CN103613664B - A kind of antibody molecule of avian influenza virus H 5 N 1 and detection kit thereof and purposes - Google Patents
A kind of antibody molecule of avian influenza virus H 5 N 1 and detection kit thereof and purposes Download PDFInfo
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- CN103613664B CN103613664B CN201310489280.2A CN201310489280A CN103613664B CN 103613664 B CN103613664 B CN 103613664B CN 201310489280 A CN201310489280 A CN 201310489280A CN 103613664 B CN103613664 B CN 103613664B
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Abstract
This application discloses a kind of antibody molecule of bird flu H 5 N 1, the variable region of heavy chain of wherein said antibody molecule comprises SEQ ID NO:1 and SEQ ID NO:2, and the variable region of light chain of described antibody molecule comprises SEQ ID NO:3 and SEQ ID NO:4.This antibody molecule can be prepared into detection reagent, detects bird flu H 5 N 1.
Description
Technical field
The application belongs to biomedical sector, specifically containing specific antibodies molecular sequences pattern, the antibody molecule of specific binding activity can be had, as the application of detection reagent in the ELISA detection method of high pathogenic avian influenza H5N1 virus with high pathogenic avian influenza H5N1 virus.
Background technology
High pathogenic avian influenza H5N1 virus belongs to orthomyxoviridae family's Influenza Virus, is the sub-thread minus-stranded rna virus with capsule structure, containing 8 RNA fragments, and known codified 11 kinds of virus proteins.H5N1 virus is high variant viral, at present China popular 4 kinds of representative strains: A/H5N1/Anhui/1/2005, A/Guangdong-Shenzhen/1/2011, A/Hubei/1/2010and A/Chicken/Hong Kong/AP156/2008, the antibody of these 4 kinds of representative strains of energy specific combination, just can be used as the reagent detecting China's H5N1 virus.
Current, enzyme linked immunoassay (ELISA) based on antigen-antibody specific binding activity be a kind of fast, convenient, the method for detecting virus that is suitable for Site Detection, it does not need accurate plant and instrument and complicated technology, specificity and sensitivity better, be easy to promote, scene reply when being particularly suitable for epidemic outbreak and the rapid screening of disease.Most domestic reagent is with H5N1 inactivation of viruses for detectable antigens goes to detect serum antibody, and principle is that application indirect ELISA principle detects the antibody of anti-avian influenza H5N1 in serum using the H5N1 inactivation of viruses after concentrated as antigen coated microwell plate.In addition, the H5N1 antibody ELISA detection kit being detectable antigens with H5N1 virus structural protein HA albumen is also had.And go the method for detectable antigens also there is no commercial test kit at home with antibody.Although by sensitivity and the specificity too late conventional amplification of nucleic acid detection virus of the ELISA method of antibody test antigen, but ELISA method is easy and simple to handle, do not need accurate plant and instrument, being applicable to the area that extensive examination, the site disposal of epidemic situation and experiment condition are poor, is a kind of effective method for detecting virus.But because H5N1 virus antigenic variation is fast, the antibody prepared for a certain year epidemic strain is not likely in conjunction with emerging epidemic strain, need constantly to prepare new antibody as detection reagent, limit commercial exploitation and popularization, and the standby antibody of this project system possesses the characteristic in conjunction with the current 4 kind H5N1 viruss popular in China, can be used as the detection reagent of ELISA method.
Phage antibody library technology is a kind of new technology of Dispersal risk, and phage antibody library can be used for Dispersal risk.By the gene recombination of the various antibody fragment of coding in phage gene, antibody molecule is made to be presented on phage surface with the form of fusion rotein, form one to collect and have numerous not homotactic antibody library, therefrom screen the antibody molecule of target protein, this technology possess easy, fast, do not need feature such as preparation immunogenic protein etc., obtain more application in antibody research field.
Summary of the invention
This application discloses a kind of antibody molecule of bird flu H 5 N 1, wherein the variable region of heavy chain of antibody molecule comprises SEQ ID NO:1 and SEQ ID NO:2, and the variable region of light chain of described antibody molecule comprises SEQ ID NO:3 and SEQ ID NO:4.
Preferably, the heavy chain 52-61 amino acids sequence of antibody molecule is SEQ ID NO:1; Heavy chain 101-104 amino acids is SEQ ID NO:2; Light chain 46-49 amino acids sequence is SEQ ID NO:3; Light chain 87-92 amino acids sequence is SEQ ID NO:4.
Preferred antibody molecule has the aminoacid sequence of SEQ ID NO:5.
Antibody molecule can be N end, inner or C is terminal modified, such as oligomerization and/or glycosylation and/or the modification of puting together with marker.
Disclosed herein as well is a kind of detection kit of the detection bird flu H 5 N 1 containing above-mentioned antibody molecule.
The application again discloses above-mentioned antibody molecule and is preparing the purposes in bird flu H 5 N 1 detection reagent.
Using the application's antibody molecule as ELISA detection reagent, can be specific with four kinds of H5N1 virus type strain lysates (A/Anhui/1/2005, A/Hubei/1/2010, A/Guangdong-Shenzhen/1/2011, with A/Chicken/Hong Kong/AP156/2008) combine, and be not combined with enterovirus EV 71, I type dengue virus, new A (H 1 N 1) virus (pdmH1N1), seasonal H1N1 virus, seasonal H3N2 virus and Type B influenza virus, there is very high specificity.
Accompanying drawing explanation
Fig. 1. the ELISA binding activities of 7 positive bacteriophage antibody molecules and A/H5N1/ANHUI/1/2005 strain is described.
Fig. 2. the specificity of the detection kit based on embodiment 1 antibody is described.
Fig. 3 illustrates the susceptibility of the detection kit based on embodiment 1 antibody.
Fig. 4 illustrates the amino acid sequence patterns of an antibody molecule in embodiment 1.
Embodiment
These feature and advantage of the application and other feature and advantage will become apparent after with reference to following embodiment.
The simple and efficient advantage that the application utilizes phage antibody library to possess in Dispersal risk, and consider in virus evolution process and also exist because height makes a variation the features such as the easy inactivation of murine antibody, the again Dispersal risk that cause be loaded down with trivial details, with deactivation A/H5N1/Anhui/1/2005 strain for target protein, the jumbo Human phage antibody library of screening chemosynthesis, therefrom obtain energy specific combination H5N1 virus antibody, and prepare the detection reagent of H5N1 virus with this antibody.
Specifically, first with A/H5N1/Anhui/1/2005 strain for target protein, screen business-like phage antibody library Tomlison I+J library, obtain secreting positive bacteriophage to H5N1 virus high specificity.Positive bacteriophage is carried out nucleic acid sequencing, obtains the nucleotide sequence of antibody molecule, thus obtain the aminoacid sequence of antibody molecule.By contrasting the aminoacid sequence of multiple antibody molecule, we find, there are best specific 3 positive antibody molecules there is identical aminoacid sequence, there is the heavy chain, the connection peptides that are positioned at 1-119 amino acids and be positioned at the light chain of 138-242 amino acids.Its variable region of heavy chain comprises SEQ ID NO:1 and SEQ ID NO:2, and variable region of light chain comprises SEQ ID NO:3 and SEQ ID NO:4.Concrete aminoacid sequence is as shown in SEQ ID NO:5.
Preferably, the heavy chain 52-61 amino acids sequence of antibody molecule is SEQ ID NO:1; Heavy chain 101-104 amino acids is SEQ ID NO:2; Light chain 46-49 amino acids sequence is SEQ ID NO:3; Light chain 87-92 amino acids sequence is SEQ ID NO:4.
Antibody molecule can be N end, inner or C is terminal modified, such as oligomerization and/or glycosylation and/or and marker, the such as modification of puting together of enzyme, fluorescence or emissivity marker.
The detection reagent prepared based on this antibody molecule, has good specificity and susceptibility to H5N1 virus type strain.
The phage secretion that this antibody molecule not only can be obtained by phage library screening obtains, and also by engineered means, can be obtained by the secretion of other expression systems.See embodiment 2.
Describe in detail below by embodiment.
Unless stated otherwise, below embodiment use reagent be analytical pure, coating buffer is 0.1MNaHCO
3solution (pH8.6); PBS is phosphate buffered saline buffer, containing 0.1M Na
2hPO
4-NaH
2pO
4; Confining liquid is the PBS solution containing 3%BSA.
Embodiment 1: preparation and A/H5N1/Anhui/1/2005 strain have the antibody of specific binding activity
1. screen phage antibody library, obtain positive colony.
Dissolve A/H5N1/Anhui/1/2005 strain to 100 μ g/ml with coating buffer, add (the coating buffer pH8.6NaHCO on the little culture dish of Nunc company production of 1ml
3), be placed in 4 DEG C, wet box and spend the night.Abandon coating buffer, wash 3 times with PBS, add 3.5ml confining liquid (PBS is containing 3%BSA), incubated at room 2hr.
Abandon confining liquid, wash 3 times with PBS, add 500 μ l Tomlison I+J libraries (purchased from Britain Camb MRC HGMP resource center) and 3.5ml confining liquid, incubated at room 2hr.
Abandon supernatant liquor, wash with PBS-0.1%TWEEN20.1st takes turns and washes 10 times, the 2nd, and 3 take turns and wash 20 times.
Add 500 μ lPBS-trypsin and add 50 μ l of10mg/ml trypsinase storage liquid to 450 μ l PBS), room temperature gently shakes 10min, sucking-off solution, is the positive bacteriophage elutriant screening and obtain.
Draw 250 μ l elutriants to be added to 1.75ml and to be in (purchased from TAKARA company) in the TG1 bacterium of logarithmic phase, hatch 30min, according to 1:10 for 37 DEG C
2, 1:10
4dilution bacterial suspension, is coated with 10 μ l original bacteria liquid and 10 μ l diluents (100 μ g/ml ampicillin and 1%glucose) on TYE flat board, 37 DEG C of overnight growth, counting colony count titre.Remaining bacterium liquid continues 37 DEG C, and 250rpm shakes grow overnight, collects bacterium liquid, the centrifugal 20min of 4000rpm, collects the elutriant that supernatant is amplification, can drop into the screening of next round.
Continue with the A/H5N1/Anhui/1/2005 strain bag of 10 μ g/ml by culture dish (the same to first round), the elutriant after amplification is dropped into the 2nd, 3 screening taken turns, screening step is same as described above.Adopt identical method, measure 3 take turns screening after elutriant titre results display after one to take turns the titre of elutriant all high than the titre of previous round elutriant, illustrate to take turns screening through 3, the phage clone containing positive antibody molecule obtains enrichment.
2. qualification and A/H5N1/Anhui/1/2005 strain have the positive antibody of specific binding activity to clone
Select the 3rd and take turns the bacterium colony 96 that elutriant grows, be inoculated on 96 well culture plates, add 100 μ l2 × TY agar powder nutrient solutions (containing 100 μ g/ml ammonia benzyl and 1% glucose) 37 DEG C, 250rpm incubated overnight.Draw 2 μ l overnight culture in 2 × TY agar powder nutrient solution (containing 100 μ g/ml ammonia benzyl and 1% glucose) of 200 μ l, 37 DEG C, 250rpm continues to cultivate 2hr.25 μ l2 × TY agar powder nutrient solutions (containing 100 μ g/ml ammonia benzyl and 1% glucose) and 10 are added in nutrient solution
9individual helper phage, 37 DEG C, 250rpm continues to cultivate 1hr, 1, the centrifugal 10min of 800g, abandon supernatant, resuspended bacterium liquid precipitate in 200 μ l2 × TY (containing 100 μ g/ml ammonia benzyls and 50 μ g/ml kantlex), 30 DEG C, 250rpm overnight incubation, the centrifugal 10min of 1,800g, is ELISA with 50 μ l supernatant liquors and measures.Use coating buffer, to wrap by 1 μ g/100 μ l A/Anhui/1/2005 strain in 96 hole elisa plates, be placed in 4 DEG C, wet box and spend the night.Abandon coating buffer, with PBS washing hole 3 times, add 350 μ l confining liquids, incubated at room 2hr closes.
Abandon confining liquid, with PBS washing hole 3 times, every hole adds 50 μ l phage supernatants and 50 μ l confining liquids, incubated at room 1hr.
Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add anti-M13 phage antibody 100 μ l, the incubated at room 1hr of the HRP coupling that 1:5000 doubly dilutes.
Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add 100 μ l tmb substrate nitrite ions, room temperature places 2-15min, treats blue appearance, adds 50 μ l1MH
2sO
4color development stopping, 450nm measures OD value.
Result as shown in Figure 1, has 7 to have very strong specific binding activity with A/H5N1/ANHUI/1/2005 strain in 96 phage antibody clones.
The sequencing of 7 positive antibody molecules.According to gene order synthesis sequencing primer, antibody gene segments is obtained with primer pcr amplification from phage clone, the nucleotide sequence of antibody molecule is measured by Hua Da genome company, applied biology software analysis sequence, show that in above-mentioned 7 clones, 3,5 is identical with 7 aminoacid sequences, is same antibody molecule, and this antibody molecule OD value is maximum, the strongest with the binding activities of target protein.
3. sequential analysis
By analysis, active 3 higher antibody amino acids sequence are as shown in sequence 5 (SEQ ID NO:5), and the amino acid sequence patterns of high reactivity antibody is: heavy chain 52-61 amino acids is: Threonine-Ile-Pro-serine-threonine-glycine-Arg-Pro-Threonine-Serine (T-I-P-S-T-G-R-P-T-S); Heavy chain 101-104 amino acids sequence is: the antibody molecule of Arg-Pro-glycine-Threonine (R-P-G-T).Light chain 46-49 amino acids sequence is: Leu-Ala-Vitro By Serine/arginine (L-A-S-R); Light chain 87-92 amino acids is: Ser-Ser-glutamine-Ser-Pro-Pro (S-S-Q-S-P-P); As shown in Figure 4, the aminoacid sequence in frame is the part in antibody molecule, immune response being had to keying action.Amino acid whose Position Number determines according to the coding reading frame on expression vector, contrasted by homology, as long as occur that in homology region the H5N1 antibody molecule of corresponding sequence is all in the application's protection domain.
Embodiment 2: the detection kit set up based on the antibody molecule of embodiment 1
1. the solubility expression of positive bacteriophage antibody molecule
Infect the HB2151 bacterium (OD600 is 0.4) of 200 μ l logarithmic phases with positive bacteriophage nutrient solution 10 μ l, place 30min for 37 DEG C.By bacterium liquid according to 1:10
2, 1:10
4and 1:10
6ratio is diluted, and is coated with (containing 100 μ g/ml ammonia benzyl and 1% glucose) the 37 DEG C of incubated overnight growths on TYE (solid agar medium) flat board of 50 μ l diluents.
Picking individual colonies in 100 μ l2 × TY (containing 100 μ g/ml ammonia benzyl and 1% glucose) 37 DEG C, 250rpm overnight growth.Inoculate 2 μ l in 200 μ l2 × TY (containing 100 μ g/ml ammonia benzyl and 1% glucose), 37 DEG C, it is 0.9 that 250rpm cultivates about 3hr to OD600.
Add 25 μ l2 × TY (containing 100 μ g/ml ammonia benzyl and 1% glucose), 30 DEG C, 250rpm shakes and spends the night.1,800g centrifugal bacterium liquid 10min, does ELISA binding activities with 50 μ l supernatants and measures.
Infect and have the HB2151 bacterium of positive bacteriophage to can be used as the engineering bacteria producing positive antibody molecule, directly carry out amplification cultivation, positive antibody can be obtained in bacterium liquid supernatant.
2, detection kit
The antibody-solutions obtained coordinates with suitable signal display system, can obtain the detection kit detecting bird flu H 5 N 1.The most frequently used signal display system is horseradish peroxidase system, comprises anti-, the substrate nitrite ion of phage-resistance two of horseradish peroxidase, as 3,3 ', and 5,5 '-tetramethyl benzidine (TMB), hydrogen peroxide and sulfuric acid.
Embodiment 3: the specificity of the detection kit of embodiment 2
Use coating buffer, to wrap by 50 μ l positive bacteriophage antibody molecules in 96 hole elisa plates, be placed in 4 DEG C, wet box and spend the night.Abandon coating buffer, with PBS washing hole 3 times, add 350 μ l confining liquids (PBS is containing 3%BSA), incubated at room 2hr closes.
Abandon confining liquid, with PBS washing hole 3 times, every hole adds following 10 μ l virus stain lysates respectively, comprise: H5N1 virus type strain (being so kind as to give by national Disease Control and Prevention Center): Anhui/1/2005 and A/Hubei/2010, A/Guangdong-Shenzhen/1/2011, A/Chicken/HongKong/AP156/2008, enterovirus EV 71, I type dengue virus, new H1N1, seasonal H1N1, seasonal H3N2 and Type B virus and 90 μ l confining liquids, incubated at room 1hr.Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add 50 μ l positive bacteriophage antibody molecules and 50 μ l confining liquids, incubated at room 1hr.Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, this albumin A of albumin A 100 μ l adding the HRP coupling that 1:5000 doubly dilutes is two to resist, incubated at room 1hr.Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add 100 μ l tmb substrate nitrite ions, room temperature places 2-15min, treats blue appearance, adds 50 μ l1M H
2sO
4color development stopping, 450nm measures OD value.As shown in Figure 2, antibody is only combined with H5N1 type strain result, and is not combined with all the other viruses, and specificity is better.
Embodiment 4: the susceptibility of embodiment 2 detection kit
Wrap by 50 μ l positive bacteriophage antibody molecules in 96 hole elisa plate (coating buffer pH8.6NaHCO
3), be placed in 4 DEG C, wet box and spend the night.
Abandon coating buffer, with PBS washing hole 3 times, add 350 μ l confining liquids (PBS is containing 3%BSA), incubated at room 2hr closes.
The A/Hubei/2010 tired by viral hemoagglutination as 1:32 carries out the serial dilution of 2 times of gradients, dilutes 8 gradients altogether, and adds lysate lytic virus.
Abandon confining liquid, with PBS washing hole 3 times, every hole add respectively 100 μ l dilute after H5N1 virus strain lysate, incubated at room 1hr.
Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add 50 μ l positive bacteriophage antibody molecules and 50 μ l confining liquids, incubated at room 1hr.
Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add the albumin A 100 μ l (this albumin A be two resist) of the HRP coupling that 1:5000 doubly dilutes, incubated at room 1hr.
Abandon supernatant liquor, with PBS-0.1%TWEEN20 washing hole 3 times, add 100 μ l tmb substrate nitrite ions, room temperature places 2-15min, treats blue appearance, adds 50 μ l1M H
2sO
4color development stopping, 450nm measures OD value.
Result shows as shown in Figure 3, and when the strain by hemagglutinative titer being 1:32 is diluted to 256 times, antibody still can specific combination H5N1 virus, and susceptibility is relatively good.
Claims (4)
1. an antibody molecule for bird flu H 5 N 1, wherein said antibody molecule has the aminoacid sequence of SEQ ID NO:5.
2. detect a detection kit for bird flu H 5 N 1, described detection kit comprises the antibody molecule of bird flu H 5 N 1 according to claim 1.
3. detection kit according to claim 2, the phage-resistance two that described test kit also comprises horseradish peroxidase coupling resists, substrate nitrite ion, hydrogen peroxide and sulfuric acid.
4. the antibody molecule of bird flu H 5 N 1 is preparing the purposes in bird flu H 5 N 1 detection reagent, and described antibody molecule has the aminoacid sequence of SEQ ID NO:5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101092456A (en) * | 2007-06-15 | 2007-12-26 | 中国疾病预防控制中心病毒病预防控制所 | Gene engineering antibody of human source neutrality for anti virus H5N1 of bird flu |
| CN101220097A (en) * | 2007-01-26 | 2008-07-16 | 厦门大学 | Monoclone antibody of H5 subtype avian influenza virus haemagglutinin protein, or its combination liveness fragment and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101220097A (en) * | 2007-01-26 | 2008-07-16 | 厦门大学 | Monoclone antibody of H5 subtype avian influenza virus haemagglutinin protein, or its combination liveness fragment and uses thereof |
| CN101092456A (en) * | 2007-06-15 | 2007-12-26 | 中国疾病预防控制中心病毒病预防控制所 | Gene engineering antibody of human source neutrality for anti virus H5N1 of bird flu |
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