CN103616509A - EIII-indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) antibody detection kit for detecting swine Japanese encephalitis virus and application thereof - Google Patents
EIII-indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) antibody detection kit for detecting swine Japanese encephalitis virus and application thereof Download PDFInfo
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Abstract
The invention discloses an EIII-indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) antibody detection kit for detecting a swine Japanese encephalitis virus and an application thereof. The kit disclosed by the invention consists of an elisa plate which takes a purified JEV-EIII protein as an envelope antigen, a cleaning liquid, a serum diluent, rabbit anti-pig elisa secondary antibody, a primer developing liquid, a stop buffer, a JEV positive serum and a JEV negative serum. The envelope antigen used by the invention is easy to be obtained stably and massively, the purifying method is simple and easy to realize, and concentration of recombinant proteins is easy to test and control, thereby facilitating industrialized production on a large scale. The kit disclosed by the invention is used for detecting the swine Japanese encephalitis virus antibody, and the detection coincidence rate with the ELISA kit in the prior art is 90%. The kit disclosed by the invention is convenient to operate, high in sensitivity, good in specificity, low in using cost, good in repeatability and suitable for popularization and application, and provides a reliable means for clinical rapid detection of the JEV antibody.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of E III-indirect ELISA antibody assay kit and application that detects Latex agglutination test.
Background technology
Japanese B encephalitis virus (Japanese encephalitis virus, JEV) belongs to flaviviridae Flavivirus, and its Japanese B encephalitis causing is a kind of acute infectious disease of central nervous system infecting both domestic animals and human.According to estimates, nearly 50000 people in the annual whole world infect JEV, and nearly 15000 examples are dead, mortality ratio 30% left and right; JEV is one of important virus of serious harm pig industry simultaneously, to pig industry, causes huge economic loss, limits greatly the foreign exchange earning of meat product.Within 2008, China Ministry of Agriculture is attributed to two class animal epidemics by pig Japanese B encephalitis disease.
This disease in Japanese reported first, and is separated to Japanese B encephalitis virus in nineteen twenty-four in 19 century 70s first.China adopted serology to make a definite diagnosis Japanese B encephalitis case with the separated method of virus between 1938～1940 years, was separated to first this virus in 1949 in Beijing.Since the nineties in 20th century, this sick epidemic regions expands to some extent, and this sick distributive province is North gets Russian Siberia, Hokkaido, Japan at present, and south is to Australian ，Dong Dao U.S. Guam ，Xi Dao India West Coast.Asia is the area that JE number of the infected is maximum, and China has accounted for again great majority wherein.
The method that is usually used at present detecting encephalitis B virus comprises separation evaluation, RT-PCR and the serological method of cause of disease.It is the most traditional detection method that the separation of cause of disease is identified, accurately and reliably, but its influence factor is many for its result, and practical operation is got up quite wastes time and energy, and therefore in clinical practice, has certain limitation; The operation that the instrument and equipment (as PCR instrument and gel imaging system etc.) that RT-PCR Technology Need is special and professional are correlated with; Enzyme immunoassay (EIA) is one of method the most frequently used in serological method, and enzyme immunoassay (EIA) combines height selectivity, the specificity of the amplification of enzymic catalytic reaction and antigen-antibody compatible reaction, antigen or the antibody of enzyme labeling of usining carries out immunoassay as main agents, there is very high sensitivity and specificity, but the sensitivity of immunoassay, specificity depend on the selection of antigen.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the present invention is to provide a kind of E III-indirect ELISA antibody assay kit that detects Latex agglutination test.This kit has high sensitivity, high specific, the simple feature of method of operating.
Another object of the present invention is to provide the application of E III-indirect ELISA antibody assay kit of described detection Latex agglutination test.
Object of the present invention is achieved through the following technical solutions: a kind of E III-indirect ELISA antibody assay kit that detects Latex agglutination test, specifically comprises following composition:
Using the JEV-E III albumen of purifying as the anti-pig ELIAS secondary antibody of ELISA Plate, cleansing solution, serum dilution, rabbit, substrate nitrite ion, stop buffer, JEV positive serum, the JEV negative serum of envelope antigen.
The JEV-E III albumen of described purifying prepares by following steps:
(1) extracting RNA from Latex agglutination test GZ0409-31 strain, after 42 ℃ of reverse transcription 1h, obtains cDNA, and the cDNA obtaining of take is template, by primer P1 and P2, carries out PCR; The PCR product obtaining and carrier pET-32a are carried out to double digestion by BamH I and HindIII respectively; Again the PCR product after double digestion is connected with pET-32a, obtains recombinant plasmid pET-32a-JEV-EIII;
P1：5'-CGC
GGATCCAAAAATCCGGCGGACACTG-3'；
P2：5'-CCC
AAGCTTTTACAAAGTTGTTGAAAAGGCCTTG-3'；
Wherein, CGC or CCC are protection base,
gGATCCfor BamH I restriction enzyme site,
aAGCTTfor Hind III restriction enzyme site, TTA is the reverse complementary sequence of terminator codon TAA;
(2) recombinant plasmid pET-32a-JEV-EIII is transformed in Escherichia coli, by the recombinant bacterial strain of test positive, by IPTG, carries out abduction delivering;
(3) collect the bacterium liquid after expressing, use His-TRAP
tMfF Crude post carries out purifying, obtains the JEV-E III albumen of purifying;
The reaction system of the reverse transcription described in step (1) is: total RNA12 μ L, dNTPs2 μ L, random primer 1 μ L, RRI2 μ L, AMV1 μ L, add ddH
2o to cumulative volume be 20 μ L;
The reaction system of PCR described in step (1) is: 10 * Buffer5 μ L, the dNTPs5 μ L that concentration is 2mM, the MgSO that concentration is 25mM
44 μ L, primer P11.5 μ L, primer P21.5 μ L, cDNA2 μ L, KOD enzyme 1 μ L, adds ddH
2o to 50 μ L;
The reaction conditions of PCR described in step (1) is: 94 ℃ of denaturation 2min; 94 ℃ of thermal denaturation 15s, 55 ℃ of 30s, 68 ℃ of 1min, totally 30 circulations; 68 ℃ of 8min extend eventually;
Described JEV-E III albumen of usining purifying, as the ELISA Plate of envelope antigen, prepares: the JEV-E III albumen of purifying is diluted to 4.7 μ g/mL with coated damping fluid, and the 100 every holes of μ L are added to ELISA Plate, and 4 ℃ are spent the night as follows; The ELISA Plate that getting spends the night has been coated with, 200 μ L/ hole cleansing solutions are washed plate 3～5 times, each 3～5min; 200 μ L/ hole confining liquids, 37 ℃ of incubation 2.5h, get the ELISA Plate of having sealed, and 200 μ L/ hole cleansing solutions are washed plate 3～5 times, each 3～5min; Obtain usining the JEV-E III albumen of purifying as the ELISA Plate of envelope antigen;
Described coated damping fluid is preferably the carbonate buffer solution of pH9.6,0.05mol/L;
Described pH9.6, the carbonate buffer solution of 0.05mol/L preferably prepare as follows: sodium carbonate 1.5g and sodium bicarbonate 2.93g are dissolved in to 600mL ddH
2o, adjust pH to 9.6, ddH
2o is settled to 1000mL, obtains coated damping fluid;
Described confining liquid is preferably the soybean lecithin solution with the mass volume ratio (g/mL) 0.5% of cleansing solution preparation;
Described ELISA Plate is preferably JET company product;
Described cleansing solution prepares as follows: by 3.579g Na
2hPO
412H
2o, 1.56gNaH
2pO
42H
2o, NaCl29.215g and 600mL ddH
2o fully dissolves, and adds 0.5mL Tween-20, with 10mol/L HCl solution adjust pH to 7.4, ddH
2o is settled to 1000mL;
Described serum dilution is preferably the soybean lecithin solution with the mass volume ratio (g/mL) 0.5% of cleansing solution preparation;
It is anti-that the anti-pig ELIAS secondary antibody of described rabbit is preferably the anti-pig IgG two of horseradish peroxidase (HRP)-rabbit;
Described substrate nitrite ion is preferably TMB nitrite ion;
Described stop buffer is 2M sulfuric acid;
The application of E III-indirect ELISA antibody assay kit of described detection Latex agglutination test, comprises following steps:
(1) sample to be checked is diluted with serum dilution, then by the amount in 100 μ L/ holes, be added to and using in the JEV-E III albumen of the purifying ELISA Plate as envelope antigen, simultaneously also by JEV positive serum and JEV negative serum respectively by the amount in 100 μ L/ holes be added to using purifying JEV-E III albumen as in the ELISA Plate of envelope antigen in contrast, 37 ℃ of incubation 40min;
(2) with cleansing solution, wash plate 3 times, 200 μ L/ holes, each 3min;
(3) the anti-pig ELIAS secondary antibody of rabbit is diluted with cleansing solution, each reacting hole adds 100 μ L, 37 ℃ of incubation 55min;
(4) with cleansing solution, wash plate 4 times, 200 μ L/ holes, each 3min;
(5) under lucifuge condition, each reacting hole adds 100 μ L substrate nitrite ions, room temperature lucifuge colour developing 20min;
(6) each reacting hole adds 50 μ L stop buffer cessation reactions;
(7) under the mono-wavelength of 450nm, survey OD value;
(8) result interpretation: OD450nm (sample) >=0.255 is judged to the positive; OD450nm is between 0.231～0.255(OD450nm (sample)+2SD) be judged to suspiciously, OD450nm (sample) < 0.231 is judged to feminine gender.
Described in step (1) by sample to be checked with serum dilution dilution be preferably by sample to be checked with serum dilution by volume 1:160 doubly dilute;
Described in step (3) by the anti-pig ELIAS secondary antibody of rabbit with cleansing solution dilution be preferably by the anti-pig ELIAS secondary antibody of rabbit with cleansing solution by volume 1:5000 doubly dilute.
The present invention, with respect to prior art, has following advantage and effect:
(1) envelope antigen that the present invention selects is Latex agglutination test E III albumen, and E albumen is the important structural protein of JEV, belongs to glycoprotein, has hemagglutinin activity, and adhesion and the film of mediation virus and host cell merge, and are important virulence factors; And E albumen has very strong immunogenicity, can induce body to produce virucidin, escape the attack of host immune monitor system.The ectodomain of E albumen is comprised of 3 domains, between 3 domains, connects each other, is exercising the various functions of E albumen.Wherein E protein structure domain III is the folding of stable immunoglobulin-like, there is the effect with host cell surface receptors bind, the adhesion of mediation virus and host cell, it is the antigen active center of epidemic encephalitis b of swine virus, on it, contain the specific epitope of JEV, its main neutralizing epitope is positioned on the residue of domain III outside surface, this region is the region of receptors bind, compare and directly select E protein-specific stronger, avoided the combination of other antibody in non-antigenic substance and serum.Antibody contacting of blocking virus and cell most effectively after being combined with E protein structure domain III.
(2) the epidemic encephalitis b of swine virus E III albumen of kit application of the present invention, on selection material, there is new meaning, pig is the antibody for E albumen what be subject to producing at first when JEV infects, and E III albumen is the antigen active center of epidemic encephalitis b of swine virus E protein, guaranteed that this kit has the specificity of height.
(3) detecting aspect epidemic encephalitis b of swine antiviral antibody at present, mainly containing by the coated indirect hemagglutination of E albumen coated ELISA kit, totivirus and suppress experiment with blood clotting and latex agglutination is tested.Due to virus need to be in cell culture propagation in a large number, and purified virus is truly difficult to obtain.The envelope antigen that the present invention uses is the prokaryotic expression product of epidemic encephalitis b of swine virus E III gene, its advantage is to recombinate, and E III albumen is easy to stablize, acquisition in a large number, purification process is simply easy to realize (application His-bind affinity column carries out purifying), the concentration of recombinant protein is easy to measure and control, and is conducive to industrial mass production.Although E albumen is also the method by prokaryotic expression, obtain, E albumen is the material that holoprotein carries many non-antigens itself, has increased the probability that detects non-specific antibody.
(4) kit of the present invention is for detection of Latex agglutination test antibody, and detection method, after the check of the indexs such as specificity, susceptibility, repeatability, is used it for the check of blood serum sample, with the detection coincidence rate of ELISA kit in prior art be 90%.Kit convenient operation of the present invention, highly sensitive, specificity is good, and use cost is low, reproducible, is applicable to applying, for fast detecting JEV antibody clinically provides reliable means.
(5) it is less that this kit is used antigen protein package amount, and protein concentration used is only 4.7 μ g/mL, and sensitivity is higher.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of JEV-E III gene magnification fragment; Swimming lane M is DNA MarkerDL2000, and swimming lane 1 and 2 is respectively take the RT-PCR amplified production that P1, P2 be primer.
Fig. 2 is the agarose gel electrophoresis figure that the double digestion of pET-32a-JEV-E III recombinant plasmid is identified; Swimming lane M1 is DNA Marker DL2000, and swimming lane 1 and 2 is that recombinant plasmid pET-32a-JEV-E III is carried out the product after double digestion with BamH I and Hind III respectively, and swimming lane M2 is DNA Marker DL10000.
The SDS-PAGE figure of the product that Fig. 3 obtains through cracking for the thalline of restructuring pET-32a-JEV-E III carrier in Bacillus coli expression bacterium liquid; Swimming lane M is albumen Marker116KDa, and swimming lane 1 is the pET-32a empty carrier of not inducing, and swimming lane 2 is the pET-32a empty carrier of induction, and swimming lane 3 is the recombinant plasmid pET-32a-JEV-E III of not inducing, the recombinant plasmid pET-32a-JEV-E III of swimming lane 4 for inducing.
The SDS-PAGE result figure of the product that the thalline in the bacterium liquid that Fig. 4 obtains at different induction times for restructuring pET-32a-JEV-E III carrier obtains through cracking; Swimming lane M is albumen Marker116KDa, and swimming lane 1～7 is for inducing successively 0h, 1h, 2h, 3h, 4h, 5h and 6h.
Fig. 5 is the SDS-PAGE result figure of recombinant plasmid different IP TG concentration induction; Swimming lane M is albumen Marker116KDa, swimming lane 1～7th, and IPTG concentration is followed successively by 0mM, 0.5mM, 1mM, 2mM, 3mM, 4mM and 5mM.
Fig. 6 is the SDS-PAGE figure of recombinant protein and the result figure of Western Blotting; Wherein, the figure A SDS-PAGE figure that is recombinant protein, figure B be the result figure of the Western Blotting corresponding with scheming A; Swimming lane M is albumen Marker116KDa, and swimming lane 1 and 2 is respectively recombinant protein.
Fig. 7 is the SDS-PAGE result figure of protein purification condition imidazoles binding buffer liquid concentration optimization; Swimming lane M is albumen Marker116KDa, and swimming lane 1 is 30mM, and swimming lane 2 is 40mM, and swimming lane 3 is 50mM.
Fig. 8 is that protein purification condition is used after the combination of 30mmol/L imidazoles binding buffer liquid, the SDS-PAGE result figure of imidazoles elution buffer concentration optimization; Swimming lane M is albumen Marker116KDa, and swimming lane 1 is 200mM, and swimming lane 2 is 100mM, and swimming lane 3 is 500mM.
Fig. 9 is that protein purification condition is used after the combination of 30mmol/L imidazoles binding buffer liquid, the SDS-PAGE result figure that 100mM imidazoles elution buffer elution volume is optimized; Swimming lane M is albumen Marker116KDa, and swimming lane 1～9 is respectively 5mL, 6mL, 7mL, 8mL, 9mL, 10mL, 11mL, 12mL, 13mL.
Figure 10 is the SDS-PAGE figure of recombinant protein after purifying and the result figure of Western Blotting; Swimming lane M is albumen Marker116KDa; Wherein, figure A is the SDS-PAGE figure of recombinant protein after purifying, and figure B be the result figure of the Western Blotting corresponding with scheming A; Swimming lane M is egg Marker116KDa, and swimming lane 1 and 2 is respectively recombinant protein after purifying.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
I, material
KODMei Wei TOYOBO used company product in following examples;
JEV-E III albumen is at BL21(DE3) concentration of expression in escherichia coli purifying is the albumen of 4.7 μ g/mL;
ELISA Plate WeiJET company product;
Cleansing solution prepares as follows: by 3.579g Na
2hPO
412H
2o, 1.56gNaH
2pO
42H
2o, 29.215g NaCl and 600mL ddH
2o fully dissolves, and adds 0.5mL Tween-20, with 10mol/L HCl solution adjust pH to 7.4, ddH
2o is settled to 1000mL;
Coated damping fluid (carbonate buffer solution of pH9.60.05mol/L): sodium carbonate 1.5g, sodium bicarbonate 2.93g are dissolved in 600mL ddH
2o, adjust pH to 9.6, ddH
2o is settled to 1000mL;
Serum dilution is the soybean lecithin solution of mass volume ratio (g/mL) 0.5%, and solvent is cleansing solution;
Confining liquid is the soybean lecithin solution of mass volume ratio (g/mL) 0.5%, and solvent is cleansing solution;
The anti-pig IgG two of horseradish peroxidase (HRP)-rabbit resists the product for Guangzhou Wei Jia company;
TMB nitrite ion is Guangzhou UCANDO biotech firm product;
Stop buffer is 2M sulfuric acid;
JEV positive serum, JEV negative serum are with reference to the JEV negative serum in the ELISA of IDEXX company kit and the positive pig serum of JEV;
Restriction enzyme BamH I, Hind III, T4DNA ligase, DNA Marker, albumen Marker, bacillus coli DH 5 alpha competent cell, e. coli bl21 competent cell etc. are all purchased from TaKaRa company;
Prokaryotic expression carrier pET-32a is purchased from Novagen company;
RNA extraction agent box, PCR reclaim kit and are all purchased from OMEGA company.
Following biomaterial is open at document " preparation of 3 kinds of inactivated vaccines of Latex agglutination test and immune effect comparison, Agricultural University Of South China's journal, 2011,32(02): 85-88 ": Latex agglutination test GZ0409-31 strain.
The concrete preparation process of II
One, the preparation of the JEV-E III albumen of purifying, comprises following steps:
(1) design of primers and synthetic: according to Latex agglutination test in GenBank (GZ0409-31 strain) gene order design primer (KF297916.1), introduce BamH I and Hind III restriction enzyme enzyme recognition site at upstream and downstream respectively.Above-mentioned primer is synthetic by Shanghai Sheng Gong biotechnology company limited.
P1：5'-CGC
GGATCCAAAAATCCGGCGGACACTG-3'；
P2：5'-CCC
AAGCTTTTACAAAGTTGTTGAAAAGGCCTTG-3'；
Wherein, CGC or CCC are protection base,
gGATCCfor BamH I restriction enzyme site,
aAGCTTfor Hind III restriction enzyme site, TTA is the reverse complementary sequence of terminator codon TAA;
(2) target fragment gene magnification: with reference to RNA extraction agent box (purchased from OMEGA company) extracting RNA from Latex agglutination test GZ0409-31 strain, the reaction system of reverse transcription is: total RNA12 μ L, dNTPs2 μ L, random primer 1 μ L, RRI2 μ L, AMV1 μ L, adds ddH
2o to cumulative volume be 20 μ L; After 42 ℃ of reverse transcription 1h, obtain cDNA, the cDNA obtaining of take is template, applies above-mentioned primer and carries out pcr amplification, reaction system (50 μ L): 10 * Buffer5 μ L, dNTPs(2mM) 5 μ L, MgSO
4(25mM) 4 μ L, primer P11.5 μ L, primer P21.5 μ L, cDNA2 μ L, KOD enzyme 1 μ L, adds ddH
2o to 50 μ L.Reaction conditions is: 94 ℃ of denaturation 2min; (94 ℃ of 15s, 55 ℃ of 30s, 68 ℃ of 1min) * 30 circulations; 68 ℃, 8min, 4 ℃ of preservations.After PCR finishes, get amplified production 5 μ L electrophoresis on the Ago-Gel of mass volume ratio 1%, utilize gel imaging system scanning to carry out Preliminary Identification.Electrophoresis result as shown in Figure 1.
(3) structure of recombinant expression carrier: with BamH I and Hind III restriction enzyme respectively to pcr amplification product and plasmid pET-32a(Novagen company) carry out double digestion, the reaction system of double digestion is: each 1 μ L of BamH I and Hind III, 10 * K buffer2 μ L, pcr amplification product or plasmid pET-32a1 μ g, add ddH
2o to 20 μ L.With the agarose gel electrophoresis of mass volume ratio 1.5%, identify, with reference to PCR, reclaim kit (purchased from OMEGA company), by kit instructions, reclaim double digestion product, the two reclaims product with T4DNA ligase (Takara for mol ratio 10:1 ratio, by specification operation) in 16 ℃ of connections, spend the night, connection product is converted in bacillus coli DH 5 alpha competent cell (TaKaRa company), and is inoculated in the Amp containing 100mg/L
+nutrient agar double dish (tryptone 2.0g, yeast extract 1.0g, sodium chloride 2.0g, agar powder 3.0g, be dissolved in 200mL ddH
2in O, pH value is adjusted to 7.4,121 ℃ of autoclaving 15min, adds Amp when 50 ℃ of left and right fall in nutrient culture media temperature
+it is 100mg/L that mother liquor makes its final concentration, after mixing, is poured into glass dish, and 4 ℃ save backup.) in, 37 ℃ of constant temperature are inverted and are cultivated 12～16h, random choose monoclonal bacterium colony, identifying correct positive colony extracting plasmid with primer P1 and P2 through bacterium colony PCR, with BamH I and the checking of Hind III double digestion, as shown in Figure 2, the fragment that enzyme is cut rear acquisition is identical with object clip size for result; Picking enzyme is cut being cloned in containing 100mg/L Amp of test positive
+lB broth bouillon (tryptone 1.0g, yeast extract 0.5g, sodium chloride 1.0g, be dissolved in 100mL ddH
2in O, pH value is adjusted to 7.4,121 ℃ of autoclaving 15min, and nutrient culture media temperature adds Amp after being down to normal temperature
+it is 100mg/L that mother liquor makes its final concentration, and 4 ℃ save backup.) in increase bacterium, draw bacterium liquid and entrust Shanghai Li Fei company to check order, the sequence obtaining and genes of interest sequence are in full accord, with hard objectives gene with correct reading frame insertion expression vector.Obtain positive recombinant plasmid, by its called after pET-32a-JEV-E III.
(4) recombinant plasmid abduction delivering and condition optimizing
The abduction delivering of recombinant protein: order-checking is identified to correct recombinant plasmid pET-32a-JEV-E III is converted into e. coli bl21 competent cell (TaKaRa company), and the positive recombinant vector bacterium colony of picking, is inoculated in fresh LB meat soup (Amp
+final concentration is 100mg/L) in nutrient culture media, in 37 ℃, 200r/min overnight incubation, obtain the bacterium liquid of activation; Next day, the bacterium liquid of activation is inoculated in to fresh LB meat soup (Amp according to volume ratio 1:50
+final concentration is 100mg/L) in nutrient culture media, when OD600=0.4～0.6, adding final concentration is the IPTG of 0.5mM, in 37 ℃ of abduction delivering 3h, collect bacterium liquid, get the centrifugal collection thalline of 1mL bacterium liquid, add 2 * SDS-PAGE sample-loading buffer of the resuspended and same volume of 100 μ LddH2O, boiling lysis 10min in water, carry out SDS-PAGE(and use discontinuous buffer system, the concentration of separation gel is 12%, and concentrated glue is 3%, wherein first carry out 80V30min, the rear voltage conditions with 120V35min) test.Set up empty carrier pET-32a transformed bacteria and recombinant plasmid pET-32a-JEV-E III not to induce bacterium is contrast simultaneously.Result as shown in Figure 3.
Different induction time expression of recombinant proteins situations are inoculated the bacterium liquid of activation according to the method described above, and the final concentration of derivant IPTG is 0.5mM, and induction time is respectively 0h, 1h, 2h, 3h, 4h, 5h, 6h.The bacterium liquid of collecting different time sections, carries out SDS-PAGE(and uses discontinuous buffer system, and the concentration of separation gel is 12%, concentrated glue is 3%, wherein first carries out 80V30min, the rear voltage conditions with 120V40min) analyze, as shown in Figure 4, result shows that best induction time is 3h to result.
Different derivant IPTG concentration expression of recombinant proteins situations are inoculated the bacterium liquid of activation according to the method described above, and the final concentration of derivant IPTG is 0.5mM, 1mM, 2mM, 3mM, 4mM and 5mM, 37 ℃ of inductions, the Best Times that induction time is above-mentioned optimization.Collect respectively bacterium liquid, carry out SDS-PAGE(and use discontinuous buffer system, the concentration of separation gel is 12%, concentrated glue is 3%, wherein first carries out 80V30min, the rear voltage conditions with 120V40min) analyze, as shown in Figure 5, result shows that best induction IPTG concentration is 2mM to result.
Recombinant protein great expression: choose the positive bacterium colony of recombinant strains BL21-pET32a-JEV-E III, be inoculated in 1L LB(Amp
+final concentration is 100mg/L) in nutrient culture media, by the inductive condition of above-mentioned optimization, carry out abduction delivering, centrifugal collection thalline, adds 5mL sodium salt damping fluid (Na by the thalline of the centrifugal rear collection of every 100mL bacterium liquid
2hPO
412H
2o1.7895g, NaH
2pO
42H
2o0.78g, NaCl14.6075g, is settled to 500mL, regulates pH to 7.4.), resuspended bacterial sediment, ultrasonication is centrifugal, is divided into cleer and peaceful precipitation (inclusion body).5mL8M urea sodium salt damping fluid for inclusion body (the urea sodium salt damping fluid that urea and above-mentioned sodium salt damping fluid are mixed to get) dissolves, 4 ℃ of dissolvings of spending the night.Dissolve latter 4 ℃, the centrifugal 15min of 8000r/min, get supernatant; Again supernatant is carried out to gradient dialysis renaturation; As the pretreatment sample of crossing post purifying.
Adopt above-mentioned pretreatment sample, Western Blotting detects the antigenicity (as shown in Figure 6) of recombinant protein.Result shows that recombinant protein has antigenicity.
(5) purifying of recombinant protein and evaluation
The optimization of purification condition: according to His-TRAP
tMfF Crude(is purchased from GE Healthcare company) operation steps of instructions carries out purifying, adopts respectively 30mM, 40mM, each 5mL volumetric balance purification column of 50mM imidazoles binding buffer liquid; After balance, add 1mL pretreatment sample, adsorbed after pretreatment sample, rinse purification column respectively with the imidazoles binding buffer liquid of the same concentration of 10mL, flow velocity 1mL/min, adopts 1.5mL EP pipe to collect the liquid under each flushing; Adopt respectively again 100mM, 200mM, each 5mL of 500mM imidazoles elution buffer to carry out gradient elution, flow velocity 1mL/min, 1.5mL EP pipe is collected each concentration imidazoles elution buffer.With SDS-PAGE(, use discontinuous buffer system, the concentration of separation gel is 12%, concentrated glue is 3%, wherein first carry out 80V30min, use afterwards the voltage conditions of 120V40min) to collecting sample detection, determine the suitableeest imidazoles binding buffer liquid concentration (as shown in Figure 7), the suitableeest imidazoles elution buffer concentration (as shown in Figure 8), the suitableeest imidazoles elution buffer volume (as shown in Figure 9).Result shows 5mL30mM imidazoles binding buffer liquid combination for 1mL sample, and the imidazoles elution buffer wash-out purification effect of 7mL100mM is best.
By after the recombinant protein sample preparation after purifying, carry out SDS-PAGE(and use discontinuous buffer system, the concentration of separation gel is 12%, concentrated glue is 3%, wherein first carry out 80V30min, use afterwards the voltage conditions of 120V40min) detect, the Westernbloting that observes electrophoretic effects (seeing shown in Figure 10 A) and purifying protein detects (seeing shown in Figure 10 B), and result shows that the JEV-E III albumen of purifying has antigenicity.
Two, the preparation of the coated plate of recombinant protein
(1) the best coated concentration of antigen and serum optimum dilution degree selection
By the best coated concentration of square formation titration method defined antigen and serum optimum dilution degree, result is as table 1; As can be seen from Table 1, with coated damping fluid, dilute antigen, with serum dilution, carry out dilute serum, along with the minimizing of antigen concentration and the increase of serum dilution, P/N value constantly reduces.When recombinant protein 1:80 dilution, the best coated concentration of recombinant antigen is 4.7 μ g/mL, and during control serum 1:160 dilution, P/N is maximum.Wherein, coated damping fluid is the carbonate buffer solution of pH9.6,0.05mol/L, and serum dilution is the soybean lecithin solution with the mass volume ratio (g/mL) 0.5% of cleansing solution preparation.
Table 1
(2) confining liquid and concentration are selected
The bovine serum albumin(BSA) (BSA), skimmed milk power (demulsification), soybean lecithin (LHP) of using respectively variable concentrations seals the ELISA Plate of envelope antigen, and result is as shown in table 2; P/N value, substantially along with confining liquid concentration reduces and reduce, selects mass volume ratio (g/mL) 0.5% soybean lecithin solution (cleansing solution preparation) as confining liquid then as can be seen from Table 2.
Table 2
(3) optimize off-period
After the coated antigen coated ELISA Plate of concentration of the best, with mass volume ratio (g/mL) 0.5% soybean lecithin solution (cleansing solution preparation) sealase target, under 37 ℃ of conditions, adopt respectively 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h to seal off-period, test findings is as shown in table 3; As can be seen from Table 3, when be 2.5h off-period, P/N value is maximum, therefore determines that be 2.5h best off-period.
Table 3
In sum, the preparation process of the coated plate of recombinant protein is as follows: the JEV-E III albumen of purifying is diluted to 4.7 μ g/mL with coated damping fluid, and the 100 every holes of μ L are added to ELISA Plate, and 4 ℃ are spent the night.The ELISA Plate that getting spends the night has been coated with, 200 μ L/ hole cleansing solutions are washed plate 3 times, each 3min.200 μ L/ hole mass volume ratio (g/mL) 0.5% soybean lecithin solution (cleansing solution preparation) seal, and 37 ℃ of incubation 2.5h, get the ELISA Plate of having sealed, and 200 μ L/ hole cleansing solutions are washed plate 3 times, each 3min; Obtain usining the JEV-E III albumen of purifying as the ELISA Plate of coating protein.
Three, detect the application of E III-indirect ELISA antibody assay kit of Latex agglutination test, the Optimization Steps of each condition comprises as follows:
(1) primary antibodie incubative time is optimized
Use above-mentioned JEV-E III albumen of usining purifying as the ELISA Plate of envelope antigen, primary antibodie (pig encephalitis standard serum: JEV positive serum and JEV negative serum) is at 37 ℃ of difference incubation different times, as can be seen from Table 4, when primary antibodie incubative time is 40min, P/N value is maximum, therefore determines that best primary antibodie incubative time is 40min.
Table 4
| The primary antibodie working time | P | N | P/N |
| 10min | 0.225 | 0.155 | 1.448 |
| 15min | 0.252 | 0.167 | 1.513 |
| 20min | 0.275 | 0.165 | 1.669 |
| 25min | 0.334 | 0.171 | 1.955 |
| 30min | 0.361 | 0.175 | 2.062 |
| 35min | 0.363 | 0.172 | 2.114 |
[0110]?
| 40min | 0.406 | 0.182 | 2.230 |
| 45min | 0.388 | 0.182 | 2.131 |
| 50min | 0.373 | 0.178 | 2.093 |
| 55min | 0.376 | 0.182 | 2.067 |
| 60min | 0.377 | 0.181 | 2.077 |
| 65min | 0.365 | 0.180 | 2.030 |
(2) two anti-concentration are selected
Use above-mentioned JEV-E III albumen of usining purifying as the ELISA Plate of envelope antigen, incubation primary antibodie under optimum condition, by the anti-coated elisa plate of the anti-pig IgG two of HRP-rabbit of the different proportion dilution according in table 5, test findings is as shown in table 5; As can be seen from Table 5, during the anti-dilute concentration 1:5000 dilution of the anti-pig IgG two of HRP-rabbit, P/N value is maximum, therefore 1:5000 is defined as to the anti-best effort concentration of the anti-pig IgG two of HRP-rabbit of recombinant protein.
Table 5
| ELIAS secondary antibody best effort concentration | P | N | P/N |
| 1:5000 | 0.979 | 0.190 | 5.164 |
| 1:8000 | 0.652 | 0.181 | 3.613 |
| 1:10000 | 0.359 | 0.179 | 2.011 |
| 1:20000 | 0.230 | 0.117 | 1.970 |
| 1:30000 | 0.216 | 0.110 | 1.958 |
| 1:40000 | 0.163 | 0.095 | 1.711 |
| 1:50000 | 0.133 | 0.081 | 1.654 |
| 1:60000 | 0.127 | 0.078 | 1.622 |
| 1:70000 | 0.107 | 0.078 | 1.368 |
| 1:80000 | 0.098 | 0.076 | 1.294 |
| 1:90000 | 0.091 | 0.074 | 1.229 |
| 1:100000 | 0.092 | 0.074 | 1.246 |
(3) two is anti-time-optimized
Use above-mentioned JEV-E III albumen of usining purifying as the ELISA Plate of envelope antigen, incubation primary antibodie under the suitableeest condition, according to the anti-coated elisa plate of the anti-pig IgG two of HRP-rabbit of 1:5000 example dilution, under 37 ℃ of conditions, the anti-pig IgG two of HRP-rabbit adopts respectively as time incubations different in table 6 anti-action time, and test findings is as table 6, therefrom can find out, during the anti-incubation time 55min of the anti-pig IgG two of HRP-rabbit, P/N value is maximum, therefore determines that the anti-incubative time of the anti-pig IgG two of HRP-rabbit is 55min.
Table 6
| The ELIAS secondary antibody working time | P | N | P/N |
| 10min | 0.303 | 0.211 | 1.435 |
| 15min | 0.318 | 0.181 | 1.759 |
| 20min | 0.288 | 0.159 | 1.814 |
| 25min | 0.298 | 0.146 | 2.038 |
| 30min | 0.274 | 0.128 | 2.142 |
| 35min | 0.326 | 0.149 | 2.188 |
| 40min | 0.308 | 0.137 | 2.241 |
| 45min | 0.325 | 0.126 | 2.583 |
| 50min | 0.348 | 0.125 | 2.781 |
| 55min | 0.314 | 0.094 | 3.330 |
| 60min | 0.287 | 0.107 | 2.678 |
| 65min | 0.279 | 0.112 | 2.487 |
(4) developing time optimization
Use above-mentioned JEV-E III albumen of usining purifying as the ELISA Plate of envelope antigen, after under the suitableeest condition, the anti-pig IgG two of incubation primary antibodie and HRP-rabbit resists, after adding substrate nitrite ion TMB, act on respectively different time as shown in table 7, test findings is as shown in table 7, as seen from Table 7, during 20min, P/N value is maximum, therefore determines that the optimum reacting time of substrate nitrite ion is room temperature reaction 20min.
Table 7
| TMB action time | P | N | P/N |
| 10min | 0.320 | 0.158 | 2.024 |
| 15min | 0.341 | 0.171 | 1.993 |
| 20min | 0.420 | 0.131 | 3.221 |
| 25min | 0.329 | 0.131 | 2.513 |
| 30min | 0.310 | 0.134 | 2.313 |
| 35min | 0.297 | 0.129 | 2.305 |
| 40min | 0.292 | 0.133 | 2.187 |
| 45min | 0.277 | 0.147 | 1.878 |
| 50min | 0.278 | 0.144 | 1.930 |
| 55min | 0.273 | 0.147 | 1.865 |
| 60min | 0.292 | 0.158 | 1.847 |
| 65min | 0.283 | 0.155 | 1.830 |
[0123](5) critical value defines and sensitivity tests
Utilize the kit (American I DEXX company kit) of buying to identify that the negative 20 parts of pig serum (Guangdong Province) of JEV detect, test findings is as shown in table 8, result is carried out to statistical analysis, draw mean value x=0.183, standard deviation SD=0.024, thereby calculate x+3SD=0.255, be critical value, OD450nm (sample) >=0.255 is judged to the positive; Between 0.255～0.231(OD450nm (sample)+2SD) be judged to suspiciously, OD450nm (sample) < 0.231 is judged to feminine gender.Meanwhile the positive pig serum (Guangdong Province) of JEV is carried out to doubling dilution, its OD450 value while being diluted to as seen 6400 times (the P value in table 9 is corresponding one by one with OD450 value) is for 0.307(is in Table 9), be greater than 0.255 positive decision content.
Table 8
Table 9
| Serum diluting multiple | P | N | P/N |
| 1：100 | 2.458 | 0.228 | 10.78 |
| 1：200 | 2.031 | 0.225 | 9.024 |
| 1：400 | 1.868 | 0.209 | 8.938 |
| 1：800 | 1.324 | 0.212 | 6.245 |
| 1：1600 | 0.901 | 0.210 | 4.293 |
| 1：3200 | 0.510 | 0.127 | 4.012 |
| 1：6400 | 0.307 | 0.130 | 2.362 |
| 1：12800 | 0.209 | 0.121 | 1.723 |
| 1：25600 | 0.152 | 0.118 | 1.284 |
| 1：51200 | 0.146 | 0.118 | 1.237 |
| 1：102400 | 0.146 | 0.114 | 1.281 |
| 1：204800 | 0.153 | 0.162 | 0.944 |
(6) specific test
The JEV-E III protein ELISA method of setting up with this test detects swine fever, encephalitis, pseudo-mad dog, aftosa, brucella, Actinobacillus, diarrhoea, transmissible gastroenteritis positive serum (above serum is all from academy of agricultural sciences, Guangdong Province veterinary institute).Result shows as shown in table 10, and except encephalitis positive serum, the OD450nm value of all the other serum, all lower than 0.231, is judged to be negative serum, shows that the ELISA kit specificity of setting up is relatively good.
Table 10
| Standard serum | OD450 | Result is judged |
| Swine fever | 0.068 | — |
| Encephalitis | 2.659 | + |
| Pseudo-mad dog | 0.154 | — |
| Aftosa | 0.175 | — |
| Brucella | 0.145 | — |
| Actinobacillus | 0.082 | — |
| PCV-II | 0.037 | — |
| Diarrhoea | 0.077 | — |
| Transmissible gastroenteritis | 0.213 | — |
(7) replica test
1. criticize interior replica test
By same batch of coated ELISA Plate to the serum of 8 parts of known background (7 parts of positive serums and 1 part of negative serum sample, above serum is all from academy of agricultural sciences, Guangdong Province veterinary institute) do and criticize interior revision test, every part of 12, serum work is parallel, result is as shown in table 11, variation within batch coefficient is 1.83%～5.50% as can be seen from the table, all, in 10%, illustrate that this detection method has batch interior repeatability preferably.
Replica test result in table 11 batch
2. between criticizing, repeat examination
With with batch albumen in the coated ELISA Plate of 8 different times to the serum of 8 parts of known background (7 parts of positive serums and 1 part of feminine gender, above serum is all from academy of agricultural sciences, Guangdong Province veterinary institute) do batch between revision test, every part of 12, serum work is parallel, result is as shown in table 12, interassay coefficient of variation is between 3.86%～9.08% as can be seen from the table, all, in 10%, illustrate that this detection method has repeatability between good batch.
Table 12
(8) clinical blood serum sample detects
The kit of applying this test assembling detects 224 parts of pig serum samples from pig farm, In Guangdong Province, and with certain ELISA kit (Latex agglutination test antibody assay kit, purchased from equation bio tech ltd, Beijing) result comparison, result shows both coincidence rates 90.7%.
Table 13
In sum, detect the application of the indirect ELISA reagent kit of epidemic encephalitis b of swine antiviral antibody, comprise following steps:
(1) by sample to be checked (pig serum to be measured, academy of agricultural sciences, Guangdong Province animal health research institute) with serum dilution by volume 1:160 doubly dilute, then the amount in 100 μ L/ holes is added to and usings in the JEV-E III albumen of the purifying ELISA Plate as envelope antigen, simultaneously also JEV positive serum and JEV negative serum are added to and are usingd in the JEV-E III albumen of the purifying ELISA Plate as envelope antigen by the amount in 100 μ L/ holes respectively, 37 ℃ of incubation 40min;
(2) with cleansing solution, wash plate 3 times, 200 μ L/ holes, each 3min;
(3) by the anti-pig IgG two of horseradish peroxidase (HRP)-rabbit anti-with cleansing solution by volume 1:5000 doubly dilute, each reacting hole adds 100 μ L, 37 ℃ of incubation 55min;
(4) with cleansing solution, wash plate 4 times, 200 μ L/ holes, each 3min;
(5) under lucifuge condition, each reacting hole adds 100 μ LTMB nitrite ions, room temperature lucifuge colour developing 20min;
(6) each reacting hole adds 50 μ L stop buffer cessation reactions;
(7) under the mono-wavelength of 450nm, survey OD value;
(8) result interpretation: OD450nm (sample) >=0.255 is judged to the positive; OD450nm is between 0.231～0.255(OD450nm (sample)+2SD) be judged to suspiciously, OD450nm (sample) < 0.231 is judged to feminine gender.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. the E III-indirect ELISA antibody assay kit that detects Latex agglutination test, is characterized in that comprising following composition:
Using the JEV-E III albumen of purifying as the anti-pig ELIAS secondary antibody of ELISA Plate, cleansing solution, serum dilution, rabbit, substrate nitrite ion, stop buffer, JEV positive serum, the JEV negative serum of envelope antigen.
2. the E of detection Latex agglutination test according to claim 1 III-indirect ELISA antibody assay kit, is characterized in that:
The JEV-E III albumen of described purifying prepares by following steps:
(1) extracting RNA from Latex agglutination test GZ0409-31 strain, after 42 ℃ of reverse transcription 1h, obtains cDNA, and the cDNA obtaining of take is template, by primer P1 and P2, carries out PCR; The PCR product obtaining and carrier pET-32a are carried out to double digestion by BamH I and HindIII respectively; Again the PCR product after double digestion is connected with pET-32a, obtains recombinant plasmid pET-32a-JEV-EIII;
P1：5'-CGC
GGATCCAAAAATCCGGCGGACACTG-3'；
P2：5'-CCC
AAGCTTTTACAAAGTTGTTGAAAAGGCCTTG-3'；
(2) recombinant plasmid pET-32a-JEV-EIII is transformed in Escherichia coli, by the recombinant bacterial strain of test positive, by IPTG, carries out abduction delivering;
(3) collect the bacterium liquid after expressing, use His-TRAP
tMfF Crude post carries out purifying, obtains the JEV-E III albumen of purifying.
3. the E of detection Latex agglutination test according to claim 2 III-indirect ELISA antibody assay kit, it is characterized in that: the reaction system of the reverse transcription described in step (1) is: total RNA12 μ L, dNTPs2 μ L, random primer 1 μ L, RRI2 μ L, AMV1 μ L, add ddH
2o to cumulative volume be 20 μ L.
4. the E of detection Latex agglutination test according to claim 2 III-indirect ELISA antibody assay kit, it is characterized in that: the reaction system of the PCR described in step (1) is: 10 * Buffer5 μ L, concentration is the dNTPs5 μ L of 2mM, the MgSO that concentration is 25mM
44 μ L, primer P11.5 μ L, primer P21.5 μ L, cDNA2 μ L, KOD enzyme 1 μ L, adds ddH
2o to 50 μ L;
The reaction conditions of PCR described in step (1) is: 94 ℃ of denaturation 2min; 94 ℃ of thermal denaturation 15s, 55 ℃ of 30s, 68 ℃ of 1min, totally 30 circulations; 68 ℃ of 8min extend eventually.
5. the E of detection Latex agglutination test according to claim 1 III-indirect ELISA antibody assay kit, is characterized in that:
Described JEV-E III albumen of usining purifying, as the ELISA Plate of envelope antigen, prepares: the JEV-E III albumen of purifying is diluted to 4.7 μ g/mL with coated damping fluid, and the 100 every holes of μ L are added to ELISA Plate, and 4 ℃ are spent the night as follows; The ELISA Plate that getting spends the night has been coated with, 200 μ L/ hole cleansing solutions are washed plate 3～5 times, each 3～5min; 200 μ L/ hole confining liquids, 37 ℃ of incubation 2.5h, get the ELISA Plate of having sealed, and 200 μ L/ hole cleansing solutions are washed plate 3～5 times, each 3～5min; Obtain usining the JEV-E III albumen of purifying as the ELISA Plate of envelope antigen.
6. the E of detection Latex agglutination test according to claim 5 III-indirect ELISA antibody assay kit, is characterized in that:
Described confining liquid is the soybean lecithin solution with the mass volume ratio 0.5% of cleansing solution preparation.
7. the E of detection Latex agglutination test according to claim 5 III-indirect ELISA antibody assay kit, is characterized in that:
Described coated damping fluid is the carbonate buffer solution of pH9.6,0.05mol/L.
8. the E of detection Latex agglutination test according to claim 1 III-indirect ELISA antibody assay kit, is characterized in that: described serum dilution is the soybean lecithin solution of the mass volume ratio 0.5% with cleansing solution preparation.
9. the E of detection Latex agglutination test according to claim 1 III-indirect ELISA antibody assay kit, is characterized in that: described ELISA Plate WeiJET company product;
Described cleansing solution prepares as follows: by 3.579g Na
2hPO
412H
2o, 1.56gNaH
2pO
42H
2o, NaCl29.215g and 600mL ddH
2o fully dissolves, and adds 0.5mL Tween-20, with 10mol/L HCl solution adjust pH to 7.4, ddH
2o is settled to 1000mL;
The anti-pig ELIAS secondary antibody of described rabbit is that the anti-pig IgG two of horseradish peroxidase-rabbit is anti-;
Described substrate nitrite ion is TMB nitrite ion;
Described stop buffer is 2M sulfuric acid.
10. the application of E III-indirect ELISA antibody assay kit of the detection Latex agglutination test described in claim 1～9 any one, is characterized in that comprising following steps:
(1) sample to be checked is diluted with serum dilution, then by the amount in 100 μ L/ holes, be added to and using in the JEV-E III albumen of the purifying ELISA Plate as envelope antigen, simultaneously also by JEV positive serum and JEV negative serum respectively by the amount in 100 μ L/ holes be added to using purifying JEV-E III albumen as in the ELISA Plate of envelope antigen in contrast, 37 ℃ of incubation 40min;
(2) with cleansing solution, wash plate 3 times, 200 μ L/ holes, each 3min;
(3) the anti-pig ELIAS secondary antibody of rabbit is diluted with cleansing solution, each reacting hole adds 100 μ L, 37 ℃ of incubation 55min;
(4) with cleansing solution, wash plate 4 times, 200 μ L/ holes, each 3min;
(5) under lucifuge condition, each reacting hole adds 100 μ L substrate nitrite ions, room temperature lucifuge colour developing 20min;
(6) each reacting hole adds 50 μ L stop buffer cessation reactions;
(7) under the mono-wavelength of 450nm, survey OD value;
(8) result interpretation: OD450nm >=0.255 is judged to the positive; OD450nm is judged to suspicious between 0.231～0.255, OD450nm < 0.231 is judged to feminine gender.
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