CN108660114A - One plant of secretion resists the hybridoma cell strain and preparation method thereof of 2 type Streptococcus suis surface protein Lmb monoclonal antibodies - Google Patents
One plant of secretion resists the hybridoma cell strain and preparation method thereof of 2 type Streptococcus suis surface protein Lmb monoclonal antibodies Download PDFInfo
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- CN108660114A CN108660114A CN201710187180.2A CN201710187180A CN108660114A CN 108660114 A CN108660114 A CN 108660114A CN 201710187180 A CN201710187180 A CN 201710187180A CN 108660114 A CN108660114 A CN 108660114A
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- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
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Abstract
The invention belongs to technical field of pathogenic microorganism immunological detection, are related to the hybridoma cell strain and preparation method thereof that one plant of secretion resists 2 type Streptococcus suis surface protein Lmb monoclonal antibodies.Present invention secretion resists 2 type Streptococcus suis surface protein Lmb mouse resource monoclonal antibody cell strains to be the separating spleen bone-marrow-derived lymphocyte when mouse internal antibody reaches top using the surface protein Lmb of 2 type Streptococcus suis 05ZYH33 of purifying as immunogen immune female Balb/c mouse；It is merged with murine myeloma cell SP2/0, the hybridoma cell strain that secretion resists 2 type Streptococcus suis surface protein Lmb mouse resource monoclonal antibodies is filtered out with ELISA method.The antibody titer of the present invention is very high, has specificity well.
Description
Technical field
The invention belongs to technical field of pathogenic microorganism immunological detection, are related to one plant of secretion and resist 2 type Streptococcus suis surfaces
The hybridoma cell strain and preparation method thereof of albumen Lmb monoclonal antibodies.
Background technology
Streptococcus suis (Streptococcus suis, S.suis) is a kind of pathogen of important zoonosis, can
Infected pigs or people cause the various clinicals disease such as acute sepsis, pneumonia, meningitis, arthritis, endocarditis and acute death,
Massive losses are brought to pig breeding industry, while public health service is caused to seriously threaten.According to the antigenicity of capsular polysaccharide,
S.suis can be divided into 33 serotypes, wherein 1,2,1/2,7,9,14 types have it is pathogenic, it is pathogenic most strong with S.Suis 2.I
The S.suis epidemic situations that state broke out in Jiangsu and Sichuan in succession respectively in 1998 and 2005 result in serious public health thing
Part causes the concern infected S.suis in world wide.The appearance of 2 multi-drug resistant bacterial strains of currently reported S.suis,
So that the prevention and control of S.suis 2 are faced with formidable challenges, screen pathogenic relevant surface protein and fast and accurately examine
It surveys and identifies that the research of bacterium infection method becomes the hot spot studied now.Early stage is in relation to 2 protective antigens molecules of S.suis and divides
The research of sub- detection means focuses primarily upon virulence factor.The virulence factor found before includes mainly capsular polysaccharide
(capsular polysaccharide, CPS), muramidase-released protein (muramidase releasedprotein, MRP),
Extracellular factor (extracellular factor, EF), hemolysin (suilysin, SLY) etc., studies have found that a great deal of
2 pathogenic strains of S.suis and be free of the above virulence factor, so that them is restricted as the use of vaccine.Therefore, carry out
The immunogenicity research of 2 newfound virulence relevant surfaces protein components of S.suis is Future Development safely and effectively novel epidemic disease
Seedling is of great significance.
Currently, the application range of authentic monoclonal antibody is more and more extensive, it is deep into each of entire biomedicine
A field, such as biochemistry, molecular biology, immunology, pharmacology, virology, bacteriology, parasitology, oncology, something lost
Pass, materia medica, haematol, endocrinology etc..In fact, antibody is used not only for medicine and biology, it applies model
It encloses and has been expanded to the various aspects such as agricultural, industry, basic research, environmental protection and food inspection.Especially in recent years, antibody
It is more and more active in terms of the prevention of animals and plants disease.It is excellent more to give full play to its as medical test reagent for monoclonal antibody
Gesture, the high specificity of monoclonal antibody substantially increase the specificity of antigen-antibody reaction, reduce and handed over other materials
The possibility for pitching reaction, makes test result Feasible degree bigger.Lmb albumen (Laminin binding protein) be one
The surface protein identified in Streptococcus suis, Lmb protein immunogenics are strong and are widely present in Streptococcus suis majority serotype, class
Topic group previous work also demonstrates that Lmb is a good diagnostic antigen.The successful invention of Lmb monoclonal antibodies is Streptococcus suis
Fast and accurately solid experiment basis has been established in diagnosis.
Invention content
The purpose of the invention is to establish quick, reliable 2 type Streptococcus suis detection method, one plant of secretion anti-2 is provided
The hybridoma cell strain of type Streptococcus suis surface protein Lmb monoclonal antibodies.
It is a further object of the present invention to provide the preparation methods of the hybridoma.
The purpose of the present invention can be achieved through the following technical solutions：
The hybridoma cell strain of one plant of anti-2 type Streptococcus suis surface protein Lmb monoclonal antibody, the hybridoma cell strain are logical
Following method is crossed to prepare：
(1) using tetra- immunization Female Balb/c mouse of surface protein Lmb of 2 type Streptococcus suis 05ZYH33 of purifying, make
Separating spleen bone-marrow-derived lymphocyte when mouse internal antibody reaches top；
(2) by the spleen bone-marrow-derived lymphocyte of step (1) separation and murine myeloma cell SP2/0 fusions；
(3) using recombination Lmb albumen after purification as antigen coat elisa plate, positive gram is screened using indirect elisa method
It is grand；Further to recombinate Lmb albumen as antigen, the positive colony that screening obtains is verified again using Western blot methods；
The positive colony that screening obtains is proved finally by chromosome counting, it is final to obtain hybridoma cell strain 2E3.
Wherein, step (1) is characterized in that：Select female 6 weeks Balb/c mouse, the recombination emulsified using complete Freund's adjuvant
Lmb albumen is immunogene, and injection protein content is 100 μ g/ mouse；It is spaced 2 weeks supplementary immunizations totally 3 times, and is changed to incomplete Freund
Adjuvant emulsion, injection protein content remain unchanged；3 days booster immunizations are primary before fusion, and injection protein content is 100 μ g/ mouse.
A method of preparing the hybridoma cell strain of secretion 2 type surface protein Lmb monoclonal antibodies of anti-streptococcus suis, packet
Containing following steps：
(1) using Freund's adjuvant, 2 tetra- immunization Female Balb/c of type Streptococcus suis surface protein Lmb of emulsification are small in equal volume
Mouse, separating spleen bone-marrow-derived lymphocyte when mouse internal antibody being made to reach top；
(2) by the spleen bone-marrow-derived lymphocyte of step (1) separation and murine myeloma cell SP2/0 fusions；
(3) it using recombination Lmb albumen after purification as antigen coat elisa plate, is used as envelope antigen using recombinating Lmb albumen
Indirect elisa method screening positive clone；Further verified again using recombinant protein Lmb as antigen with Western blot methods
The positive colony that ELISA is screened；The positive colony that screening obtains is proved finally by chromosome counting, is finally secreted
The hybridoma cell strain 2E3 of anti-2 type Streptococcus suis surface protein Lmb.
Wherein, step (1) is characterized in that：Select female 6 weeks Balb/c mouse, the recombination emulsified using complete Freund's adjuvant
Lmb albumen is immunogene, and injection protein content is 100 μ g/ mouse；It is spaced 2 weeks supplementary immunizations totally 3 times, and is changed to incomplete Freund
Adjuvant emulsion, injection protein content remain unchanged；3 days booster immunizations are primary before fusion, and injection protein content is 100 μ g/ mouse.
Advantageous effect
1. the present invention uses cell-fusion techniques, the splenocyte of mouse after recombinating Lmb protein immunizations is taken to melt with SP2/0 cells
It closes, through ELISA, Western Blot, Observation on Chromosome Number, successfully obtains one plant of secretion 2 type surface protein of anti-streptococcus suis
The hybridoma cell strain of Lmb monoclonal antibodies, is named as 2E3.
2. the hybridoma cell strain that the present invention obtains is injected into mouse peritoneal, ascites antibody is obtained, is obtained after purifying ascites
Monoclonal antibody, identified monoclonal antibody hypotype are IgG1 types, are 1: 819200 with indirect ELISA detection antibody titer, resist
Body potency is high.
3. monoclonal antibody prepared by the present invention is incubated altogether with Streptococcus suis, hence it is evident that the speed of growth of Streptococcus suis is influenced,
The chaining situation of Streptococcus suis is significantly affected by the Gram's staining scientific discovery antibody；Streptococcus suis 2-type surface protein Lmb
With preferable immunogenicity, the conservative of height and gene distribution popularity.The present invention is that the development of vaccine is laid a good foundation.
4. the 2 type surface protein Lmb monoclonal antibody specifics of anti-streptococcus suis of the present invention have specificity well, right
Streptococcus suis have recognition reaction, can be used as identify the cause of disease powerful, for the streptococcic quick diagnosis of people infected pigs,
Epidemiology detects and experiment basis is established in prevention and control.
Description of the drawings
Fig. 1 Lmb antibody titer testing results
The Western Blot results of Fig. 2 Lmb monoclonal antibodies and recombination Lmb albumen
The monoclonal antibody-purified results of Fig. 3 Lmb
Specific embodiment
Embodiment 1：Immune and cell fusion, screening and cloning
1. selecting 6 week old female BAl BIcs/c mouse.Use the recombination Lmb albumen that complete Freund's adjuvant emulsifies for immunogene,
Injection protein content is 100 μ g/ mouse；It is spaced 2 weeks supplementary immunizations totally 3 times, and is changed to incomplete Freund's adjuvant emulsification, inject albumen
Amount remains unchanged；3 days booster immunizations are primary before fusion, and injection protein content is 100 μ g/ mouse.
2. cell fusion, screening and cloning
Mouse booster immunization is sterile after 3 days to take spleen cell (2 × 108) and SP2/0 cells (4 × 107), add preheating
50%PEG merged, fused cell is suspended in HAT culture mediums, is distributed in 96 orifice plates containing feeder cells, 100 μ L/
Hole is set 37 DEG C, is cultivated in 5%CO2 incubators, and per 3-4 days, half measured and change HAT culture mediums, HT culture mediums was used after 7 days instead, after 14 days
Use complete 1640 culture medium instead.With recombination Lmb albumen coating, takes Hybridoma Cell Culture supernatant to be ELISA and carries out antibody test,
Positive colony carries out 3-4 time clonings by limiting dilution assay.
Embodiment 2：Indirect ELASA methods detect antibody
The recombination Lmb albumen of 100 μ l, 5 μ g/ml is added per hole, 4 DEG C three times, 200 μ l are added in coating, PBST board-washings overnight
37 DEG C of confining liquid (3%BSA) closing 1h, abandon confining liquid, and three times, 100 μ l Hybridoma Cell Culture supernatants are added in PBST board-washings, and 37
DEG C it is incubated 0.5h, then three times with PBST board-washings, ELIAS secondary antibody, 37 DEG C of incubation 0.5h is added, PBST board-washings three times, are added TMB and show
Color.
The results are shown in Figure 1, as a result shows：The hybridoma of one plant of stably excreting antibody is obtained through screening and cloning
Strain, is named as 2E3.The ascites prepared using this plant of hybridoma is detected through ELASA, antibody titer 1: 819200.
Embodiment 3：Western blot detections
The SDS-PAGE electrophoresis that Lmb albumen carries out 12% will be recombinated, electrotransfer is to pvdf membrane after electrophoresis；Film is taken out,
37 DEG C are closed 60 minutes, and TBST is washed three times；By film and 1:1000 4 DEG C of diluted ascites antibodies are incubated overnight, on second day shaking table
37 DEG C are reacted 60 minutes, and TBST is washed three times；Film is immersed in HRP-sheep anti mouse (1:5000) in, 37 DEG C are reacted 60 minutes, and TBST is washed
Three times；Exposure apparatus exposes, and shows band.
It can obtain：After recombinating Lmb albumen progress SDS-PAGE electrophoresis and transferring film, carried out with 2E3 ascitic type antibody
Western blot react band the results show that antibody has with recombination Lmb albumen at about 45KD.Antibody and Lmb albumen
Western blot results such as Fig. 2.
Embodiment 4：The preliminary purification of monoclonal antibody
Octanoic acid-ammonium sulfate method preliminary purification antibody.4 DEG C of ascites, 12000rpm centrifuge 15min, removal cell fragment and big
Protein aggregate.Ascites supernatant filter paper filters, after 60mM acetate buffer solutions (pH4.0) dilution of 4 times of volumes of filtrate, dropwise
Octanoic acid is added, 30min is stirred at room temperature, then 4 DEG C of standing 2h or more, make it fully precipitate, 12000r/min, 4 DEG C, 30min, receive
Collect supernatant, filtering supernatant is added the 10*PBS (0.1M pH7.4) of 1/10 volume, adds 0.277g according to the above-mentioned mixed liquors of every ml
Solid ammonium sulfate (under the conditions of 0 DEG C, 45% saturated ammonium sulfate be 0.291g/ml) is slowly added to sulphur under condition of ice bath while stirring
Sour ammonium, and stir frequently, in order to avoid causing local concentration excessively high, after ammonium sulfate is added entirely, it is stirred for 10-30min, keeps solution complete
Balance, stands at least 60min (general to stay overnight), 12000r/min, and 4 DEG C of centrifugation 30min are discarded supernatant, of short duration centrifugation will precipitate
It is dissolved in a small amount of PBS, is put into the bag filter handled well, dialyse 3 hours in 1000ml deionized waters, change liquid 3 times.Finally exist
Dialysed overnight in 0.01M PBS.The sample dialysed is added to the glycerine of final concentration 10%, -20 DEG C save backup, as a result as schemed
Shown in 3.
Claims (4)
1. one plant of secretion resists the hybridoma cell strain 2F1 of the monoclonal antibody of 2 type Streptococcus suis surface protein Lmb, feature to exist
It is prepared via a method which in the hybridoma cell strain：
(1) using tetra- immunization Female Balb/c mouse of surface protein Lmb of 2 type Streptococcus suis 05ZYH33 of purifying, make mouse
Separating spleen bone-marrow-derived lymphocyte when internal antibody reaches top；
(2) by the spleen bone-marrow-derived lymphocyte of step (1) separation and murine myeloma cell SP2/0 fusions；
(3) using recombination Lmb albumen after purification as antigen coat elisa plate, indirect elisa method screening positive clone is used；Again
Further to recombinate Sao albumen as antigen, the positive colony that screening obtains is verified again using Western blot methods；Finally lead to
Crossing chromosome counting proves the positive colony that screening obtains, final to obtain hybridoma cell strain 2F1.
2. secretion according to claim 1 resists the hybridoma cell strain of 2 type Streptococcus suis surface protein Lmb monoclonal antibodies
2F1, it is characterised in that：Female 6 weeks Balb/c mouse, the recombination Lmb eggs emulsified using complete Freund's adjuvant are selected in step (1)
It is immunogene in vain, injection protein content is 100 μ g/ mouse；It is spaced 2 weeks supplementary immunizations totally 3 times, and is changed to incomplete Freund's adjuvant breast
Change, injection protein content remains unchanged；3 days booster immunizations are primary before fusion, and injection protein content is 100 μ g/ mouse.
3. a kind of method for preparing secretion and resisting the hybridoma cell strain of 2 type Streptococcus suis surface protein Lmb monoclonal antibodies, special
Sign is to comprise the following steps：
(1) Freund's adjuvant 2 tetra- immunization Female Balb/c mouse of type Streptococcus suis surface protein Lmb of emulsification in equal volume are used, are made
Separating spleen bone-marrow-derived lymphocyte when mouse internal antibody reaches top；
(2) by the spleen bone-marrow-derived lymphocyte of step (1) separation and murine myeloma cell SP2/0 fusions；
(3) it is that envelope antigen uses indirectly to recombinate Lmb albumen using recombination Lmb albumen after purification as antigen coat elisa plate
ELISA method screening positive clone；Further ELISA sieves are verified again using recombinant protein Lmb as antigen with Western blot methods
Select obtained positive colony；Prove that the positive colony that screening obtains, final acquisition secretion resist 2 type pigs finally by chromosome counting
The hybridoma cell strain 2F1 of streptococcus surface protein Lmb.
4. the hybridoma according to claim 3 for preparing secretion and resisting the monoclonal antibody of 2 type Streptococcus suis surface protein Lmb
The method of cell strain, it is characterised in that：6 weeks female Balb/c mouse are selected in step (1), using complete Freund's adjuvant emulsification
Recombination Lmb albumen is immunogene, and injection protein content is 100 μ g/ mouse；It is spaced 2 weeks supplementary immunizations totally 3 times, and is changed to not exclusively
Freund's adjuvant emulsifies, and injection protein content remains unchanged；3 days booster immunizations are primary before fusion, and injection protein content is 100 μ g/
Mouse.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957057A (en) * | 2021-11-16 | 2022-01-21 | 西南大学 | Hybridoma cell, monoclonal antibody capable of neutralizing porcine lysin and detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113957057A (en) * | 2021-11-16 | 2022-01-21 | 西南大学 | Hybridoma cell, monoclonal antibody capable of neutralizing porcine lysin and detection kit |
| CN113957057B (en) * | 2021-11-16 | 2023-10-20 | 西南大学 | Hybridoma cell, monoclonal antibody capable of neutralizing pig lysin and detection kit |
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