CN106290866A - The indirect ELISA detection method of bovine papilloma virus 13 type antibody - Google Patents
The indirect ELISA detection method of bovine papilloma virus 13 type antibody Download PDFInfo
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Abstract
The invention discloses the indirect ELISA detection method of a kind of bovine papilloma virus 13 type antibody, step includes: step 1, structure recombinant expression plasmid pET28a L1, with BPV13 genome as template, for BPV13 capsid protein L 1 gene sequential design specific primer；Obtain purpose fragment by PCR reaction, to prokaryotic expression carrier pET28a and purpose fragment double digestion simultaneously, after connection, obtain recombinant expression plasmid pET28a L1；Step 2, destination protein is expressed and purification；Step 3, set up the indirect ELISA detection system of BPV13 antibody, for BPV13L1 albumen sample yin and yang attribute marginal value, judge.The method of the present invention, step is simple, and accuracy rate is high.
Description
Technical field
The invention belongs to animal virus detection technique field, relate to the indirect of a kind of bovine papilloma virus 13 type antibody
ELISA detection method.
Background technology
Bovine papilloma (bovine papillomatosis, BP) is to be caused tumor to send out by bovine papilloma virus (BPV)
The chronic hyperplastic disease of life, all has popular all over the world in vaccary, Chinese, Japanese, Brazilian, Canadian, Australian,
Turkey, Britain, the U.S. and India all have been reported that.This disease is the common benign neoplastic disease of milch cow, multiple is born in body surface, mucosa
Deng, this disease can damage animal's leather quality, reduces animal body constitution.BPV may result in the benign and malignant neoplastic lesion of many animals, good
Property tumor can disappear in the ordinary course of things voluntarily, but can cause canceration when environmental factors and inherited genetic factors synergism, and then
Cause animal dead, ultimately cause the economic loss that meat, milk industry and leather-like industry are serious, and when disease betides genitals
The reproductive function of bull and cow can be damaged, reduce production performance, cause damage.
It is not difficult to make preliminary examining to bovine papilloma disease according to modes such as clinical manifestation, epidemiology, pathological observations
Disconnected；But bovine papilloma is in addition to occurring at skin mucosa, also occurs at the internal organs such as digestive tract, bladder, also have some hidden
The cattle of sexy dye or atypical case, then lack effective diagnostic method for these, and making a definite diagnosis must be by laboratory diagnosis.
But owing to BPV typing is more, the most variant to the specificity of tissue, therefore, applied molecular biology technology carries out type to BPV
Qualification is the most necessary.Specifically include the inspection methods such as Electronic Speculum, immunofluorescence, hemagglutination test, can obtain in conjunction with facing the data of examining
To making a definite diagnosis.But the shortcoming of these methods is that complex operation, expensive, susceptiveness and specificity are the highest.Enzyme-linked Immunosorbent Assay
Test (ELISA) is easy to operate because of it, quickly the advantage such as sensitivity, high specificity be widely used in many pathogen antigens or
The detection of antibody.
At present, the prevention and controls to bovine papilloma has vaccine prevention, treatment by Chinese herbs, surgical removal, CO2Laser is cut
Remove, burn and iron treatment and tumor body liquid nitrogen freezing carries out the report of the prevention to bovine papilloma and treatment.Develop now
Go out the vaccine for BPV2 and BPV4, body can have been protected to exempt to be infected by the virus, produce virucidin simultaneously.But,
These vaccines have certain specificity, and body can only be made from the virus infection of syngeneic type.Therefore, a kind of energy it is badly in need of fast
Speed detects and evaluates the serodiagnosis of antibody horizontal after vaccine immunity effectively.
Summary of the invention
It is an object of the invention to provide the indirect ELISA detection method of a kind of bovine papilloma virus 13 type antibody, solve
Prior art during detection bovine papilloma virus 13, complex operation, testing cost are high, susceptiveness and specificity all
Undesirable problem.
The technical solution used in the present invention is, the indirect ELISA detection method of a kind of bovine papilloma virus 13 type antibody,
Implement according to following steps:
Step 1, structure recombinant expression plasmid pET28a-L1
With BPV13 genome as template, for BPV13 capsid protein L 1 gene sequential design specific primer；Pass through PCR
Reaction obtains purpose fragment, to prokaryotic expression carrier pET28a and purpose fragment double digestion simultaneously, obtains recombinant expressed after connection
Plasmid pET28a-L1；
Step 2, destination protein is expressed and purification；
Step 3, set up the indirect ELISA detection system of BPV13 antibody, critical for BPV13 L1 albumen sample yin and yang attribute
Value, judges, obtains final testing result.
The invention has the beneficial effects as follows, with bovine papilloma virus 13 type (bovine papillomavirus type
13, BPV13) L1 gene expression albumen is as antigen, constructs recombinant expression plasmid pET28a-L1, then enters destination protein
Row is expressed and purification, finally sets up the indirect ELISA detection system of BPV13 antibody, for BPV13 L1 albumen sample yin and yang attribute
Marginal value judges, obtains final testing result；The method is applicable to detect on a large scale the indirect ELISA of BPV13 antibody
Differentiate, it is possible to the discriminating for BPV13 antibody provides a kind of low cost, efficiently detection technique means.
Accompanying drawing explanation
Fig. 1 is the L1 gene PCR amplification electrophoretogram in the inventive method；
Fig. 2 is that the recombinant expression plasmid pET28a-L1 double digestion in the inventive method identifies electrophoretogram；
Fig. 3 is the His-L1 fusion protein soluble analysis electrophoretogram in the inventive method；
Fig. 4 is the His-L1 expressing fusion protein purification electrophoretogram in the inventive method.
Detailed description of the invention
The present invention is described in detail with detailed description of the invention below in conjunction with the accompanying drawings.
The indirect ELISA detection method of the bovine papilloma virus 13 type antibody of the present invention, implements according to following steps:
Step 1, structure recombinant expression plasmid pET28a-L1
With BPV13 genome as template, for BPV13 capsid protein L 1 gene sequential design specific primer, it is shown in Table 1,
Table 1, the expression formula of specific primer
In table 1, single file underscore part is the restriction enzyme site of BamH I and Hind III double digestion, wave underscore part
Being that 2 kinds of primers 5 ' hold complementary series, duplicate rows underscore part is the base after sudden change；
Obtain purpose fragment by PCR reaction, to prokaryotic expression carrier pET28a and purpose fragment double digestion simultaneously, connect
After obtain recombinant expression plasmid pET28a-L1.
Step 2, destination protein is expressed and purification
Positive recombination bacillus coli BL21 (DE3) is inoculated in the LB fluid medium containing kanamycin and carries out IPTG and lure
Lead expression, under the conditions of IPTG concentration is 1mM, after inducing 6 hours, collect the positive recombination bacillus coli BL21 of great expression
And ultrasonic degradation (DE3), dissolving inclusion body and with membrane filtration with 8M carbamide, employing nickel ion affinity chromatograph method is to expression
His-L1 fusion protein is purified, and obtains the His-L1 fusion protein of purification.
Step 3, set up the indirect ELISA detection system of BPV13 antibody
Using the His-L1 fusion protein of purification as envelope antigen coated elisa plate, determine that His-L1 fusion protein is coated dense
Degree is 92.5 μ g/mL；Antigen coated condition be 4 DEG C overnight；5% defatted milk powder is used to close, 1 hour off-period；Serum is not
Dilution, 90 minutes action time；The anti-concentration of HRP-protein A IgG bis-is 1:8000,30 minutes action time；Often
Under temperature, 20 minutes substrate lucifuge response time；
For BPV13 L1 albumen sample yin and yang attribute marginal value, judge:
When sample OD450nm >=0.329, it is determined that for the positive；During sample OD450nm ＜ 0.264, it is determined that for feminine gender；It is situated between
It is judged in therebetween suspicious, obtains final testing result.
Finding when verifying the specificity of this ELISA detection method, BPV13 does not sends out with IBRV, FMDV, BVDV positive serum
Raw cross reaction, illustrates that specificity is preferable.In batch, the repetition result of the test coefficient of variation is between 1.734%～7.769%, explanation
Same sample degree of variation in a collection of test is less, has good batch interior repeatability.Result of the test variation is repeated between Pi
Coefficient, between 1.589%～10.563%, shows that same sample degree of variation in different batches is tested is less, has preferably
Repeatability.
Embodiment
Step 1, the clone of bovine papilloma virus 13 type L1 gene and the structure of prokaryotic expression carrier
According to the BPV13 Strain complete genome sequence of report in GenBanK, (Hainan Province tropical animal breeds and epidemic disease research
Key lab submits to, accession number: KM258443), utilize DNAMAN software design primer, the ORF district of amplification L1 gene, for increasing
Add the expression of L1 albumen, its codon is optimized, further according to the primers after optimizing.The present embodiment is used for expanding
The specific primer increasing L1 gene sees the above table 1.
By the L1 specificity genes of interest that Overlap extension PCR amplification size is 1494bp, see Fig. 1, in Fig. 1 from left to right
Indicate respectively: M refers to that D2000DNA Marker, sequence number 1 refer to L1 gene PCR product.
By reclaim the L1 gene of purification and prokaryotic expression carrier pET28a (+) carry out BamH I and Hind III double digestion simultaneously,
Reclaim kits with DNA glue and reclaim L1 gene and linearized vector, then the two is overnight connected at 4 DEG C, build restructuring table
Reaching plasmid pET28a-L1, enzyme action is identified and is seen Fig. 2, from left to right indicates respectively: M1 refers to λ Hind III DNA in Fig. 2
Marker, sequence number 1 refers to that recombiant plasmid pET28a-L1, sequence number 2 refer to that recombiant plasmid pET28a-L1 is through BamH I and Hind III couple
Digestion products, M2 refers to D2000DNA Marker.
Step 2, the expression of His-L1 fusion protein and purification
Recombinant expression plasmid pET28a-L1 is converted BL21 (DE3) competent cell, and picking positive recombinant bacterium is inoculated in and contains
In the LB fluid medium of kanamycin, 37 DEG C, rotating speed overnight shake cultivation under conditions of 200r/ minute, after amplification culture, when
When bacterium solution OD600 reaches 0.5, the IPTG adding final concentration of 1mM carries out abduction delivering.The centrifugal host collecting great expression
Bacterium, suspension bacterial sediment, the suspended bacterial of final concentration of for lysozyme 0.1g/L is placed in ice bath and does ultrasonication, ultrasonic 3
Second, intermittently 9 seconds, ultrasonic 5 minutes altogether, wave amplitude 30%；When ultrasonic bacterium solution is limpid, 4 DEG C, rotating speed is under conditions of 12000r/ minute
Centrifugal 25 minutes, collecting upper cleer and peaceful precipitation respectively, sampling carries out SDS-PAGE electrophoresis, determines that expression product is with soluble form
Or inclusion bodies exists.
Result shows, His-L1 fusion protein is present in inclusion body (precipitation), sees Fig. 3, from left to right indicates and divide in Fig. 3
Not: M1 refers to that protein standard, sequence number 1 refer to that recombiant plasmid pET28a-L1 does not induces thalline expression product, sequence
Numbers 2 refer to that recombiant plasmid pET28a-L1 induces thalline expression product, and sequence number 3 refers to supernatant after expression product ultrasonic Treatment, sequence
Numbers 4 refer to expression product ultrasonic Treatment postprecipitation.
After the precipitation ultrasonic degradation collected, dissolve inclusion body and with 0.45 μm membrane filtration with 8M carbamide, with nickel ion parent
With chromatography purification recombiant protein, being identified by SDS-PAGE, obtaining recombiant protein size is 60kDa, sees Fig. 4, in Fig. 4 from left to right
Indicate respectively: M1 refers to that protein standard, sequence number 1 refer to that recombiant plasmid pET28a-L1 does not induces thalline to express
Product, sequence number 2 refers to supernatant after recombiant plasmid pET28a-L1 induction thalline carrying out ultrasonic bacteria breaking, and sequence number 3 refers to purified product.
Step 3, the determination of BPV13 antibody test indirect ELISA optimal detection condition and specificity, sensitivity and repetition
Property experiment
The fundamental procedure of indirect ELISA detection includes following little step:
1) it is coated: His-L1 fusion protein good for purification is diluted to certain concentration with being coated liquid, is coated on ELISA Plate
On, 100 μ L/ holes, 4 DEG C of overnight incubation；
2) wash plate: discard and be coated liquid, wash 3 times with PBST cleaning mixture, each 3 minutes, pat dry；
3) close: add the confining liquid containing 5% defatted milk powder, 100 μ L/ holes, close 2 hours at 37 DEG C；
4) testing sample: serum-dilution to be checked becomes finite concentration, each sample are repeated twice, 100 μ L/ holes, the most every
Block ELISA Plate sets blank and yin and yang attribute comparison, hatches 1 hour for 37 DEG C, and mode of washing is ibid；
5) ELIAS secondary antibody: the protein A (HRP-SPA) of horseradish peroxidase-labeled is diluted to certain concentration, 100 μ
L/ hole, hatches 1 hour at 37 DEG C, and mode of washing is ibid；
6) colour developing: every hole adds the TMB nitrite ion of 100 μ L, and vibration shook up, 37 DEG C of lucifuge effects 10 minutes；
7) terminate: every hole adds the 2mol/L sulphuric acid stop buffer of 100 μ L, carry out terminating reaction；
8) reading: measure the OD value at 450nm wavelength by microplate reader.
Above 8 little steps are that the refinement to aforesaid step 3 supplements.
Checking:
1, Checkerboard titration method is used to select antigen and the working concentration of serum
With being coated liquid by the recombinant protein antigen of purification successively according to 1:50,1:100,1:200,1:400,1:800 five
Concentraton gradient is diluted, and each dilution factor 100 μ L/ hole is coated, and 4 DEG C overnight.Washing pats dry, and closes with containing 5% defatted milk powder
Fluid-tight closes 2 hours.The most again washing pat dry, bovine papilloma virus yin and yang attribute serum is made respectively 1:20,1:40,1:80,1:
Former serum five dilution gradient composition square formation before 160 times of dilutions, with dilution, it is positive and negative that each antigen diluent degree respectively adds 1 hole
Comparison, 100 μ L/ holes, 37 DEG C are reacted 1 hour.After washing pats dry, add, according to square formation, the horseradish peroxidase that 1:5000 has diluted
The protein A (HRP-SPA) of enzyme labelling resists as two, 100 μ L/ holes, and 37 DEG C are reacted 1 hour, and washing pats dry addition TMB nitrite ion
100 μ L/ holes, shake up, and 37 DEG C of lucifuges develop the color 10 minutes, are subsequently adding 2mol/L H2SO4Reaction is terminated at 100 μ L stop buffers,
Read the value of OD450nm on enzyme connection detector, the results are shown in Table 2.
The suitableeest determination being coated concentration and serum diluting multiple of table 2, recombinant protein antigen
Note :+represent positive serum OD450nm value ,-represent negative serum OD450nm value.
2, the determination of optimal off-period
His-L1 fusion protein is diluted to optimal antigen coated concentration coated elisa plate, adds confining liquid, 37 DEG C of difference
Act on 30,60,90,120 minutes.Adding the standard BP V yin and yang attribute serum of excess, 37 DEG C are reacted 1 hour, and washing adds after patting dry
The ELIAS secondary antibody of 1:5000 dilution, 37 DEG C are reacted 1 hour, and washing adds tmb substrate reactant liquor 100 μ L/ hole, colour developing 10 after patting dry
Minute, every hole adds 2mol/L H2SO4Reaction is terminated at 100 μ L stop buffers.Enzyme connection detector reads OD450nm value, calculates
P/N value.Using maximum for P/N value one group as the Best Times closed, the results are shown in Table 3.
Table 3, optimal off-period
3, the determination of optimal serum action time
According to optimum condition determined above, according to ELISA program, add standard BP V yin and yang attribute serum.Make respectively for 37 DEG C
With 30 minutes, 60 minutes, 90 minutes, 120 minutes, carry out ELISA detection.Measure OD450nm value after termination, calculate P/N value.Will
Using maximum one group of P/N value as the Best Times of serum effect, the results are shown in Table 4.
Table 4, the OD450nm value of serum differential responses time to be checked
4, the determination of optimal ELIAS secondary antibody concentration
According to optimum condition determined above, according to ELISA program, the protein A (HRP-of horseradish peroxidase-labeled
SPA) ELIAS secondary antibody dilutes according to 1:3000,1:5000,1:8000, measures OD450nm value, calculate P/N value after termination.By P/N
One group of value maximum, as the best use of concentration of ELIAS secondary antibody, the results are shown in Table 5.
Table 5, the determination of ELIAS secondary antibody optium concentration
5, the determination of optimal ELIAS secondary antibody action time
According to optimum condition determined above, add ELIAS secondary antibody according to ELISA program.37 DEG C act on 30 respectively, 60,
90, within 120 minutes, ELISA detection is carried out.Terminate reaction, measure OD450nm value, calculate P/N value.The one group work maximum by P/N value
For the best use of time of ELIAS secondary antibody, it is shown in Table 6.
Table 6, the determination of ELIAS secondary antibody the best use of time
6, the determination of optimal substrate reactions time
According to optimum condition determined above, according to ELISA program, after adding substrate, 37 DEG C act on 5 respectively, 10,15,
20,25,30 minutes.Terminate reaction, measure OD450nm value, calculate P/N value.Using maximum for P/N value one group optimal as substrate
Action time, the results are shown in Table 7.
Table 7, the determination of substrate developing time
7, the determination of indirect ELISA method yin and yang attribute marginal value
Under optimum reaction condition, take 14 parts of negative Ox blood serum samples and carry out indirect ELISA detection analysis.According to ELISA
Operation sequence is measured, and reads OD450nm value, calculates OD450 meansigma methods X and standard deviation SD of 14 parts of serum, according to system
Principle learned by meter, is judged to the positive during OD450nm value >=X+3SD；OD450nm value ＜ X+2SD person is judged to feminine gender, therebetween
It is judged to suspicious.
Result shows, ELISA yin and yang attribute marginal value is 0.329, and OD450nm >=0.329 is the positive: negative marginal value is
Feminine gender it is judged to during 0.264, OD450nm ＜ 0.264.
8, specific test
According to the recombiant protein concentration coated elisa plate groped, respectively with recombinant antigen and bovine viral diarrhoea (BVD) sun
Property serum, infectious bovine rhinotrachetis (IBR), A type foot and mouth disease (A-FMD) and O type foot and mouth disease (O-FMD) positive serum are carried out
ELISA detects, and sets the comparison of BPV standard positive and negative simultaneously, determines whether recombinant antigen and other common cattle disease positive serum occurs
Cross reaction, the results are shown in Table 8.
Table 8, specific detection result
9, replica test
(9.1) replica test in criticizing
The recombiant protein taking same Batch purification is coated same ELISA Plate, to known 3 parts of positive serums and 3 parts of negative blood
Final proof product detect, and 3 repetitions made by every part of sample.Test by the ELISA operation sequence optimized, measure
OD450nm value.Result is carried out statistical analysis, calculates the coefficient of variation and evaluate batch interior test effect repeated, the results are shown in Table
9。
Table 9, batch interior replica test
(9.2) criticize between replica test
By the antigen coated ELISA Plate of four batches, known 3 parts of positive serums and 3 parts of negative serum samples are examined
Survey.Test by the ELISA operation sequence optimized, measure OD450nm value.Result is carried out statistical analysis, calculates
The test effect repeated between the coefficient of variation evaluation batch, the results are shown in Table 10.
Table 10, batch between replica test
10, sensitivity tests
Positive serum is done 1:40,1:80,1:160,1:320,1:640,1:l 280, seven dilution factors of 1:2560, presses
Good reaction condition carries out ELISA reaction, the results are shown in Table 11.
Table 11, sensitivity experiments result
Claims (5)
1. the indirect ELISA detection method of a bovine papilloma virus 13 type antibody, it is characterised in that real according to following steps
Execute:
Step 1, structure recombinant expression plasmid pET28a-L1
With BPV13 genome as template, for BPV13 capsid protein L 1 gene sequential design specific primer；Reacted by PCR
Obtain purpose fragment, to prokaryotic expression carrier pET28a and purpose fragment double digestion simultaneously, after connection, obtain recombinant expression plasmid
pET28a-L1；
Step 2, destination protein is expressed and purification；
Step 3, set up the indirect ELISA detection system of BPV13 antibody, for BPV13L1 albumen sample yin and yang attribute marginal value, enter
Row judges, obtains final testing result.
The indirect ELISA detection method of bovine papilloma virus 13 type antibody the most according to claim 1, its feature exists
In, in described step 1, the expression formula of specific primer is shown in Table 1,
Table 1, the expression formula of specific primer
In table 1, single file underscore part is the restriction enzyme site of BamH I and Hind III double digestion, and wave underscore part is 2
Planting primer 5 ' and hold complementary series, duplicate rows underscore part is the base after sudden change.
The indirect ELISA detection method of bovine papilloma virus 13 type antibody the most according to claim 1, its feature exists
In, in described step 2, destination protein is expressed and purification comprises the concrete steps that:
Positive recombination bacillus coli BL21 is inoculated in the LB fluid medium containing kanamycin and carries out IPTG abduction delivering, receive
The positive recombination bacillus coli BL21 of collection great expression ultrasonic degradation, dissolve inclusion body and with membrane filtration with carbamide, use
The His-L1 fusion protein expressed is purified by nickel ion affinity chromatograph method, obtains the His-L1 fusion protein of purification.
The indirect ELISA detection method of bovine papilloma virus 13 type antibody the most according to claim 1, its feature exists
In, in described step 3, set up the comprising the concrete steps that of indirect ELISA detection system of BPV13 antibody:
Using the His-L1 fusion protein of purification as envelope antigen coated elisa plate, determine that His-L1 fusion protein is coated concentration and is
92.5μg/mL；Antigen coated condition be 4 DEG C overnight；5% defatted milk powder is used to close, 1 hour off-period；Serum do not dilutes,
90 minutes action time；The anti-concentration of HRP-protein A IgG bis-is 1:8000,30 minutes action time；Under room temperature,
20 minutes substrate lucifuge response time.
The indirect ELISA detection method of bovine papilloma virus 13 type antibody the most according to claim 1, its feature exists
In, in described step 3, for BPV13L1 albumen sample yin and yang attribute marginal value, it is determined that standard is:
When sample OD450nm >=0.329, it is determined that for the positive；During sample OD450nm ＜ 0.264, it is determined that for feminine gender；Between two
Between person, person is judged to suspicious.
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Cited By (1)
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Application publication date: 20170104 |