CN106442981A - Human bocavirus type 1 antibody indirect ELISA diagnosis kit - Google Patents

Human bocavirus type 1 antibody indirect ELISA diagnosis kit Download PDF

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CN106442981A
CN106442981A CN201610782532.4A CN201610782532A CN106442981A CN 106442981 A CN106442981 A CN 106442981A CN 201610782532 A CN201610782532 A CN 201610782532A CN 106442981 A CN106442981 A CN 106442981A
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human bocavirus
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李毅
朱记平
冯小婷
王小梅
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Wuhan Bioengineering Institute
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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Abstract

The invention discloses a human bocavirus type 1 antibody indirect ELISA diagnosis kit. The kit comprises an antigen coated ELISA antibody detection plate and an enzyme-labeled secondary antibody, the antigen coated on the antibody detection plate is the truncated protein (VP2N) of 210 amino acids at the N terminal of a human bocavirus type 1 structure protein VP2, and the enzyme-labeled secondary antibody is a sheep anti-human enzyme-labeled secondary antibody. The amino acid sequence of the truncated protein VP2N is represented by SEQ ID NO.1, and the coding sequence of the truncated protein VP2N is represented by SEQ ID NO.2. The VP2N recombinant protein adopted in the invention is convenient for massive preparation and purification. The kit can be used for detecting a human bocavirus type infected antibody or diagnosing human bocavirus type infection, and has the advantages of simplicity and fastness in operation, short time, low cost, extreme suitableness for detecting a large amount of individual serum samples, and very high practicality.

Description

A kind of human bocavirus 1 type antibody indirect ELISA diagnostic kit
Technical field
The present invention relates to a kind of diagnostic kit is and in particular to a kind of diagnosis of human bocavirus 1 type antibody indirect ELISA tries Agent box.
Background technology
Human bocavirus 1 type (human bocavirus 1, HBoV1) is to carry out in children with acute respiratory tract infection sample Extensive examination and a kind of new parvovirus that find.Since human bocavirus 1 type be reported discovery after, some country and Area is in succession reported in the respiratory tract specimens of upper and lower Patients With Respiratory Tract Infection and this virus is detected it was demonstrated that human bocavirus Exist in worldwide, the popular of this virus becomes a global problem.In China, successively Zhejiang Province, Hunan Province, The place such as Beijing and Hongkong, Taiwan detects the sample of human bocavirus infection.Easily by human bocavirus The crowd of infection is mostly the infant of less than 2 years old, the related breathing tract disease causing mainly have pneumonia, bronchitis, rhinitis, Pharyngolaryngitis, also have a few studies report to lead to conjunctivitis, erythra, asthma etc. after infecting this virus.Its main clinical manifestation is to cough Cough, rhinorrhea, dyspnea and fever.
For the infection of human bocavirus, clinical symptoms can not efficient diagnosis cause of disease, making a definite diagnosis of its cause of disease must carry out Laboratory diagnosiss.The research work with regard to human bocavirus detection of oneself report is all using round pcr and Real-time at present PCR amplification technique.However, these methods exist complex operation, expensive the shortcomings of.Elisa (ELISA) Because it is easy to operate, quick, sensitive, high specificity the advantages of be widely used in the antigen of many cause of diseases or the inspection of antibody Survey.VP2 is the main antigen gene of human bocavirus 1 type, is also the focus of human bocavirus serodiagnosiss research.But VP2 albumen preparation difficulty is very big, and screening can be stablized and the strong antigen gene of the immunogenicity of great expression is to set up people Bo Ka disease The key of the reliable ELISA diagnostic method of poison infection.There is presently no a kind of stable and reliable human bocavirus serodiagnosiss Method, this is current human bocavirus preventing and treating urgent problem, sets up rapidly and effectively human bocavirus diagnostic method, Preventing and treating for human bocavirus has very important significance.
Content of the invention
It is an object of the invention to overcoming shortcoming and the deficiency of prior art presence, there is provided a kind of human bocavirus 1 type Antibody indirect ELISA diagnostic kit, this test kit energy specific detection goes out the antibody of human bocavirus 1 type, can be used for people and wins Block the metainfective antibody test of viral 1 type.
The purpose of the present invention is achieved through the following technical solutions：
A kind of human bocavirus 1 type antibody indirect ELISA diagnostic kit, including the ELISA antibody test of envelope antigen Plate and ELIAS secondary antibody.On described antibody test plate, coated antigen is the N-terminal 210 of human bocavirus 1 type Viral structural protein VP2 The truncated protein (VP2N) of aminoacid, its package amount is preferably 1 μ g/ hole.Described ELIAS secondary antibody is goat-anti people's ELIAS secondary antibody.Institute The aminoacid sequence of truncated protein of 210 aminoacid of N-terminal of human bocavirus 1 type Viral structural protein VP2 stated and its code sequence Row are respectively as shown in SEQ ID NO.1,2.
For convenience of using, described human bocavirus 1 type antibody indirect ELISA diagnostic kit can also include other Related reagent, such as cleaning mixture, nitrite ion, terminate liquid etc..
The truncated protein of described 210 aminoacid of N-terminal of human bocavirus 1 type Viral structural protein VP2 preferably passes through to include The method preparation of following steps：
(1) with human bocavirus 1 type genomic DNA as template, enter performing PCR amplification with following primer：
Forward primer：CGAGCTCTCTGACACTGACATTCAAGA,
Downstream primer：CCCAAGCTTATTGCCTCCAGCTGCA.
(2) by the PCR primer of amplification, expression vector pET28a (+) with reconnecting after Sac I, Hind III digestion, build Prokaryotic expression carrier pET28a-VP2N.
(3) use prokaryotic expression carrier pET28a-VP2N conversion e. coli bl21 (DE3), positive restructuring bacterium is lured Lead expression, purification obtains destination protein.Described induction, expression are specially：Positive restructuring bacterium is inoculated in containing kanamycin Carry out IPTG abduction delivering in LB culture medium；Collect Host Strains the ultrasonic degradation of expression, be used in combination with 8M carbamide dissolving inclusion body Membrane filtration, obtains destination protein through nickel agarose gel affinitive layer purification.
In test kit of the present invention, envelope antigen used is 210 amino of N-terminal of human bocavirus 1 type Viral structural protein VP2 The truncated protein (VP2N) of acid, VP2N be one section of antigenicity in human bocavirus 1 type Viral structural protein VP2 gene and hydrophilic all Very strong target sequence.VP2 gene code human bocavirus 1 type capsid protein, is concentrated mainly on new at present for the research of VP2 Recombinant vaccine aspect.Testing through the present invention proves, between being set up by the recombinant VP 2 N protein of prokaryotic expression Connect ELISA detection method, can be infected by antibody test specific human bocavirus 1 type, there is very strong practicality and popularization Property, there is boundless market prospect.
Compared with prior art, the invention has the advantages that：
(1) VP2N recombiant protein of the present invention is easy to prepare and purify in a large number, and VP2N gene is in pET prokaryotic expression Can be stablized in system and efficiently be expressed, the recombiant protein carrying histidine mark is easy to purification.
(2) diagnostic kit of the present invention has very strong practicality, can be used for the inspection of human bocavirus metainfective antibody Survey or the diagnosis of human bocavirus infection.
(3) kit results judgement of the present invention is convenient, sensitive, accurate, reliable, is developed the color using tmb substrate, uses enzyme mark Instrument measures OD value, judges the testing result of measuring samples；Yin and yang attribute result difference substantially, than OPD develop the color sensitiveer, reliable and Stable.
(4) test kit of the present invention is easy and simple to handle quick, can complete sample detection in 1.5h, takes short, low cost, very It is suitable for the detection of individual blood serum sample in a large number.
Brief description
Fig. 1 is the nucleic acid electrophoresis qualification figure of VP2N gene.Wherein, 1 and 2 swimming lanes are VP2N gene, and M is DL2000Plus.
Fig. 2 is the expression SDS-PAGE electroresis appraisal figure of VP2N recombiant protein.Wherein each swimming lane is：1：Protein Marker, 2：BSA(12μg)；3：The IPTG abduction delivering of pET28 empty vector control；4：2 times of VP2N recombiant protein inclusion body is dilute Release (applied sample amount 30 μ L)；5：5 times of dilutions (applied sample amount 30 μ L) of VP2N recombiant protein inclusion body.
Specific embodiment
With reference to embodiment, further detailed description is done to the present invention, but embodiments of the present invention not limited to this. If not specializing, the conventional meanses that in embodiment, technological means used are well known to those skilled in the art.
Embodiment 1
1st, the structure of the clone of human bocavirus 1 type VP2N gene and prokaryotic expression carrier
With reference to human bocavirus 1 type genome sequence (GU139423.1) design VP2N genetic fragment (coding in GenBank The genetic fragment of 210 aminoacid truncated proteins of N-terminal of human bocavirus 1 type Viral structural protein VP2) specific primer, used Primer sequence is：
Forward primer：CGAGCTCTCTGACACTGACATTCAAGA, underscore part is Sac I restriction enzyme site,
Downstream primer：CCCAAGCTTATTGCCTCCAGCTGCA, underscore part is Hind III digestion site.
By PCR expand size be 630bp VP2N specificity genes of interest, after enzyme action with pET28a (+) expression vector Connect, build prokaryotic expression carrier pET28a-VP2N, shown by PCR, double digestion and sequencing identification, successfully build VP2N base Because of prokaryotic expression carrier, its electrophoretogram is shown in Fig. 1.
2nd, the high efficient expression of VP2N recombiant protein and purification
By prokaryotic expression carrier pET28a-VP2N conversion BL21 (DE3), picking positive restructuring bacterium is inoculated in containing 50 μ g/mL 37 DEG C of shaken cultivation in the LB culture medium of kanamycin, when bacterium solution OD600When reaching 0.6, add the IPTG of final concentration of 0.5mM Carry out abduction delivering.Host Strains the ultrasonic degradation of great expression after abduction delivering 4 hours, are collected by centrifugation, with the dissolving of 8M carbamide Inclusion body with 0.45 μm of membrane filtration, through nickel agarose gel affinitive layer purification recombiant protein.SDS-PAGE electrophoresis reflects Determine to show, VP2N recombiant protein size is about 35kD, and its electrophoretogram is shown in Fig. 2.
Embodiment 2
1st, the preparation of ELISA antibody test plate
With the carbonate buffer solution of pH9.6 as being coated liquid, the VP2N recombiant protein of purification is diluted to finite concentration, presses 100 μ L/ holes add in ELISA Plate, and 4 DEG C are coated overnight.Wash plate four times with PBST, every hole adds 200 μ L 1 × blocking Close 1 hour for buffer37 DEG C, wash plate four times with PBS, room temperature is dried, load desiccant and carry out ELISA antibody is vacuum-packed to obtain Detection plate, puts 4 DEG C of preservations.
2nd, ELISA method operation sequence
(1) 10 × concentrated cleaning solution is diluted 10 times and be cleaning mixture.
(2) serum to be checked, VP2N antibody positive control serum and negative control sera all with antibody diluent (1 × Blocking buffer) make 1:100 times of dilutions, add in ELISA antibody test plate by every hole 100 μ L, 37 DEG C of incubation 40min, Dry.
(3) every hole adds 200 μ L cleaning mixture, washs 4 times, each 1min, dries.
(4) every hole adds 100 μ L ELIAS secondary antibody working solutions, 37 DEG C of incubation 40min, dries.
(5) every hole adds 200 μ L cleaning mixture, washs 4 times, each 1min, dries.
(6) every hole adds 100 μ L TMB nitrite ions, and 37 DEG C of lucifuges are incubated 5min.
(7) every hole adds 50 μ L terminate liquids, reads absorbance value (OD with microplate reader under 630nm wavelength630Value).
Above-mentioned cleaning mixture, 1 × blocking buffer, TMB nitrite ion, terminate liquid etc. are the conventional examination of ELISA method Agent.ELIAS secondary antibody working solution is to dilute 40,000 times of goat-anti people ELIAS secondary antibody (HRP- through 1 × blocking buffer Conjugated Affinipure Goat Anti-Human IgG (H+L), PTG).
Preparing of VP2N antibody positive control serum and negative control sera is as follows：The system of VP2N antibody positive control serum Standby：Select 3 monthly age large ear rabbits of health, the VP2N recombiant protein of intradermal injection 0.8 μ g/ μ L inside root of the tail or inside stock 0.5mL, head carried out a booster immunization every 2 weeks using same procedure after exempting from, and the 5th after the 4th immunity day carries out neck Arterial blood drawing, separate serum, add ten thousand/ thimerosal anti-corrosion, aseptic filtration.Negative control sera is not inject VP2N The negative rabbit anteserum of recombiant protein, add ten thousand/ thimerosal anti-corrosion, aseptic filtration.
3rd, the optimization of antigen coat amount
VP2N recombiant protein presses every hole 0.01 μ g, 0.03 μ g, 0.0625 μ g, 0.125 μ g, 0.25 μ g, 0.5 μ g, 1 μ g and 2 μ The concentration that is coated of g carries out square formation test, operation ibid, positive control serum under different antigen coat amounts, negative control sera OD630Value is shown in Table 1.Square formation test shows that the concentration that is most preferably coated of antigen is 1 μ g/ hole.
Table 1
4th, the criterion of testing result
Antibody test, testing result (OD are carried out to 36 parts of negative human serum samples630Value) it is shown in Table 2.Negative by calculating Meansigma methodss and standard deviation (X+2SD) obtain the cut-off value of this ELISA detection method.Treated by the cut-off value definition calculating The criterion of sample product is：As measuring samples OD630>=0.29 is judged to the positive；OD630When≤0.24, sample is judged to feminine gender；When 0.29 ＞ OD630It is judged to suspicious during ＞ 0.24, now sample need to be rechecked once, if OD630>=0.27 is judged to the positive, OD630≤ 0.27 is judged to feminine gender.
Table 2
0.225 0.203 0.234 0.229 0.229 0.242
0.252 0.247 0.231 0.256 0.239 0.231
0.265 0.28 0.273 0.262 0.267 0.266
0.229 0.229 0.242 0.275 0.27 0.284
0.252 0.247 0.231 0.268 0.256 0.273
0.265 0.28 0.273 0.248 0.251 0.258
5th, clinical serum detection
To 24 parts collecting, clinical human serum sample enters performing PCR detection, altogether the positive sample of 4 parts of human bocavirus infection of detection This 4 parts of serum are detected, testing result (OD by this through ELISA diagnostic kit630Value) it is shown in Table 3, it is consistent with PCR testing result, Test shows, the ELISA diagnostic method high specificity of the present invention, and result is reliable and stable, can specifically detect people Bo Ka disease Malicious antibody.
Table 3
Sample 1 2 3 4 5 6
OD630 0.304 0.306333 0.302667 0.347667 0.215 0.628
In table 3,1-4 infects positive sample for human bocavirus, and 5,6 are respectively VP2N antibody negative controls serum and the positive Control serum.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify, All should be equivalent substitute mode, be included within protection scope of the present invention.

Claims (6)

1. a kind of human bocavirus 1 type antibody indirect ELISA diagnostic kit it is characterised in that：ELISA including envelope antigen Antibody test plate and ELIAS secondary antibody；On described antibody test plate, coated antigen is human bocavirus 1 type Viral structural protein VP2 The truncated protein of 210 aminoacid of N-terminal.
2. human bocavirus 1 type antibody indirect ELISA diagnostic kit according to claim 1 it is characterised in that：Described Antibody test plate antigen package amount be 1 μ g/ hole.
3. human bocavirus 1 type antibody indirect ELISA diagnostic kit according to claim 1 it is characterised in that：Described ELIAS secondary antibody be goat-anti people's ELIAS secondary antibody.
4. human bocavirus 1 type antibody indirect ELISA diagnostic kit according to claim 1 it is characterised in that：Including Cleaning mixture, nitrite ion, terminate liquid.
5. human bocavirus 1 type antibody indirect ELISA diagnostic kit according to claim 1 it is characterised in that：Described The truncated protein of 210 aminoacid of N-terminal of human bocavirus 1 type Viral structural protein VP2 pass through the method system that comprises the steps Standby：
（1）With human bocavirus 1 type genomic DNA as template, enter performing PCR amplification with following primer：
Forward primer：CGAGCTCTCTGACACTGACATTCAAGA,
Downstream primer：CCCAAGCTTATTGCCTCCAGCTGCA；
（2）By amplification PCR primer, expression vector pET28a (+) useSacI、HindReconnect after III digestion, build protokaryon Expression vector pET28a-VP2N；
（3）Convert e. coli bl21 with prokaryotic expression carrier pET28a-VP2N, positive restructuring bacterium is carried out abduction delivering, pure Change and obtain destination protein.
6. human bocavirus 1 type antibody indirect ELISA diagnostic kit according to claim 5 it is characterised in that：Step （3）Described in induction, be expressed as：Positive restructuring bacterium is inoculated in the LB culture medium containing kanamycin and carries out IPTG induction Expression；Collect Host Strains the ultrasonic degradation of expression, dissolve inclusion body with 8M carbamide and use membrane filtration, through nickel agarose gel Affinitive layer purification obtains destination protein.
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CN108508217A (en) * 2018-06-29 2018-09-07 北京博奥森生物技术有限公司 A kind of indirect ELISA method kit and its application for detecting people's lower respiratory tract bocavirus
CN114703319A (en) * 2022-01-11 2022-07-05 武汉明了生物科技有限公司 Primer group and kit for identifying human bocavirus and application of primer group and kit
CN116953234A (en) * 2023-08-25 2023-10-27 中国农业科学院兰州兽医研究所 Pig bocavirus G3 type polypeptide-ELISA antibody detection kit
CN117129675A (en) * 2023-10-25 2023-11-28 首都儿科研究所 Reagent or kit for human bocavirus type specific detection or diagnosis
CN117126269A (en) * 2023-10-25 2023-11-28 首都儿科研究所 Type 1 human bocavirus type specific antibody and application thereof

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108508217A (en) * 2018-06-29 2018-09-07 北京博奥森生物技术有限公司 A kind of indirect ELISA method kit and its application for detecting people's lower respiratory tract bocavirus
CN114703319A (en) * 2022-01-11 2022-07-05 武汉明了生物科技有限公司 Primer group and kit for identifying human bocavirus and application of primer group and kit
CN114703319B (en) * 2022-01-11 2024-02-09 武汉明了生物科技有限公司 Primer group and kit for identifying human bocavirus and application
CN116953234A (en) * 2023-08-25 2023-10-27 中国农业科学院兰州兽医研究所 Pig bocavirus G3 type polypeptide-ELISA antibody detection kit
CN116953234B (en) * 2023-08-25 2024-05-17 中国农业科学院兰州兽医研究所 Pig bocavirus G3 type polypeptide-ELISA antibody detection kit
CN117129675A (en) * 2023-10-25 2023-11-28 首都儿科研究所 Reagent or kit for human bocavirus type specific detection or diagnosis
CN117126269A (en) * 2023-10-25 2023-11-28 首都儿科研究所 Type 1 human bocavirus type specific antibody and application thereof
CN117126269B (en) * 2023-10-25 2024-02-13 首都儿科研究所 Type 1 human bocavirus type specific antibody and application thereof
CN117129675B (en) * 2023-10-25 2024-02-13 首都儿科研究所 Reagent or kit for human bocavirus type specific detection or diagnosis

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