, takes and precipitate for the first time, 75% ethyl alcohol, 4 DEG C of conditions are added
Under, 8000g centrifuges 5min, then takes second of precipitation 75% ethyl alcohol of addition, and under the conditions of 4 DEG C, 8000g centrifuges 5min, obtains yellow heat
Viral RNA, it is after drying 6min at room temperature, yellow fever virus RNA is soluble in water；
(13), PCR amplification
Yellow fever virus RNA reverse transcriptions obtain cDNA, then carry out PCR amplification：5 μ L of cDNA2 μ L, 10 × PCR buffer solution are taken, it is positive
Each 2 μ L of primer, reverse primer, 8 dNTP μ L, LA Taq 2U, moisturizing to 50 μ L of total volume carry out PCR amplification, amplified fragments warp
After purifying, recycling, -20 DEG C save backup；
(14), the structure of prokaryotic expression carrier
Restricted digestion and connection are carried out to the amplified fragments after pET28a carriers and recycling by BamH I and Hind III, obtained
To recombinant plasmid；
(15), the induced expression of recombinant plasmid
Set temperature is 37 DEG C, mixing speed 250rpm, the coli strain Top10F containing pET28a-17D containing
It is grown in the LB culture solutions of kanamycins, 1mM IPTG is added during protein growth and carry out induced expression, induction time is
3h；Carry out 12%SDS-PAGE electrophoretic analysis；
Step 2, the protein that step 1 obtains is purified：The coli strain Top10F culture solutions of pET28a-17D connect
Kind is in the LB culture solutions containing kanamycins, 37 DEG C of cultures to OD450=0.6, IPTG shaken cultivation 5h at 37 DEG C are added,
12000g centrifuging and taking bacterial precipitation objects, bacterial precipitation object, using affinity chromatography column purification, obtain yellow fever virus after PBS is washed
Recombinant antigen protein；
Step 3, in the case where humidity is 45~65%, 1mg/mL sheep anti mouses are prepared with the PBS solution that pH value is 8.2, a concentration of 10mM
Anti-TNF-α liquid solution and 1mg/mL yellow fever virus recombinant antigen protein solution, it is prepared by being respectively charged into spray film instrument
10mL yellow fever virus recombinant antigen protein solution and 10mL people's IgM solution start to spray film, and the nitrocellulose filter after spray moves into 37
It is taken out after dry 10h in DEG C thermostatic drying chamber, obtains the nitrocellulose filter that membrane aperture is 3 μm；
Step 4, the isoamyl alcohol solution that mass fraction is 1% is prepared, in the case where humidity is 75%, by the nitric acid prepared by step 3
Cellulose membrane is soaked in the mixed solution of 100mL isoamyls alcohol solution and acetone, the volume ratio of isoamyl alcohol solution and acetone
It is 5:1,40 DEG C are slowly heated to, 6min is kept, temperature is promoted to 65 DEG C later, keeps 5min, takes out nitrocellulose filter
It is at least washed 3 times with deionized water afterwards, then impregnates 15min, room in the polyvinyl alcohol water solution that 60mL mass fractions are 5%
It is preserved after temperature is dry；
Step 5, nitrocellulose filter is pasted onto PVC board upper surface, obtains detection diaphragm plate, is equipped with successively on nitrocellulose filter
Detection line and control line are coated with yellow fever virus recombinant antigen at detection line, human IgM antibody are coated at control line, with slitting
Machine is cut into the detection film item of strip, the monoclonal antibodies directed against human μ-chain with horseradish peroxidase-labeled and its bottom by diaphragm plate is detected
Object is assembled into yellow fever virus recombinant antigen kit jointly.
6. a kind of production method of yellow fever virus recombinant antigen kit according to claim 5, it is characterised in that：Step
(12) in, the additive amount of chloroform is the 1/5 of Trizol liquid product, and the additive amount of water-saturated phenol is the 1/3 of Trizol liquid product, different
The additive amount of propyl alcohol is the 1/2 of Trizol liquid product, and the first time additive amount of 75% ethyl alcohol is 1.5 times of Trizol liquid product,
Second of additive amount of 75% ethyl alcohol is 1.2 times of Trizol liquid product.
7. a kind of production method of yellow fever virus recombinant antigen kit according to claim 5, it is characterised in that：Step
(13) response parameter of PCR is in：Then 95 DEG C of pre-degeneration 5min are denaturalized 30s, 57 DEG C of annealing 40s, 72 DEG C of extension 90s for 94 DEG C,
Totally 32 cycles, 72 DEG C of extension 7min after last time recycles.
8. a kind of production method of yellow fever virus recombinant antigen kit according to claim 5, it is characterised in that：With matter
Amount/volume percentage, the substrate of horseradish peroxidase is by 0.03% hydrogen peroxide and 0.07% diamino in step 5
Benzidine forms.
9. a kind of production method of yellow fever virus recombinant antigen kit according to claim 5, it is characterised in that：Yellow heat
Virus recombinant antigen detection box further includes PVC board, further includes that PVC board is put into treatment fluid to impregnate 20min before step 5, leaching
Be put into after bubble after 37 DEG C of thermostatic drying chambers, 10h it is dry take out, treatment fluid is by a concentration of 1% bovine serum albumin, a concentration of
0.3% odium stearate, a concentration of 0.1% dodecyldimethylammonium hydroxide inner salt, is mixed a concentration of 1% Tritonx-1000
The filtrate of gained after even filtering.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN109799334A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | Fluorescent microsphere joint-detection device of cardiac marker and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435732A (en) * | 2011-09-19 | 2012-05-02 | 厦门市仙岳医院 | Toxoplasma IgM antibody immunoblotting kit and preparation method thereof |
| CN102827260A (en) * | 2012-08-31 | 2012-12-19 | 任瑞文 | Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein |
| CN107201370A (en) * | 2016-03-17 | 2017-09-26 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of DNA molecular and recombinant virus and their preparation method and purposes |
-
2018
- 2018-03-29 CN CN201810270771.0A patent/CN108546706A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435732A (en) * | 2011-09-19 | 2012-05-02 | 厦门市仙岳医院 | Toxoplasma IgM antibody immunoblotting kit and preparation method thereof |
| CN102827260A (en) * | 2012-08-31 | 2012-12-19 | 任瑞文 | Recombinant antigen protein for detecting yellow fever virus antibody, kit and application of recombinant antigen protein |
| CN107201370A (en) * | 2016-03-17 | 2017-09-26 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of DNA molecular and recombinant virus and their preparation method and purposes |
Non-Patent Citations (3)
| Title |
|---|
| ＨILL等: "Mａｐｐｉｎｇ ｔｈｅ ｒｉｓｋ ｏｆ ｙｅｌｌｏｗ ｆｅｖｅｒ ｉｎｆｅｃｔｉｏｎ", 《ＣＵＲＲ ＩＮＦＥＣＴ ＤＩＳ ＲＥＰ》 * |
| 侯爵等: "黄热病毒的基因组及其蛋白研究进展", 《病毒学报》 * |
| 胡文龙等: "黄热病毒特异性抗原片段的筛选与鉴定", 《中国预防医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN109799334A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | Fluorescent microsphere joint-detection device of cardiac marker and preparation method thereof |
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