CN106093385A - The indirect ELISA method of detection gene IV type hepatitis E virus antibody - Google Patents

The indirect ELISA method of detection gene IV type hepatitis E virus antibody Download PDF

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CN106093385A
CN106093385A CN201610703423.9A CN201610703423A CN106093385A CN 106093385 A CN106093385 A CN 106093385A CN 201610703423 A CN201610703423 A CN 201610703423A CN 106093385 A CN106093385 A CN 106093385A
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orf3
pet42a
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王凤阳
杜丽
赵天靖
聂鑫
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Hainan University
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Abstract

The invention discloses a kind of indirect ELISA method detecting gene IV type hepatitis E virus antibody, step includes: 1) build recombinant expression plasmid pET42a ORF3, by pMD18T ORF3 plasmid and pET42a (+) prokaryotic expression carrier converts DH5a competent cell, coats on LB solid medium overnight；Picking list colony inoculation in LB culture fluid overnight；It is centrifuged after 36 hours, collects thalline, extract plasmid DNA；Under the effect of ligase, by the ORF3 fragment after double digestion and pET42a (+) prokaryotic expression carrier is attached, it is thus achieved that recombinant expression plasmid pET42a ORF3；2) to recombinant expression plasmid pET42a ORF3 expression and purity；3) succeed and have expressed His/GST ORF3 fusion protein, i.e. can be used for serosurvey.The method of the present invention, low cost, accuracy rate is high.

Description

The indirect ELISA method of detection gene IV type hepatitis E virus antibody
Technical field
The invention belongs to technical field of biological, relate to a kind of detecting the indirect of gene IV type hepatitis E virus antibody ELISA method.
Background technology
Hepatitis E (hepatitis E, HE) is that one is drawn by hepatitis E virus (hepatitis E virus, HEV) The Amphixenosis that the main acute infectious disease propagated through fecal oral route risen is a kind of people mutually to be propagated with animal, as The reservoir host of HEV, pig HE propagation, play important role in the groove.Therefore, the prevention to pig hepatitis E is strengthened Control extremely necessary, and detecting fast and accurately and vaccinating is maximally efficient means.But, due to the most business-like HEV antibody test reagent, still based on I type HEV antigen, there may be different genotype sample detection leakage phenomenon in Clinical detection, It is necessary to develop the detectable using IV type HEV antigen to prepare for reducing missing inspection.HEV is positive 20 bodies, sick without the RNA of cyst membrane Poison, the unique member belonged to for Hepatitis E virus section Hepatitis E virus.Similar with other RNA viruses, that HEV RNA relies on RNA polymerase Do not replicate proofreading function so that it is be susceptible to variation, produce virus groups relevant on hereditism and antigenicity, show as There is different genotype and gene hypotype in HEV.
The HEV Strain having been found that in the world at present is divided into four main genotypes: I type from Asia and Africa, with Burma's strain prototype is representative, including Southeast Asia and the Pakistani strain in the Central Asia, the Xinjiang Strain of China, India's strain, the Ji of the former Soviet Union Er Jisitan strain and Africa strain；II type is from Mexico and Africa, and representative strains is Mexico's strain；III type is from the U.S., including U.S. State strain USl, US2 separate strain with U.S. SHEV；IV type is essentially from Asian countries such as China, Japan, including China's Mainland and platform The Strain in area, gulf；Additionally birds HEV is new HEV genotype, i.e. a gene V-type.
HEV is single strand plus RNA virus, full-length genome about 7.2kb, encodes 2400-2533 aminoacid, and structure is 5 '-NCR-NS-S-NCR-Poly (A)-3 ', full-length genome contains 3 opening code-reading frames (open reading frame, ORF). Wherein the genome of 5 ' ends 2/3 is ORF1, and length is about 5kb, about 1694 aminoacid of coding, and mainly viral RNA is replicated with The non-structural protein closed, including: transmethylase, the Y structure territory of Unknown Function, papain-like cystein protease, The RNA polymerase that " hinge " domain of proline rich, the X architecture territory of Unknown Function, DBPA and RNA rely on.ORF2 Being capsid gene, be positioned at genome 3 ' end, length is about 2kb, about 660 aminoacid of coding, for virus structural protein [13]. ORF3 is positioned at genome 3 ' end, and its coded product is one contains the phosphoprotein that 123 aminoacid is relevant with immunity, containing multiple The recognition sequence of protein kinase, its concrete function is the most not clear.
Up to now, being how to invade hepatocyte to HEV, the mechanism how causing hepatocyte injury is the most not clear.Body Outer Binding experiment shows, HEV ORF3 albumen can be combined with multi-signal transducin, illustrates that it may be with host cell signal Albumen interacts.Foreign scholar studies discovery, and pORF3 is affecting the signal transduction path of infection cell, and then impact Target cell function aspects plays important role, and pORF3 may (albumen that mitogen activates swashs by activating MAPK Enzyme) signal pathway, mediated cell is to intracellular, the hypertrophy of the reactivity of external stimulus, cell, vicious transformation and the tune of stress response Joint.Confirm the signal protein interaction in pORF3 Yu MAPK signal pathway.Additionally in the cell of process LAN pORF3, carefully The protein kinase (extracellular signal-regulated protein kinases, ERK) of extracellular signal regulation and control Activity raises to be increased relevant to the transhipment in core, thus shows that pORF3 is a kind of virus protein relevant with cell signal regulation and control, And it is in close relations with MAPK signal pathway.
Summary of the invention
It is an object of the invention to provide a kind of indirect ELISA method detecting gene IV type hepatitis E virus antibody, solve The problem that prior art of having determined is low to Hepatitis E virus antibody test accuracy rate.
The technical solution adopted in the present invention is, a kind of indirect ELISA detecting gene IV type hepatitis E virus antibody Method, implements according to following steps:
Step 1, structure recombinant expression plasmid pET42a-ORF3
By pMD18T-ORF3 plasmid and pET42a (+) prokaryotic expression carrier converts DH5a competent cell, takes 50uL bacterium solution Coat on LB solid medium, be inverted overnight incubation for 37 DEG C；
After 12 hours, picking list colony inoculation in the LB culture fluid of 3mL, 37 DEG C of shaken overnight；
After 36 hours, it is centrifuged 2 minutes under 12000rpm state, collects thalline, extract plasmid DNA；To pMD18T-ORF3 Plasmid and pET42a (+) prokaryotic expression carrier uses EcoR I and Sal I to carry out double digestion, at the work of T4DNA ligase ligase Under with, by the ORF3 fragment after double digestion and pET42a (+) prokaryotic expression carrier is attached, it is thus achieved that recombinant expression plasmid pET42a-ORF3；
Step 2, to recombinant expression plasmid pET42a-ORF3 expression and purity
The recombinant expression plasmid pET42a-ORF3 being correctly inserted into genes of interest is converted e. coli bl21 competence thin Born of the same parents, picking list bacterium colony, simultaneously set convert empty carrier plasmid e. coli bl21 for compare, be placed in the LB culture fluid of 5mL, 37 DEG C shaking overnight；
After 12 hours, when it is shaken cultivation to OD600=0.4-0.6 with the ratio of 1:100, aseptic taking-up lmL bacterium solution As comparison before induction, to adding IPTG to final concentration of 1mmol/L in remaining bacterium solution, 30 DEG C are continued shaking and cultivate 4 hours, Within centrifugal 1 minute under 12000rpm state, collect thalline；
Abandoning supernatant, precipitation adds isopyknic 2 times of sample buffers, fully mixes, and heats 5 minutes in boiling water, It is centrifuged 1 minute under 12000rpm state；
Take supernatant, not induce thalline for comparison, carry out SDS-PAGE electrophoresis detection；
Expression condition is optimized, determines the optimum condition of abduction delivering；
Step 3, utilize nickel ion affinity chromatograph column purification His/GST-ORF3 fusion protein, collect eluent respectively, take Each several part sample carries out SDS-PAGE analysis, observes protein purification effect, succeeds and have expressed His/GST-ORF3 fusion egg In vain, i.e. can be used for serosurvey.
The invention has the beneficial effects as follows, by genetic engineering recombinant technique, SHEV-ORF3 protein gene cloning is arrived PET42a (+) in expression vector, and make it obtain high efficient expression in e. coli bl21 (DE3) system, with the fusion of purification Albumen is antigen, establishes the indirect ELISA method of detection pig HEV antibody test, and carries out excellent to the technical parameter of the method Change, determine optimum reaction condition, further the method is carried out specific test, sensitivity tests, replica test, to having a competition Test, with specificity, sensitivity and the stability of clear and definite the method；Finally the method is applied to blood serum sample HEV antibody test. The method testing cost is low, and accuracy rate is high, it is simple to promote.
Accompanying drawing explanation
Fig. 1 is that the enzyme action of the recombinant expression plasmid pET42a-ORF3 in the inventive method identifies photo；
Fig. 2 is to use the inventive method by His/GST-ORF3 fusion protein different IPTG concentration, temperature, the luring of time Lead expression product；
Fig. 3 is to use the inventive method purification result to His/GST-ORF3 expressing fusion protein；
Fig. 4 is to use the inventive method to analyze the Western Blot of His/GST-ORF3 expressing fusion protein.
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 present invention detects the indirect ELISA method of gene IV type hepatitis E virus antibody, implements according to following steps:
Step 1, structure recombinant expression plasmid pET42a-ORF3
By existing pMD18T-ORF3 plasmid and pET42a (+) prokaryotic expression carrier converts DH5a competent cell, take 50uL bacterium solution coats (ampicillin and kanamycin concentration are 50ug/mL) on LB solid medium, is inverted for 37 DEG C and cultivates Overnight；
After 12 hours, picking list colony inoculation (wherein includes kanamycin 50ug/L) in the LB culture fluid of 3mL, and 37 DEG C shaken overnight；
After 36 hours, it is centrifuged 2 minutes under 12000rpm state, collects thalline, extract plasmid DNA；To pMD18T-ORF3 Plasmid and pET42a (+) prokaryotic expression carrier uses EcoR I and Sal I to carry out double digestion, at the work of T4DNA ligase ligase Under with, by the ORF3 fragment (purpose fragment) after double digestion and pET42a (+) prokaryotic expression carrier is attached, it is thus achieved that restructuring table Reach plasmid pET42a-ORF3.
Enzyme action is identified and is seen Fig. 1, and in Fig. 1, M is DNA molecular quality standard；1 is positive control fragment；2 is recombinant expression plasmid Double digestion product；3 be prokaryotic expression carrier pET42a (+) double digestion product.
Step 2, to recombinant expression plasmid pET42a-ORF3 expression and purity
The recombinant expression plasmid pET42a-ORF3 being correctly inserted into genes of interest is converted e. coli bl21 (DE3) impression State cell, picking list bacterium colony, simultaneously set convert empty carrier plasmid e. coli bl21 for compare, be placed in the LB culture fluid of 5mL In (wherein including kanamycin 50ug/L), 37 DEG C shaking overnight；
After 12 hours, when it is shaken cultivation to OD600=0.4-0.6 with the ratio of 1:100, aseptic taking-up lmL bacterium solution As comparison before induction, to adding IPTG to final concentration of 1mmol/L in remaining bacterium solution, 30 DEG C are continued shaking and cultivate 4 hours, Within centrifugal 1 minute under 12000rpm state, collect thalline；
Abandoning supernatant, precipitation adds isopyknic 2 times of sample buffers, fully mixes, and heats 5 minutes in boiling water, It is centrifuged 1 minute under 12000rpm state；
Take supernatant, not induce thalline for comparison, carry out SDS-PAGE electrophoresis detection；
Expression condition is optimized, different IPTG inducer concentrations (0.1,0.5,1.0,2.0mmol/L), different Inducing temperature (25 DEG C, 30 DEG C, 37 DEG C), different induction time (1 hour, 2 hours, 3 hours, 4 hours, 5 hours) conditions Under carry out abduction delivering, collect thalline, carry out SDS-PAGE electrophoretic analysis, determine the optimum condition of abduction delivering.
Abduction delivering is shown in that Fig. 2, M therein are protein standards；1-4 be respectively 30 DEG C, 0.1,0.5,1, 2mmol/L IPTG, 4 hours expression products of induction；5-6 be respectively 25 DEG C, 37 DEG C, 1mmol/L IPTG, induction 4 hours express Product；7-11 is respectively 30 DEG C, 1mmol/L IPTG, 1-5 hour abduction delivering product；12 be pET42a (+) empty carrier induction； 13 be pET42a (+) empty carrier do not induces；14 is the comparison of BL21 sky bacterium.
Step 3, utilize nickel ion affinity chromatograph post (Ni2+-NTA) the purification His/GST-that Qiagen company of the U.S. produces ORF3 fusion protein, the operation instructions with reference to this product are carried out, and process is summarized as follows:
First, being centrifuged 5 minutes under 10000rpm state, collect 500mL abduction delivering bacterium, ice bath 15 minutes, by 5mL/g The ratio of wet bacterium (bacterium solution precipitate) adds Buffer B, and (Buffer B is by the carbamide of 8M, the NaH of 100mM2PO4, 100mM Tris Cl forms, pH8.0), under room temperature, it is gently mixed, makes bacteria lysis translucent to solution；
Then, 4 DEG C, be centrifuged 25 minutes in 10000rpm state, collect supernatant, discard precipitation；By column volume 10 times amount Buffer B (pH8.0) balances pillar, and by the flow velocity of 5mL/min, supernatant is crossed post, and (Buffer C is by 8M's in Buffer C washing Carbamide, the NaH of 100mM2PO4, 100mM Tris Cl composition, pH6.3), (Buffer E is by the urine of 8M for Buffer E eluting Element, the NaH of 100mM2PO4, 100mM Tris Cl composition, pH4.5)；
Finally, collect eluent respectively, take each several part sample and carry out SDS-PAGE analysis, observe protein purification effect, pure Changing result and see Fig. 3, in Fig. 3, M is protein standard；1-9 is the purified product that different time is collected；
The final products that step 3 the obtains His/GST-ORF3 fusion protein that has been successful expression, utilizes its (optimum product) Serosurvey can be carried out.
Embodiment
1, the indirect ELISA method of detection gene IV type hepatitis E virus antibody is set up
After diluting GST-ORF3 fusion protein after purification with the carbonate buffer solution (pH9.6) of 0.05mol/L, it is coated enzyme Target, every hole 100 μ L, 4 DEG C overnight；
Discarding and be coated liquid, PBST washs three times, adds the sealer containing 5% defatted milk powder, and every hole 100 μ L, in 37 DEG C of envelopes Close 2 hours；After PBST washing, it is separately added into the serum to be checked with PBS dilution, and the HEV positive serum of standard and negative blood Clearly, μ L hole, every hole 100, set blank simultaneously, 37 DEG C act on 1 hour, and PBST washs；Add horseradish peroxidase-labeled Protein A (HRP-SPA) resists as two, every hole 100 μ L, and 37 DEG C act on 1 hour；PBST washs, and adds TMB nitrite ion 100 μ L, shakes Even, 37 DEG C of lucifuges develop the color 10 minutes；Finally add the H of 2mol/L2SO4Stop buffer 100 μ L terminates reaction, measures OD450Nm value；Logical Cross optimal conditions when determining optimal off-period, serum the best use of time, ELIAS secondary antibody the best use of time, substrate-function Between, optimal antigen coated concentration and two anti-working concentrations.
Through the optimization to the condition such as antigen and the selection of antibody optimum dilution degree and off-period, serum action time, Finally determine following parameters optimization: antigen coated concentration is 2 μ g/mL, an antiserum is 90 minutes action time, ELIAS secondary antibody is made With the time be 60 minutes, off-period be 120 minutes, the substrate-function time be 20 minutes, two to resist optimal diluted concentration be 1: 8000, the optimum dilution degree of yin and yang attribute comparison be 1:400, ELISA yin and yang attribute marginal value be 0.31, OD450nm >=0.31 be positive Property；Negative marginal value is 0.23, and OD450nm < is judged to feminine gender when 0.23.
2, serosurvey
Haikou City each age group general population serum specimen 235 parts, year is come from the indirect ELISA method detection set up It is distributed as 19-85 year, wherein, male's serum totally 148 parts age, accounts for 63.0%；Women serum totally 87 parts, accounts for 37.0%.Will Tested serum 1:50 times each adds in reacting hole after diluting, and measures OD450nm value by standardization program, more than 0.31 after reacting It is judged to the positive.
Result: positive number of cases is 7, and positive rate is 3.0%, this numbers illustrated Haikou City general population gene IV type penta type Hepatites virus infections level is relatively low.
Fig. 4 shows GST-ORF3 fusion protein success specifically expressing, and M therein is pre-dyed protein standards；1 is purpose Albumen；2 be pET42a (+) negative control.

Claims (4)

1. the indirect ELISA method detecting gene IV type hepatitis E virus antibody, it is characterised in that according to following steps Implement:
Step 1, structure recombinant expression plasmid pET42a-ORF3
By pMD18T-ORF3 plasmid and pET42a (+) prokaryotic expression carrier converts DH5a competent cell, takes the coating of 50uL bacterium solution On LB solid medium, it is inverted overnight incubation for 37 DEG C；
After 12 hours, picking list colony inoculation in the LB culture fluid of 3mL, 37 DEG C of shaken overnight；
After 36 hours, it is centrifuged 2 minutes under 12000rpm state, collects thalline, extract plasmid DNA；To pMD18T-ORF3 plasmid With pET42a (+) prokaryotic expression carrier use EcoR I and Sal I carry out double digestion, at the work of T4 DNA ligase ligase Under with, by the ORF3 fragment after double digestion and pET42a (+) prokaryotic expression carrier is attached, it is thus achieved that recombinant expression plasmid pET42a-ORF3；
Step 2, to recombinant expression plasmid pET42a-ORF3 expression and purity
The recombinant expression plasmid pET42a-ORF3 being correctly inserted into genes of interest is converted e. coli bl21 competent cell, chooses Taking single bacterium colony, set the e. coli bl21 converting empty carrier plasmid for compareing simultaneously, be placed in the LB culture fluid of 5mL, 37 DEG C are shaken Shake overnight；
After 12 hours, when it is shaken cultivation to OD600=0.4-0.6 with the ratio of 1:100, aseptic taking-up lmL bacterium solution conduct Comparison before induction, to adding IPTG to final concentration of 1mmol/L in remaining bacterium solution, 30 DEG C are continued shaking and cultivate 4 hours, Within under 12000rpm state centrifugal 1 minute, collect thalline；
Abandoning supernatant, precipitation adds isopyknic 2 times of sample buffers, fully mixes, and heats 5 minutes, at 12000rpm in boiling water It is centrifuged 1 minute under state；
Take supernatant, not induce thalline for comparison, carry out SDS-PAGE electrophoresis detection；
Expression condition is optimized, determines the optimum condition of abduction delivering；
Step 3, utilize nickel ion affinity chromatograph column purification His/GST-ORF3 fusion protein, collect eluent respectively, take each portion Divide sample to carry out SDS-PAGE analysis, observe protein purification effect, succeed and have expressed His/GST-ORF3 fusion protein, i.e. Can be used for serosurvey.
The indirect ELISA method of detection gene IV type hepatitis E virus antibody the most according to claim 1, its feature exists In: in described step 1, ampicillin and kanamycin concentration in LB solid medium are 50ug/mL；LB culture fluid In kanamycin be 50ug/L.
The indirect ELISA method of detection gene IV type hepatitis E virus antibody the most according to claim 1, its feature exists In: in described step 3, utilizing nickel ion affinity chromatograph column purification His/GST-ORF3 fusion protein, process is as follows:
First, being centrifuged 5 minutes under 10000rpm state, collect 500mL abduction delivering bacterium, ice bath 15 minutes, by the wet bacterium of 5mL/g Ratio add Buffer B, under room temperature, be gently mixed, make bacteria lysis translucent to solution；
Then, 4 DEG C, be centrifuged 25 minutes in 10000rpm state, collect supernatant, discard precipitation；By column volume 10 times amount Buffer B balances pillar, and by the flow velocity of 5mL/min, supernatant is crossed post, and BufferC washs, Buffer E eluting.
The indirect ELISA method of detection gene IV type hepatitis E virus antibody the most according to claim 3, its feature exists In: described Buffer B is by the carbamide of 8M, the NaH of 100mM2PO4, 100mM Tris Cl composition, pH8.0；Described Buffer C is by the carbamide of 8M, the NaH of 100mM2PO4, 100mM Tris Cl composition, pH6.3；Described Buffer E is by 8M Carbamide, the NaH of 100mM2PO4, 100mM Tris Cl composition, pH4.5.
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CN106699895A (en) * 2016-12-05 2017-05-24 上海科华生物工程股份有限公司 Novel fusion antigen and detection kit comprising same and application
CN106699895B (en) * 2016-12-05 2020-08-04 上海科华生物工程股份有限公司 Novel fusion antigen, detection kit containing same and application
CN108085290A (en) * 2018-01-04 2018-05-29 哈尔滨瀚邦医疗科技有限公司 A kind of detection method of Hepatitis E virus genetic stability and application

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Application publication date: 20161109