CN105294848B - A kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody and its preparation and application - Google Patents
A kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody and its preparation and application Download PDFInfo
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- CN105294848B CN105294848B CN201410283323.6A CN201410283323A CN105294848B CN 105294848 B CN105294848 B CN 105294848B CN 201410283323 A CN201410283323 A CN 201410283323A CN 105294848 B CN105294848 B CN 105294848B
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- monoclonal antibody
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Abstract
The present invention relates to bioengineering technical fields, the present invention provides a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody, the polypeptide for being used to prepare the monoclonal antibody, the hybridoma cell strain for secreting the monoclonal antibody and the monoclonal antibodies for detecting application of the Gigantoxin-4 in and in Gigantoxin-4 haemocylolysis.
Description
Technical field
The present invention relates to bioengineering technical fields, and in particular to a kind of sea anemone cytolysin Gigantoxin-4 is mono-
Clonal antibody, the polypeptide for being used to prepare the monoclonal antibody, the hybridoma cell strain for secreting the monoclonal antibody and the Dan Ke
Grand antibody is for detecting application of the Gigantoxin-4 in and in Gigantoxin-4 haemocylolysis.
Background technique
China sea area is vast, and living marine resources are abundant, and operation on the sea is frequent.Operating personnel injures by toxic marine organisms
The case where happen occasionally, toxic sea anemone sting is exactly a kind of common injuries from marine creature.
The tentacle and body of sea anemone contain a large amount of nematoblast, there is the cell of a special cryptomere in each nematoblast
Device, entitled nematocyst inside store venom, and thread tube is tortuous in nematocyst, and ecthoaeum tip is pricker, when by mechanical or change
When learning stimulation, ecthoaeum can be discharged and be pierced into prey body and injected toxin in prey body by thread tube.It is main after sea anemone sting
The symptom wanted is injured topoalgia, itches, and erythema, papule, nettle rash or bleb sample fash then occurs.Constitutional symptom have anxiety,
It is depressed, nauseous etc., it is serious muscle, tracheospasm, cardiovascular function failure and shock etc., or even death occur.For
Sea anemone sting can only be to symptomatic treatment currently without preferable treatment method.
Actinocongestin can be classified as neurotoxin and molten cell according to the difference of molecular weight and biological function
Plain two major classes.Gigantoxin-4 (GT-4) is a kind of sea anemone cytolysin, its maturation protein includes 178 amino acid, point
Son amount is 19kDa (GenBank:JQ353486.1).GT-4 can specifically bind the sphingomyelins on cell membrane, further in film
Upper formation hole is swollen so as to cause cell and cracks.GT-4 has very high hemolytic activity, half hemolysis score about 40ng/ml.
After the GT-4 for giving SD rat low dosage (30 μ g/kg), whole heart failure caused by myocardial contractive power declines is the master of rats death
Want reason；And after giving the GT-4 of high dose (60 μ g/kg), rat in 15min just as respiratory failure and it is dead.Pass through tissue
Pathological examination and blood parameters analysis, GT-4 can lead to the multiple organ injury of SD rat, as the heart, lung, liver, kidney occur
Different degrees of multifocal bleeding or necrosis, it is especially the most serious with lung.In conjunction with Vitro Experimental Results, GT-4 leads to the one of SD rat
Serial pathological change is all the consequence of its haemocylolysis, if it is possible to find the drug or antibody for inhibiting its haemocylolysis, it will
It is a kind of important method for treating sea anemone sting.(referring to document: Hu B, Guo W, Wang LH, Wang JG, Liu XY, Jiao
BH.Purification and characterization of gigantoxin-4,a new actinoporin from
the sea anemone Stichodactyla gigantea.Int J Biol Sci.2011；7(6):729-39).
It there is no the relevant report of sea anemone cytolysin Gigantoxin-4 antibody at present.
Summary of the invention
The purpose of the present invention is to provide a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody, it is used to prepare this
The application of the polypeptide of monoclonal antibody, the hybridoma cell strain for secreting the monoclonal antibody and the monoclonal antibody.
The first aspect of the present invention is used to prepare sea anemone cytolysin Gigantoxin-4 monoclonal there is provided one kind and resists
The polypeptide of body, amino acid sequence is as shown in SEQ ID NO:1.
Asp Val Ile Leu Pro Glu Phe Val Pro Asn Asn Lys (SEQ ID NO:1)
The second aspect of the present invention, there is provided a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody, be by
The hybridoma cell strain that deposit number is CCTCC No:C201473, which is secreted, to be generated.
The third aspect of the present invention, there is provided a strain of hybridoma strains, are named as 14853-1hz-5/C377, it
Deposit number is CCTCC No:C201473.
The fourth aspect of the present invention, there is provided a kind of preparations of sea anemone cytolysin Gigantoxin-4 monoclonal antibody
The step of method, this method, is as follows:
A) synthetic amino acid array polypeptide as shown in SEQ ID NO:1；
B) using aforementioned polypeptides as immunogen immune mouse；
C) splenocyte for taking immunized mice, is merged with murine myeloma cell；
D) cell clone for going out synthesis polypeptide reacting positive through multi-turns screen, as the miscellaneous of anti-GT-4 protein monoclonal antibody
Hand over tumor cell strain CCTCC No:C201473；
E) by producing ascites antibody in mouse Inoculation hybridoma, ascites is purified, anti-human GT- is obtained
4 monoclonal antibody.
The fifth aspect of the present invention, there is provided aforementioned polypeptides, monoclonal antibody, hybridomas in preparation Western
Application in Blot or immunohistochemistry detection reagent or kit, the Western Blot or immunohistochemistry detection reagent or reagent
Box is for detecting Gigantoxin-4.
And aforementioned polypeptides, monoclonal antibody, hybridoma are further provided in preparation and Gigantoxin-4 is molten
Application in the drug of blood effect.
The present invention provides the monoclonal antibody of anti-GT-4, monoclonal antibody identification is located at GT-4 amino acid sequence
The epitope of DVILPEFVPNNK.
The method that the art routine can be used in the acquisition of monoclonal antibody of the present invention, i.e., by mouse Inoculation
Hybridoma simultaneously generates ascites antibody, and taking-up ascites is purified and obtains.
Hybridoma cell strain of the invention is preserved in China typical culture collection center, deposit number CCTCC No:
C201473, prepared monoclonal antibody are a kind of immunoglobulins, can specifically bind GT-4 albumen.
The present invention also provides application of the monoclonal antibody in Western Blot detection GT-4.
The present invention provides new thinking for the clinical personalized treatment application of sea anemone sting.
Detailed description of the invention
Fig. 1 is monoclonal antibody Western Blot detection GT-4.
Wherein, swimming lane 1 is GT-4,2 albumen marker of swimming lane.
In Fig. 2 monoclonal antibody and the haemocylolysis of GT-4.
Hybridoma cell strain of the invention, is named as 14853-1hz-5/C377, is preserved in China typical culture collection
Center (abbreviation CCTCC), preservation date on June 19th, 2014, deposit number CCTCC No:C201473, preservation address: China
Wuhan Wuhan University.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail.But the following example should not be regarded as to the present invention
The limitation of range.Experimental method in following embodiments is unless otherwise specified conventional method.
The preparation of the polypeptide sequence selection and polypeptide of the monoclonal antibody of the anti-GT-4 of embodiment 1.
It is analyzed according to GT-4 active site and transmembrane structure, selects SEQ ID NO:1 for epitope, entrust Ai Bimate
Biological medicine (Shanghai) Co., Ltd. synthesis polypeptide, sequence are as follows:
Asp Val Ile Leu Pro Glu Phe Val Pro Asn Asn Lys (SEQ ID NO:1)
Entrust Ai Bimate biological medicine 's synthesis polypeptide that aforementioned polypeptides are connected to corresponding carrier
On, it prepares and purifying antigen is reacted for follow-up immunization.
The preparation and purification of the monoclonal antibody of the anti-GT-4 of embodiment 2.
The polypeptide antigen purified in embodiment 1 is used to be immunized, female BAl BIc/c mouse 3 of 8-12 week old is immunized, adopts
Mouse tachysynthesis mode is taken, initial immunity carries out muscle note using subcutaneous injection and rear both legs are carried out under the skin around both shoulders
It penetrates, each region is injected intraperitoneally with about 1/8 immunogene by the 1/2 of residual immunity original.50 μ g of booster immunization after 1 week,
Complete Freund's adjuvant, intraperitoneal injection.50 μ g of booster immunization after 2 weeks, incomplete Freund's adjuvant (IFA), intraperitoneal injection.3 weeks and with
Afterwards, 50 μ g booster immunizations are directly injected intraperitoneally weekly.
The splenocyte and murine myeloma cell SP2/0 cell (being purchased from ATCC) for the mouse for taking ELISA detection effect best
With 5:1 ratio, under the action of PEG1500, merged.Fused cell is by appropriate dilution, and be placed in the culture of 96 holes
It is cultivated in plate, 37 DEG C, 5%CO2Under the conditions of cultivate, cultivate progress ELISA detection in 10-14 days, select thin in the high hole of OD value
Born of the same parents carry out limiting dilution assay subclone.
The specific method is as follows: by the cell culture of limiting dilution into 96 orifice plates, when the 1/6 of clonal growth to complete opening,
Monoclonal and polyclonal is marked, ELISA detection is carried out to monoclonal.It is after ELISA detection that the highest monoclonal of OD value is limited again
It is as above subcloned described in method again in dilution 96 orifice plates of access, this process is repeated several times, until positive hole ratio is 100%, i.e.,
Think that this for monoclonal, that is, builds the successful cell strain of strain.
Above-mentioned hybridoma cell strain, is named as 14853-1hz-5/C377, is preserved in China typical culture collection center
(abbreviation CCTCC), preservation date on June 19th, 2014, deposit number CCTCC No:C201473.
The positive monoclonal that screening is obtained expands culture, and cell number presses 106/ pipe is frozen.It is collected simultaneously cell peace
Arrange ascites preparation.Cell strain prepares ascites, 10-14 days collection ascites using mice celiac inoculation, and ELISA detection ascites is
It is no to be successfully prepared.Purifying uses ProteinG affinity chromatography.ProteinG affinity column takes ascites after balancing pillar with PBS
Column is crossed, then column is washed to OD value close to zero with PBS, is eluted with the glycinate acid solution of 0.1mol/L, collect eluent, measurement
The OD value of each collecting pipe, calculating antibody concentration.
The identification of the monoclonal antibody of the anti-GT-4 of embodiment 3.
1. potency is identified
By polypeptide antigen (4 μ g/ml) be coated with elisa plate, by the anti-GT-4 monoclonal antibody of purifying press 1:6.25K, 1:
12.5K, 1:25K, 1:50K, 1:100K, 1:200K are diluted, and are added in ELISA Plate hole, and HIS antibody (HIS antibody is selected
1mg/ml1:8K uses milk to dilute) as the positive control of system operatio, 5% milk is as negative control.Sheep is added after reaction
Anti- mouse IgG-HRP, colour developing.Positive judgement standards: the ratio between sample detection hole OD450 and negative control hole OD450 (P/N) >=2.1,
The result shows that antibody titer is 200K (table 1).
1 antibody titer qualification result of table
| 6.25K | 12.5K | 25K | 50K | 100K | 200K | It is negative | It is positive | Potency |
| 3.205 | 2.711 | 2.364 | 1.475 | 0.954 | 0.607 | 0.036 | 2.917 | 200k |
2. subtype identification
Using the mouse source monoclonal antibody hypotype identification kit (the Mouse Monoclonal Antibody for being purchased from Sigma company
Isotyping Reagents) subtype identification, subtype identification knot are carried out to obtained anti-GT-4 monoclonal antibody of the invention
Fruit shows that monoclonal antibody is IgG2b hypotype, the results are shown in Table 2.
2 antibody subtype qualification result of table
| Sample dilution gradient | IgG1 | IgG2a | IgG2b | IgG3 | IgM | IgA |
| 1:10 | 0.323 | 0.321 | 2.167 | 0.579 | 0.374 | 0.363 |
| 1:50 | 0.3 | 0.296 | 1.997 | 0.318 | 0.304 | 0.288 |
| 1:250 | 0.229 | 0.272 | 1.44 | 0.228 | 0.244 | 0.193 |
| 15%HT | 0.075 | 0.088 | 0.393 | 0.088 | 0.1 | 0.093 |
| 1:10/1%BSA | 0.432 | 0.37 | 0.398 | 0.397 | 0.411 | 0.431 |
The application of the monoclonal antibody of embodiment 4.GT-4
1. the Western Blot of anti-GT-4 monoclonal antibody detects application
The GT-4 protein sample of denaturation carries out SDS-PAGE electrophoresis；By electroporation by protein delivery to nitrocellulose
Film；TBST containing 10% skimmed milk power closes 1h, and TBST is washed three times, each 5min；Antibody diluent dilutes anti-GT-4 monoclonal
Antibody primary antibody (1 μ g/ml) is incubated overnight, and TBST is washed three times, each 5min；Antibody diluent dilutes the anti-mouse two of HRP label
It is anti-, it is incubated for 1h, TBST is washed three times, each 10min；It is developed the color with enhanced HRP-DAB substrate colour reagent box, in 19kDa
There are specific band appearance, scanning result (Fig. 1) in left and right.
The result shows that the monoclonal antibody more can specifically identify GT-4 albumen.
2. in monoclonal antibody and the application of GT-4 haemocylolysis
It takes 1ml at acquaintance's blood, is rinsed with PBS, 3000g is centrifuged 5min, abandons supernatant, then rinse 2-3 times to supernatant with PBS
It is limpid, supernatant is abandoned, red blood cell and PBS are diluted by 1:50 then, that is, are prepared into 2% red cell suspension.Take red cell suspension
Then certain density sample is added in 0.5mL, react total volume 1.5mL.Every group separately sets negative control (toxin is not added), the positive
It compares (0.5%Triton X-100).In 37 DEG C of oscillation incubation 30min after sample-adding, then 3000g is centrifuged 5min, and supernatant is taken to exist
405nm absorbance value (being repeated 3 times) is detected in 96 orifice plates.Haemolysis score: haemolysis score=[(sample cell is calculated with following formula
Absorbance value-negative control pipe absorbance value)/(positive control pipe absorbance value-negative control pipe absorbance value] × 100%.
When sea anemone cytolysin GT-4 concentration is 40ng/ml, 100% erythrocyte hemolysis can be caused.
Antibody (1mg/ml) is diluted to various concentration by 1:100000,1:10000,1:5000,1:1000, takes 0.1ml
It is incubated for 5 minutes with 37 DEG C of GT-4 (40ng/ml), then carries out hemolytic activity measurement according to the above method, the results showed that antibody can be with
The haemocylolysis (Fig. 2) for neutralizing GT-4 well, can provide for haemolysis caused by clinical treatment sea anemone sting and complication
New approaches.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (7)
1. a kind of polypeptide for being used to prepare sea anemone cytolysin Gigantoxin-4 monoclonal antibody, amino acid sequence such as SEQ ID
Shown in NO:1.
2. it is CCTCC No:C201473 that a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody, which is by deposit number,
Hybridoma cell strain secretion generates.
3. a strain of hybridoma strain, its deposit number is CCTCC No:C201473.
4. a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody as claimed in claim 2 is in preparation Western
Application in Blot or immunohistochemistry detection reagent or kit.
5. one plant of hybridoma cell strain as claimed in claim 3 preparation Western Blot or immunohistochemistry detection reagent or
Application in kit.
6. a kind of sea anemone cytolysin Gigantoxin-4 monoclonal antibody as claimed in claim 2 in preparation and
Application in the drug of Gigantoxin-4 haemocylolysis.
7. one plant of hybridoma cell strain as claimed in claim 3 is in preparation and in the drug of Gigantoxin-4 haemocylolysis
Application.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020045736A1 (en) * | 2001-05-09 | 2002-04-18 | Xianxhang Yu | Lytic peptide prodrugs |
| CN102174097A (en) * | 2011-01-25 | 2011-09-07 | 中国人民解放军第二军医大学 | Actinoporin and application thereof |
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2014
- 2014-06-23 CN CN201410283323.6A patent/CN105294848B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020045736A1 (en) * | 2001-05-09 | 2002-04-18 | Xianxhang Yu | Lytic peptide prodrugs |
| CN102174097A (en) * | 2011-01-25 | 2011-09-07 | 中国人民解放军第二军医大学 | Actinoporin and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| NCBI Reference Sequence:AFC17962.1;NCBI;《NCBI》;20120303(第10期);第1页 * |
| Purification and Characterization of Gigantoxin-4, a New Actinoporin from the Sea Anemone Stichodactyla Gigantea;Bo Hu;《International Journal of Biological Sciences》;20110607;第7卷(第6期);第733页图2 * |
| 海葵溶细胞素Gigantoxin-4的分离、鉴定和功能研究;胡波;《中国优秀硕士学位论文全文数据库基础科学辑》;20111015;第65页第6段 * |
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