CN110452921A - The packaging and serum neutralizing antibody detection method of 10 type pseudovirus of Coxsackie virus A - Google Patents
The packaging and serum neutralizing antibody detection method of 10 type pseudovirus of Coxsackie virus A Download PDFInfo
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Abstract
The present invention discloses the expression and serum neutralizing antibody detection method of 10 type pseudovirus of Coxsackie virus A, including a kind of for packing the recombinant expression plasmid of CA10 type pseudovirus, recombinant expression plasmid is named as CA10-EGFP-CAPSID plasmid, and recombinant expression plasmid has nucleotide sequence shown in SEQ ID NO:1 in sequence table；A method of expression CA10-EGFP-CAPSID recombinant plasmid, comprising the following steps: 1) splice the gene order of green fluorescent protein and the gene order of all structural proteins of CA10；2) and by the restriction enzyme site of 2A protease it is inserted into the leading portion of green fluorescence protein gene；3) design primer.A kind of pseudovirus that the EV71 subgenomic RNA that 10 structural proteins of Coxsackie virus A and EV71-LUC-REPLICON replicon by CA10-EGFP-CAPSID plasmid expression are transcribed assembles and the neutralizing antibody kit comprising the pseudovirus.The present invention provide it is a kind of it is safe, sensitive, quick, special, it is easy and it is low-cost detection CA10 neutralizing antibody method, using the pseudovirus of single cycle infection, thus use live virus when safety problem.
Description
Technical field
The present invention relates to field of biotechnology, specially in the packaging of 10 type pseudovirus of Coxsackie virus A and serum and anti-
Body detecting method.
Background technique
Hand-foot-and-mouth disease (Hand, foot and mouth disease, HFMD) be as caused by a variety of enterovirus infections,
There is fash, bleb as the communicable disease of clinical manifestation using fever and hand, foot, oral cavity.Enterovirns type 71
(Enterovirus 71, EV71) and coxsackievirus A16 (Coxsackievirus A16, CA16) are main cause of diseases
Body, but in recent years has researches show that the infection rates of EV71 and CA16 in downward trend year by year, and 6 type of Coxsackie virus A group
Other serum such as (Coxsackievirus A6, CA6) and 10 type of Coxsackie virus A group (Coxsackievirus A10, CA10)
The infection rate of type enterovirus is but rising year by year.CA10 has become the HFMD cause of disease for being only second to EV71 and CA16 in some areas
Body.
10 type category Picornaviridae (Picornaviridae) of Coxsackie virus A group, enterovirus genus
(Enterovirus) A group.CA10 virus structure is relatively easy, the icosahedron being made of 60 structural proteins, diameter 23-
30nm, no coating.Genome is single-stranded positive RNA, and about 7400bp, the RNA pass through and internal ribosome is combined and translated.Core
Sour RNA is broadly divided into four parts: 5 ' noncoding regions, and polyprotein code area can be divided into P1, P2 and P3 three parts, and P1 is structure
Protein-coding region, P2 and P3 are functional protein code area, 3 ' noncoding regions.CA10 causes mild to infect more, including HFMD, blister
Rash angina (herpangina, HA) and piptonychia disease (Onychomadesis) etc., are embodied in the positions such as hand, foot, oral cavity
Fash, ulcer, fever, pharyngalgia and loss of finger-nail.CA10 epidemiological investigation and vaccine are ground in the detection of neutralizing antibody
Hair evaluation has critically important meaning.
If a kind of serum neutralizing antibody detection method for detecting CA10 is developed, to the CA10 specific detection of hand-foot-and-mouth disease
Have great importance.
Summary of the invention
To solve the above existing issue, the present invention provides in the packaging and serum of 10 type pseudovirus of Coxsackie virus A and anti-
Body detecting method.The invention is realized by the following technical scheme.
It is a kind of for packing the recombinant expression plasmid of 10 type pseudovirus of Coxsackie virus A, the recombinant expression plasmid name
For CA10-EGFP-CAPSID plasmid, the recombinant expression plasmid has nucleotides sequence shown in SEQ ID NO:1 in sequence table
Column.
Further, the recombinant plasmid uses Green fluorescent protein fusion vector (EGFP), in green fluorescent protein base
It is the encoding gene (P1 gene) of all structural proteins of CA10 after cause and 2A restriction enzyme site.
Further, protease cutting site (2A restriction enzyme site ,-AITTL-) is added between EGFP and P1 genetic fragment.
Further, a method of expression CA10-EGFP-CAPSID recombinant plasmid, comprising the following steps: 1) will be green
The gene order of color fluorescin and the gene order of all structural proteins of CA10 are spliced；2) and by the restriction enzyme site of 2A protease
It is inserted into the leading portion of green fluorescence protein gene；3) design primer；
The primer sequence are as follows:
CA10-EGFP-F ACCCAAGCTGGCTAGCGCCACCATGGTGAGCAAGGGCG
CA10-EGFP-R gacttgagctccAAGGGTAGTAATGGCCTTGTAC
CA10-P1-F GCCATTACTACCCTTGGAGCTCAAGTCTCGACGCAAAAATCC
CA10-P1-R GGTGATGATGaccggtTAACATATTAGCTTGCTTGATACTGGATCG。
A kind of pseudovirus, by 10 structural proteins of Coxsackie virus A and EV71- of CA10-EGFP-CAPSID plasmid expression
The EV71 subgenomic RNA of LUC-REPLICON replicon transcription assembles.
A kind of detection method of the neutralizing antibody comprising pseudovirus described in claim 5 is used for vitro detection enteron aisle Ke's Sa
The neutralizing antibody of odd virus A10.
It is a kind of detect 10 neutralizing antibody of Coxsackie virus A detection method comprising the steps of: 1) by the pseudovirus with
The test antibodies sample mixing being serially diluted, obtains mixture；2) sensitive with mixture infection enteron aisle Coxsackie virus B 5
Cell, Ke in sample to be tested is qualitatively or quantitatively detected according to signal caused by the representation of reporter gene in sensitive cell
The potency of Sa Qi virus B5 specificity neutralizing antibody.
Based on above scheme, the present invention provides safety, sensitive, quick, special, easy and low-cost detection CA10
The method of neutralizing antibody.Due to using the pseudovirus of single cycle infection, so safety problem when not using live virus.In
Then correspond to the viral residual volume of test into intracellular viral RNA subgenome (delete structural gene, and replace with
The viral genome of luciferase gene) in the cell expression obtain the amount of luciferase.Fluorescein is added to cell pyrolysis liquid
After chemiluminescence reaction occurs for the substrate of enzyme, the amount of luciferase can be by Chemiluminescence Apparatus accurate quantification.Luciferase chemistry hair
The logarithm of light reaction reading has good linear relationship with remaining virus quantity in a big way, and test process is easy to operate,
It is of less demanding to technical staff.For cost, this method is also cheaper, can be sieved on a large scale into the cell in 96 holes
Choosing test.
Based on the above feature, which is very suitable for quickly, detects the examination of neutralizing antibody on a large scale
It tests, the development and detection individual patients and crowd's hand-foot-and-mouth disease, CA10 specificity neutralizing antibody level for viral vaccine have
Important application value.
Detailed description of the invention
Fig. 1 is enterovirus genome schematic diagram；
Fig. 2 is single cycle infection pseudovirus schematic diagram；
Fig. 3 is the result of quantitative detection test plasma neutralizing antibody titers；
Fig. 4 is the range of linearity of CA10 pseudovirus system
Fig. 5 is that CA10 pseudovirus does the analysis of neutralization test specific outcome；
Fig. 6 is that CA10 pseudovirus does neutralization test repeatability interpretation of result；
Fig. 7 is pseudovirus detection and tradition CPE detection method results relevance；
Fig. 8 is pseudovirus detection and tradition CPE detection method result consistency.
Specific embodiment
Technical solution of the present invention work specifically, is completely illustrated below with reference to embodiment and attached drawing.In embodiment
Used experimental method is conventional method unless otherwise specified.Material, reagent used etc. are unless otherwise specified
It is commercially available.
1. experimental material: RD cell (deriving from human rhabdomyosarcoma) (being purchased from ATCC) and 293T cell (derive from people's embryo
Foetus renal cells) (be purchased from ATCC) cultivate in the DMEM of 10%FBS (fetal calf serum is purchased from GIBCO).RD cell is for detecting
The sensitive cell line of neutralizing antibody test.293T cell is used to the pseudovirus of packaging CA10.
2. 10 type expressor plasmid construction method of Coxsackie virus A group: utilizing CA10 live virus strain (CA10virus
Strain CA10-2, Genbankaccession NO.KY012321, Xiamen University professor Xia Ningshao give), pass through virus
RNA extraction agent box obtains the full-length genome (shown in Fig. 1) of CA10 Strain, and carries out RT-PCR by specific primer
(Reverse Transcription PCR) amplification coding viral capsid proteins gene P1 (shown in Fig. 2).Using containing EGFP gene
Expression vector pcDNA6.0-CA6-EGFP-Capsid be that template by PCR amplification acquisition is able to carry out the EGFP of seamless connection
Segment, in order to avoid EGFP green fluorescent protein cuts the influence of folding and pseudovirion packaging to capsid protein, in EGFP
Protease cutting site (2A restriction enzyme site ,-AITTL-) is added between P1 genetic fragment, the restriction enzyme site is in 2A albumen enzyme effect
Lower cutting separates EGFP and capsid protein P1；In order to enhance expression quantity of the capsid protein in 293T, in building capsid protein
Kozak sequence (gccacc) and initiation codon are added in front of EGFP gene segment by primer when expression vector
(ATG), kozak sequence is the optimal design sequence on eukaryon mRNA before initiation codon, and ribosomes can identify this section of sequence
To improve the translation starting efficiency of eukaryotic gene.Addition terminator codon (TAA) is ended up to tie in target fragment by primer
The expression of beam target gene.Excellent optimal eukaryotic expression vector pcDNA6.0 is selected for the purposes of enhancing capsid protein table
Up to efficiency；Meanwhile double digestion is carried out to eukaryotic expression vector pcDNA6.0 by restriction enzyme A geI, NheI.It will
PcDNA6.0 carrier after P1 segment and EGFP segment and double digestion that PCR amplification obtains carries out seamless connection reaction together.It will
The product of acquisition is converted, picking monoclonal colonies, extracts plasmid.The plasmid after connection is carried out using double digestion method preliminary
Identification, agarose gel electrophoresis figure are shown in figure, and band molecular size range meets expection after digestion.Sequence verification matching rate as the result is shown
The building of 100%, CA10 capsid protein expression plasmid is correct.The CA10 capsid protein expression plasmid of building is transfected into 293T cell,
Since in plasmid construction, by fluorescin EGFP and P1 amalgamation and expression, EGFP can be observed by fluorescence inverted microscope
Brightness judge the expression of P1.By being compared with the positive control of transfection expression EGFP plasmid, luminous efficiency >
80%, it may be determined that the capsid protein expression plasmid of building being capable of normal expression.
The primer is as follows:
3. transfection and pseudovirus packaging
The 12-14 hours tissue culture plate upper berth 293T cells in 10cm2 before transfecting.The transfection same day, the density of 293T are answered
This reaches 80-90%.Previous hour is transfected, cultivates 293T cell with 10%FBS-DMEM.With purchased from Polyplus, catalogue
Number for 114-15 transfect 10 μ g tri- kinds of plasmid mixture Cs A10-EGFP-CAPSID, EV71-LUC-REPLICON, pcDNA-T7-
Polymerase (mass mixings such as three kinds of plasmids), is cultivated in 37 DEG C of cell incubators.In fluorescence microscopy microscopic observation after for 24 hours
EGFP fluorescin is to judge the expression of structural proteins.After 48h, cell conditioned medium is collected into 15ml pipe, in cell
Surface leaves the culture medium of about 500 μ l.293T cell is resuspended with remaining culture medium, and with dry ice fast freeze-thaw two repeatedly
It is secondary.The precipitating for the culture medium supernatant freeze thawing collected before is mixed, then uses centrifuge 2 again, 500g is centrifuged 15 minutes, is abandoned
Precipitating is collected supernatant, and is dispensed into EP pipe I.5ml, and it is spare to be stored in -80 DEG C of refrigerators.
In 4.CA10 pseudovirus serum and test
Culture medium, every 50 μ l of hole is added in 2-8 row in 96 porocyte culture plates, 8 times of diluted blood to be measured are added in the 1st row
Final proof sheet and known potency standard serum, every 100 μ l of hole, each blood serum sample set 2 multiple holes；2 times are serially diluted, test serum
Sample sets 8 dilution gradients, and standard serum sets 6 dilution gradients.100 μ l culture mediums are added in cell blank control wells, if 2
A multiple holes.50 μ l culture mediums are added in pseudovirus Positive control wells, if 2 multiple holes.By the pseudovirus of 108.5Copies/50ul
Usage amount dilutes pseudovirus, and 4 DEG C of refrigerators are spare after mixing.In addition to cell blank control wells, CA10 pseudovirus, every 50 μ of hole is added
L is gently patted and is placed in culture incubation 2h in 37 DEG C of incubators after mixing.After neutralization reaction, in 96 porocyte culture plates
The RD cell suspension of the optimum cell density in 2 × 105/hole is added, it is small to be placed in culture 8 in 37 DEG C of incubators by every 100 μ l of hole
When.Culture terminates, abandon supernatant, be added 1 × cell pyrolysis liquid, every 50 μ l of hole.Multigelation 2 times, shift lysate after freeze thawing
Into 96 hole white detection plates, every 30 μ l of hole.Different cell lines are infected by chemiluminescence imaging system detection CA10 pseudovirus
Chemiluminescence signal intensity (relative light unit, RLU).As a result calculate: test serum potency=test serum exists
The corresponding standard serum potency × test serum dilution of RLU value in standard curve range.As a result as shown in Figure 3.
Test relative ease: the neutralization test of pseudovirus can carry out in 96 porocyte culture plates, and every kind of serum is not
Multiple multiple holes can be done with dilution gradient, difference between hole can be eliminated from the perspective of statistics.Pseudovirus and test sample
Mixture be added after tissue culture plate 8 hours, can will use lysate lytic cell, and and take 10ul-30ul to crack with the volley of rifle fire
Sample is used for the reading of Chemiluminescence Apparatus.When obtaining data, it is only necessary to read luciferase chemistry hair automatically with Chemiluminescence Apparatus
The reading of light reaction.Reagent required for chemiluminescence reaction includes lysate, the substrate of luciferase, luciferase
Reaction buffer, it is low in cost.Therefore pseudovirus system detection neutralizing antibody method is fast and convenient and data are reliable, at low cost
It is honest and clean.As a result as shown in Figure 4.
The advantages of single cycle infection: the packaging system of pseudovirion consists of two parts.Disease is constructed on one plasmid
The structural protein gene of poison constructs the RNA replicon of virus on another plasmid.The pseudovirus of CA10 will take off when entering cell
Capsid, and the replicon rna of virus is discharged into cell interior.5 ' ends of replicon are Internal ribosome entry site
(IRES).Therefore, the luciferase after the element and nonstructural protein gene can directly be turned over by intracellular ribosomes
It translates.Non-structural protein can help replicon to be replicated in the cell after being translated.Single stranded positive-sense RNA base in pseudovirion
Because the structural protein gene of group is deleted, and replaced by luciferase gene.Therefore, pseudovirus is in single invasion cell
Later, structural proteins can not be translated, so new infectious viral particle can not be assembled.Pseudovirus infects system and eliminates
After live virus for the first time infects, the problem of entering cell is repeated.When carrying out infection experiment in same cell line, pseudovirus is most
The reading of the whole resulting luciferase chemiluminescence reaction of infection cell is only interfered by virion when invading cell
The influence of factor (such as neutralizing antibody).
In 5.CA10 pseudovirus serum and experimental verification
The main application of the CA10 pseudovirus of building is to establish in the serum based on pseudovirus and evaluation test method
(pNT), which will be from specificity, repeatability, with traditional few cells lesion serum neutralizing antibody evaluation test method (cNT)
Correlation and consistency in terms of evaluate the new method, to judge the feasibility of CA10 pseudovirus serum neutralization test.
In order to examine the specificity of CA10 pseudovirus system, the specific antisera for CA10 in mouse source is used
(serum for the SPF grade BALB/c mouse that Lai Ziyong CA10-2 inactivation of viruses was immunized), mouse source are directed to EV-A71 (FY)
Specific antisera (serum for the SPF grade BALB/c mouse that Lai Ziyong EV-A71 (FY) inactivation of viruses was immunized), mouse come
Specific antisera (the blood for the SPF grade BALB/c mouse that Lai Ziyong CA16-G10 inactivation of viruses was immunized for CA16 in source
Clearly), specific antisera (the SPF grade BALB/ that Lai Ziyong CA6-TW inactivation of viruses was immunized for CA6-TW in mouse source
The serum of c mouse), the specific antisera for EVD68 in mouse source (exempt from by Lai Ziyong EVD68-BCH895A inactivation of viruses
The serum for the SPF grade BALB/c mouse that epidemic disease is crossed.To the obtained serum of eye socket blood sampling of SPF grades of BALB/c mouses, this is conventional
Preparation method；SPF grades of BALB/c mouses are purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.) neutralization examination is done to CA10 pseudovirus
It tests.As a result as shown in Figure 5.
For the repeatability for detecting CA10 pseudovirus serum neutralization test method, high, normal, basic each 1 part of serum is selected to carry out cape horn fever
Malicious serum neutralization test repeats detection 2 times in every piece of 96 porocyte culture plates, is operated altogether by 2 testing crews, repeatedly does 3 days,
That is every part of serum detects 12 times altogether.The serum titer result of 3 parts of serum is shown in figure, and analysis is it is found that every part of serum is being repeated 12 times examination
Under conditions of testing, serum titer variation control illustrates that carrying out serum neutralization titer with CA10 pseudovirus comments in three dilutions
The test method of valence has good repeatability.As a result as shown in Figure 6.
In order to detect CA10 based in the serum of pseudovirus and evaluation test method (pNT) and traditional few cells lesion blood
The correlation of clear neutralizing antibody evaluation test method (cNT), this test is with pNT method to 70 parts of CA10 positive source of people serum and yin
Property 20 parts of serum carried out the evaluation of serum neutralization titer, which eats from magnificent blue biology, cNT test data from China
Laboratory, product drug assay research institute testing result.The pseudovirus batch used is 20180913, viral copy number is 2.2 ×
107/ml.The result (cNT) of evaluation result (pNT) and traditional serum titer evaluation test based on euvirus is compared
Compared with；Negative serum pNT test is consistent with cNT testing inspection result, and detection serum titer, which is respectively less than, is equal to 8；Positive serum passes through
5 software of GraphPad Prism carries out statistics Spearman correlation analysis to serum test result, and comparison result is shown in figure.
Analysis is it is found that the related coefficient of two methods serum testing result is 0.78, and according to software decision principle, related coefficient is less than
0.3 is weak correlation, is related between 0.3 to 0.7, is strong correlation greater than 0.8；Statistics display illustrates that pseudovirus serum titer detects
As a result there is strong correlation with conventional method, the pseudovirus constructed with this project, which evaluates serum titer by serum neutralization test, is
It is feasible.As a result as shown in Figure 7.
In order to detect CA10 based in the serum of pseudovirus and evaluation test method (pNT) and traditional few cells lesion blood
The consistency of clear neutralizing antibody evaluation test method (cNT), what this test carried out methodology validation is magnificent blue 70 parts of biology
CA10 positive source of people serum, cNT testing result is from laboratory, National Institute for Food and Drugs Control testing result, cNT examination
It tests and is carried out with pNT test in same laboratory, therefore the serum titer that can be detected to two methods is compared, it can be to two
The consistency of kind method is analyzed.Statistics is carried out by comparing 70 parts of serum pNT serum titers and cNT serum titer
The analysis of Bland-altma consistency, comparison result are shown in figure.Analysis is it is found that pNT test and cNT test two methods serum inspection
Survey result consistency with higher.Known to detailed analysis: the mean value of two kinds of detection method result differences is 0.14,2 times of standards
Poor range is -0.37-0.65, and all the points are within the scope of 2 times of standard deviations.CNT is above by pNT testing inspection serum titer
Test serum potency it is sensitiveer to illustrate that pNT test is tested than cNT, it is contemplated that two methods result judgement standard is different, pNT
Testing inspection result is overall or has higher consistency with cNT testing inspection result.As a result as shown in Figure 8.
Based on the above embodiment, the present invention provides safety, sensitive, quickly, specifically, in easy and low-cost detection
With the method for antibody, large-scale screening test can be carried out into the cell in 96 holes.When maternity leave virus, it is only necessary to first thin to 293T
Dysuria with lower abdominal colic dye contains the plasmid of the structural gene of pseudovirus, then the RNA replicon of transfected virus again.The structural proteins base of virus
Because being structured on plasmid, can steadily be stored at -20 DEG C.Pseudovirus overcomes live virus that antigen easily occurs in succeeding generations
It drifts about the disadvantages such as low with operational safety.And pseudovirus has very high sensitivity for detection neutralizing antibody, only needs a small amount of
Serum just detects neutralizing antibody special in serum.Development and detection individual patients and crowd CV-B5 for viral vaccine is special
Anisotropic neutralizing antibody level has important application value.
The foregoing is merely the preferable specific embodiments of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art's change or replacement obtained without making creative work, are all answered
It is included within the scope of the present invention.
Claims (7)
1. a kind of for packing the recombinant expression plasmid of 10 type pseudovirus of Coxsackie virus A, it is characterised in that: the recombinant expression
Plasmid is named as CA10-EGFP-CAPSID plasmid, and the recombinant expression plasmid has in sequence table shown in SEQ ID NO:1
Nucleotide sequence.
2. recombinant expression plasmid according to claim 1, it is characterised in that: the recombinant plasmid uses green fluorescent protein
Reporter gene (EGFP) is the encoding gene of all structural proteins of CA10 after green fluorescence protein gene and 2A restriction enzyme site
(P1 gene).
3. recombinant expression plasmid according to claim 1, it is characterised in that: add egg between EGFP and P1 genetic fragment
White restriction enzyme site (2A restriction enzyme site ,-AITTL-).
4. a kind of method for expressing recombinant plasmid described in claim 1, it is characterised in that: the following steps are included: 1) will be green
The gene order of fluorescin and the gene order of all structural proteins of CA10 are spliced；2) it and by the restriction enzyme site of 2A protease inserts
Enter the leading portion of green fluorescence protein gene；3) design primer；
The primer sequence are as follows:
CA10-EGFP-F ACCCAAGCTGGCTAGCGCCACCATGGTGAGCAAGGGCG
CA10-EGFP-R gacttgagctccAAGGGTAGTAATGGCCTTGTAC
CA10-P1-F GCCATTACTACCCTTGGAGCTCAAGTCTCGACGCAAAAATCC
CA10-P1-R GGTGATGATGaccggtTAACATATTAGCTTGCTTGATACTGGATCG。
5. a kind of pseudovirus, it is characterised in that: by 10 structural proteins of Coxsackie virus A of CA10-EGFP-CAPSID plasmid expression
Assemble with the EV71 subgenomic RNA of EV71-LUC-REPLICON replicon transcription.
6. a kind of kit of the neutralizing antibody comprising pseudovirus described in claim 5, it is characterised in that: the reagent box body is used
In vitro detection enteron aisle Coxsackie virus A 10.
7. a kind of detection method for detecting 10 neutralizing antibody of Coxsackie virus A, it is characterised in that: comprise the steps of: institute 1)
It states pseudovirus to mix with the test antibodies sample being serially diluted, obtains mixture；2) sensitive with mixture infection CV-A10
Cell, qualitatively or quantitatively detected in sample to be tested according to signal caused by the representation of reporter gene in sensitive cell
The potency of CV-A10 specificity neutralizing antibody.
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