CN101096656A - Helicobacter pylori recombinant VacA monoclonal antibody and uses thereof - Google Patents
Helicobacter pylori recombinant VacA monoclonal antibody and uses thereof Download PDFInfo
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- CN101096656A CN101096656A CNA2007100784983A CN200710078498A CN101096656A CN 101096656 A CN101096656 A CN 101096656A CN A2007100784983 A CNA2007100784983 A CN A2007100784983A CN 200710078498 A CN200710078498 A CN 200710078498A CN 101096656 A CN101096656 A CN 101096656A
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Abstract
The invention discloses a monoclonal antibody of Helocobacter pylori recombinant VacA protein through induced method for hybridomas cell in the mouse, which is characterized by the following: connecting Helocobacter pylori recombinant VacA protein specificly; fitting for manufacturing the ELISA agent box infected by VacA strain as clad antibody or treating VacA strain infection as negative immune agent; researching pathogenic mechanism of VacA with huge commercial value.
Description
Technical field
The present invention relates to recombinant monoclonal antibodies, particularly a kind of helicobacter pylori recombinant VacA monoclonal antibody and application thereof.
Background technology
From mucosa tissue, find helicobacter pylori (Helicobacter pylori from nineteen eighty-three Warren and Marshall, Hp) since, big quantity research has shown that this bacterium is the common cause that causes chronic gastritis and stomach ulcer, plays an important role in the generation of the recurrence of stomach ulcer and cancer of the stomach.The infection of Hp is very general, developing country is higher than developed country, susceptible type Childhood that developing country's epidemiological features being, the newborn infant of infant even birth some months may be infected, and rises with the speed of annual 3-10%, and the inspection of Hp not being infected because of various health check-ups is as conventional index, mostly be when causing the clinical symptom prescription on individual diagnosis and just find, in case infect, will get involved all the life, the self-healing rate is almost nil.The sickness rate of chronic gastritis is that 45-85%, peptide ulceration are that 10-20%, cancer of the stomach and lymphoma are 40.61/10 ten thousand among the infected.Infection rate in China general population is 50-80%, and increases with the speed of annual 1-2%.Prevention Hp infects, and the protection population health is had important practical significance.
At present, the virulence factor of having found Hp has multiple, and (vacuolating cytotoxin A, VacA) etc., wherein VacA is a kind of important morbid substance as flagellum, adhesion factor, urease, proteolytic ferment, toxin associated protein, vacuolate cytotoxin.1988, Leunk found that at first Hp has the toxin that makes the eukaryotic cell vacuolar degeneration, and called after vacuolate cytotoxin (VacA).1992, Cover and B1aster this toxin of having purified.Most scholars thinks that VacA belongs to the bacteriotoxin of A-B type structure, and promptly the 37000Da fragment is a toxicity subunit, and the 58000Da fragment is in conjunction with subunit.Its pathogenesis is not clear fully as yet, thinks that VacA can enter in the cell by receptor mediated endocytosis more, causes the endocytosis obstacle, finally causes cavity sample sex change in the cell; Can on the endochylema film, form simultaneously passage and the perviousness that strengthens polarization epithelium monolayer cell, cause necrocytosis.Though popular data shows 50%-60% population infection Hp, only there is the infected of 15% symptom to occur clinically.Almost there are all bacterial strains in other virulence factors of Hp, therefore can not explain the metainfective different clinical manifestations of Hp.And the gene of coding VacA exists in all bacterial strains, but has only the 50%-60% bacterial strain to express, and it is relevant with the virulence of infection strain whether the infected clinical symptom occurs.Therefore it is significant clinically to diagnose VacA to produce virus strain infection, also has important value at the prophylactico-therapeutic measures of VacA.
The about 3.8kb of DNA of coding vacA gene.Atherton etc. find that ((m1 m2), can be divided into Hp different vacA gene hypotypes to s2) different with two kinds intermediate zone sequences to three kinds of different signaling zone sequences of the interior existence of the vacA gene of Hp in view of the above for s1a, s1b.The vacA genotype has very strong regional disparity, studies show that China's genotype mostly is the s1m2 type greatly.Discipline Xu Zhun etc. carry out the vacA gene sequencing to the representational Chinese Hp bacterial strain of five strains, use the methods analyst of icp gene sequence difference distance then, find that four strains China m2 type vacA gene is similar each other, and its gene difference distance obviously is different from homotype west bacterial strain.The gene of coding toxicity subunit and deferent segment is similar each other between Chinese bacterial strain, compares with the west bacterial strain then to form independently one group.Because the vacA gene is polymorphism, causes the difference of expressing protein structure.Therefore be used for diagnosis at the vacA gene product of Chinese strain and control is more suitable for domestic clinical.
Prior art has successfully been expressed the VacA recombinant protein, and identifies that it has good antigenicity and immunogenicity, because recombinant VacA monoclonal antibody is not prepared in the existence of preparation method's loaded down with trivial details and various influence factors at present as yet.
Summary of the invention
The objective of the invention is (1): provide a kind of hybridoma cell line ZYA2, ZYB5, ZYC8, ZYD4 and a kind of this hybridoma that utilizes of helicobacter pylori recombinant VacA monoclonal antibody can secreted continually and steadily with inducing the proteic monoclonal antibody of anti-helicobacter pylori recombinant VacA (at the recombinant VacA monoclonal antibody of Chinese strain) that method obtains in the homology mouse body.(2), provide application and helicobacter pylori recombinant VacA monoclonal antibody the application in the reagent of preparation corresponding vaccine and treatment VacA positive strain infection of this monoclonal antibody in the ELISA test kit that the strain of the preparation diagnosis VacA positive is infected.
The concrete technical scheme that adopts is as follows:
A kind of hybridoma cell line ZYA2, ZYB5, ZYC8, ZYD4 that can secrete helicobacter pylori recombinant VacA monoclonal antibody continually and steadily, they are made by following steps: the helicobacter pylori VacA recombinant protein that adopts the genetically engineered abduction delivering to obtain is the antigen immune BALB/c mouse, the splenocyte of immune mouse, active SP2/0 murine myeloma cell greater than 95% are carried out cytogamy, and colony screening is the hybridoma cell strain of secrete monoclonal antibody continually and steadily.
Described immune mouse spleen cell carries out cytogamy with active SP2/0 murine myeloma cell greater than 95% in the 10:1 ratio, and the molecular weight of used promotor PEG is 4000 during cytogamy, and concentration is 50%, and cytogamy is placed in 96 orifice plates and cultivates.
In its preparation process, merge the antibody that the back began to detect in the culture supernatant in 7-10 days in hybridoma.
Described hybridoma obtains the proteic monoclonal antibody of anti-helicobacter pylori recombinant VacA with inducing method in the homology mouse peritoneal inoculum.
Wherein the dosage 1 * 10 of hybridoma is inoculated in the abdominal cavity
7Milliliter, volume is 0.5 milliliter.The whole preparation process flow process as shown in Figure 1
The preparation of VacA recombinant protein
By BLAST the representational Chinese Hp bacterial strain toxicity of five strains of discipline Xu Zhun order-checking subunit is carried out homology relatively, obtaining a height homologous gene fragment is goal gene.VacA gene order with Hp (AF050320) type strain serves as according to the design primer, with the clinical strains genome is template, through pcr amplification, amplifies toxic fragment V from the vacA gene, be cloned among the prokaryotic expression carrier PQE-30, transform and express a large amount of abduction deliverings of bacterium DH5 α.
The sequential analysis of recombinant plasmid PQE-30-V shows, the gained sequence of complete is compared the homology that height is arranged with the Chinese bacterial strain of document.Recombinant plasmid transformed is used Ni after expressing a large amount of abduction deliverings of bacterium DH5 α
2+-NTA resin purification expressing protein, SDS-PAGE analyzes visible single band, and image software analysis revealed purity can reach more than 93%.Western blot identifies that NC film colour developing back corresponding band occurs at relative molecular weight 27000 places, and size is consistent with expection, shows that recombinant protein has good antigenicity; It is 0.8mg/ml that the Bradford method is measured recombinant protein content; Normal serum identifies that the rabbit anti-serum VacA antibody of visible recombinant protein is positive before the rabbit anti-serum that detects recombinant protein with Helico Blot test kit and the immunity, and contrasts negatively, shows that recombinant protein has good immunogenicity.This recombinant plasmid PQE-30-V is stored in this laboratory.
The present invention adopts hybridoma technology to prepare the monoclonal antibody of anti-helicobacter pylori reconstitution cell vacuolating cytotoxin VacA toxic fragment, has obtained success, is applied to following gordian technique in preparation process:
A. the splenocyte of immune BALB/c mouse and SP2/0 myeloma cell's integration percentage
It is 10: 1 that the present invention selects the splenocyte of immune BALB/c mouse and SP2/0 myeloma cell's integration percentage for use.
B. merge the selection of promotor
It is 4000 that the present invention selects molecular weight for use, and concentration is that 50% PEG is as merging promotor.
Principle: cytogamy is the key link of experiment.Select for use PEG as merging promotor during fusion, PEG molecular weight 1000-6000 is all available, and the most handy 4000.The typical concentration scope of PEG is 30%-50%, and it is low to be lower than 30% fusion rate, be higher than 50% o'clock toxic.The molecular weight of PEG is 4000 o'clock, and with 30%, 40%, 50% 3 kind of concentration is made comparisons, and the result shows that 50% fusion rate is higher relatively and toxicity is little relatively, can be used as the suitable concentration of cytogamy.When the concentration of PEG was increased to 50%, PEG may combine with the moisture of adjacent membrane, and the space moisture that has only several dusts between the cell is replaced, and reduced the polarity of cell surface thus, caused the lipid bilayer instability, caused the fusion of cytolemma.Simultaneously, be the success that guarantees fusion, myeloma cell's activity is also quite important, has only activity greater than 95% o'clock, could guarantee the success of merging.The used PEG molecular weight of the present invention is 4000, and concentration is 50%, obtains good syncretizing effect.It should be noted that: in operating process, it is soft that action is wanted, and notes preventing to pollute.When adding incomplete nutrient solution, it is slow to try one's best, it is too much, too fast to avoid liquid to add, it is dead to cause cell to rise brokenly, because under the effect of the PEG of centrifugal and high density, the cell of shrinkage can very fast expansion be in a kind of unsure state cell because add incomplete nutrient solution owing to dewater.Liquid is too fast, too many when adding, and cell may rise brokenly.Thereby, slowly dripping when progressively balancing each other in the incomplete nutrient solution process when being in the cell of height under oozing with external environment, the cytolemma under playing pendulum just is easy to merge.
Selection the present invention of C, fused cell culture plate preferably selects for use 96 orifice plates to cultivate fused cell.
Principle: can place 24 orifice plates and 96 orifice plates to cultivate after the cytogamy, preferably select for use 96 orifice plates to cultivate, its advantage is as follows:
1) the cell clone growth more disperses, in case form the many tentatively clonings of clone, probably just can obtain the clone of single hybrid cell in former generation growth, thereby increase the chance of building strain.
2) can reduce the competitiveness growth of different hybridomas in same hole, thereby avoid the hypertrophy of non-secretory hybridoma and constrain or cover the hybridoma of secretion monoclonal antibody ability.
3) pore capacities of 96 orifice plates has only 1/5 of 24 orifice plate capacity, thus can play certain inspissated to single hybridoma cell clone excretory antibody, thus the corresponding sensitivity that improves screening antibody.The density that cell after merging in addition distributes in 96 orifice plates also unsuitable too high (5 * 10
6Cell/ml) antibody screening and the subclone to help the later stage.In this test in 96 orifice plates every hole add 50ul, every mouse drips 2 96 orifice plates.
D, the time of getting hybrid cell culture supernatant detection antibody
The present invention adopts hybridoma to merge the antibody that the back began to detect culture supernatant in 7-10 days.
Principle: because the hybridoma colony in the hole of ELISA test positive may not be to come from individual cells, may be mixed with the not clone of secretory antibody, and the clone of secretory antibody does not have metabolic advantage, it is faster than the clonal growth of secretion antibody, so will carry out antibody test and cloning early cultivates, in case after detecting positive hole, heal cloning early better, the hybrid cell hypertrophy that is not produced antibody with the clone who avoids secretory antibody is flooded.
When E. preparing the VacA monoclonal antibody, the quantity and the concentration of mouse peritoneal inoculation hybridoma
The present invention is with mouse peritoneal inoculation preparation monoclonal antibody the time, and the hybridoma total amount of inoculation is 1 * 10
7Ml, volume are 0.5ml.Principle is as follows:
Abdominal cavity inoculation is during with a large amount of preparation monoclonal antibody, the formation of ascitic tumor and the output of monoclonal antibody are subjected to the influence of many factors, comprise the whiteruss preform injection dosage, cause the timed interval inoculating to the abdominal cavity, hybridoma quantity and concentration, the growing state of hybridoma itself and the factors such as strain, sex and age of mouse of abdominal cavity inoculation from whiteruss.Wherein, the hybridoma quantity of inoculation and concentration are important factor.Because the inoculation volume generally is 0.5-1ml, thereby inoculum density changes with the inoculating cell sum.The present invention is when preparation ascites, and the hybridoma total amount of employing is 1 * 10
7Ml, volume are 0.5ml, all form ascites, and output are higher.
The present invention has carried out the evaluation of various indexs to hybridoma cell strain and monoclonal antibody, and the result is as follows:
1. hybridoma chromosome examination
Carry out chromosome counting after getting hybrid cell strain dyeing, learn by statistics and handle, the hybridoma chromosome number is 90 ± 4 as a result, and SP2/0 myeloma cell is 3 of 64 scholars, and mouse boosting cell is 40; As seen the telocentric chromosome of myeloma cell's significant karyomit(e) (middle part and inferior middle part kinetochore) and mouse boosting cell.Show that this clone really is the hybridoma that mouse bone-marrow-derived lymphocyte and murine myeloma cell merge.
2. the evaluation of hybridoma secretion monoclonal antibody ability
It is 1: 1.28 * 10 through the tiring of monoclonal antibody that mouse ascites produces that ELISA detects cell strain
4, show that this cell strain has the ability of the high titre antibody of secretion.
3. hybridoma cell strain stability is identified
Determine that through the square formation titration the suitableeest wrapper sheet concentration of VacA is 0.3ug/0.1ml,, can guarantee the accurate detection of indirect ELISA antagonist with the rVacA wrapper sheet of this concentration.Detect 223 holes altogether, treat that gaging hole OD value/negative hole OD value 〉=2.1 have 68 holes, identical with finding of naked eye, see the hybridoma growth that the clone is arranged in the respective aperture under the mirror; Through behind two time clonings, treat that only there are 25 holes gaging hole OD value/negative hole OD value 〉=2.1 again, be respectively four strain of hybridoma sources, carry out mono-clonal once more after, all clone hole ELISA detect all positive, it is stable preferably illustrate that hybridoma has.Hybridoma cell strain in 30 generations of external continuous biography, 6 liquid nitrogen cryopreservation resuscitations and liquid nitrogen cryopreservation were recovered after 1.5 months repeatedly, measured with indirect elisa method respectively, its excretory monoclonal antibody is tired constant, it is 1: 1.28 * 10 that ascites is tired
4, show the hybridoma cell strain good stability.
4. other evaluation of the immunoglobulin class of Zhi Bei monoclonal antibody, subclass and light chain type
Be the critical index of confirming McAb, to the purifying of McAb with use and have important directive significance.Laboratory animal immunity of the present invention totally four times, the immunity time is longer relatively, and adopting the Hybridoma Cell Culture supernatant is sample, and avoids using the ascites that contains monoclonal antibody, because also contain all kinds of Ig of mouse self in ascites, can influence qualification result.Antibody subtype detection kit with HyCult biotechnology company is identified: ZYA2 is the IgG2b class as a result; ZYB5 is the IgM class; ZYC8 and ZYD4 are the IgG1 class.Light chain is the κ type.
5. the evaluation of the antigen-binding specificity of monoclonal antibody
Immunoblotting assay is on nitrocellulose filter, in VacA antigen swimming lane zone a tangible specific band (27KDa) appears only, and occur at the no specific band in contrast factor hole (being HpaA antigen swimming lane), show that this monoclonal anti physical efficiency combines with VacA antigen generation specificity.
The application of recombinant VacA monoclonal antibody
The present invention adopts indirect elisa method with prepared monoclonal antibody, has set up the antigenic test method of HpVacA on the detection stomach mucous membrane sample, and HpVacA produces virus strain infection with diagnosis, and this test method can be used for producing commercial test kit.
With the indirect elisa method of setting up to 50 parts of clinical gastric mucosas at random sample carry out the HpVacA Detection of antigen, wherein with rapid urease test (Hp detects test paper fast provides available from Zhuhai clone Science and Technology Ltd.), the direct smear microscopy is defined as 30 parts of Hp male, negative 20 parts.Compare with Helico Blot test kit immunoblotting assay (the biological limited engineering corporation of the Ai Like of Huizhou City product, by specification operation) simultaneously.Result's (seeing Table 1) is in full accord.Wherein in the Hp positive sample, the positive rate of VacA also conforms to bibliographical information, shows that the antigenic indirect elisa method of being set up of detection HpVacA can further prepare commercial kit.
50 parts of gastric mucosa samples of table 1 HpVacA Detection of antigen result
| Sample (50 parts) | ||
| Positive | Negative | |
| Helico Blot test kit | 26 | 24 |
| The self-control indirect elisa method | 26 | 24 |
Utilize monoclonal antibody provided by the invention can prepare the ELISA test kit that the positive strain of diagnosis VacA is infected, also can prepare the reagent that corresponding vaccine and the positive strain of treatment VacA are infected.
The invention has the beneficial effects as follows that reorganization HpVacA monoclonal antibody and preparation method do not appear in the newspapers both at home and abroad, this method can be used for preparing in a large number the monoclonal antibody of VacA, the VacA monoclonal antibody of preparation can be used for producing diagnosis VacA and produces the ELISA test kit of virus strain infection, also can be used as the passive immunization preparation and be used for the treatment of VacA product virus strain infection, in scientific research, also can be used for immunohistochemistry, mechanism of causing a disease with research VacA has huge commercial value.
Description of drawings
The process flow sheet of accompanying drawing 1 preparation recombinant VacA monoclonal antibody
Specific embodiment (totally 11 examples)
The detection of embodiment 1 animal immune and serum antibody titer
(1) animal immune
Get about 6～8 age in week 2 of female SPF BALB/c mouse, the VacA recombinant protein is made into the protein solution of 200 μ g/ml with physiological saline, behind Fu Shi Freund's complete adjuvant (available from Beijing ancient cooking vessel state) the fully emulsified mixing of equal proportion, get 1ml (protein content is 100 μ g/ml) suspension down and subcutaneous multi-point injection immunity such as neck, every mouse 1ml/ time respectively at belly, the fossa cubitalis, inguinal region, jaw; At interval after two weeks, the suspension that adds freund 's incomplete adjuvant with the VacA recombinant protein of same dosage carries out the subcutaneous multi-point injection of nape portion, immunity once more; In first three sky of cytogamy, carry out booster immunization with 100 a μ g/ abdominal injection with the VacA recombinant protein.Immunity is 3 times altogether.Other gets a normal BALB/c mouse and compares.
(2) detection of serum antibody titer (indirect elisa method)
The square formation volumetry is determined the best effort concentration of antigen and enzyme labelled antibody:
Respectively with 10,6,3,1.5,1 μ g/ml VacA recombinant protein is antigen coated polyethylene micropore plate, the 100ul/ hole, after spending the night in 4 ℃ of wet boxes, add immune mouse serum respectively and with sample diluting liquid sesquialter dilution (1: 10,1: 20,1: 40,1: 80,1: 160 ...) immune mouse serum do the square formation titration, with the not negative contrast of immune serum of homology, indirect ELISA detects the antibody of serum VacA recombinant protein, immune serum with high dilution reaches positive, and the antigen concentration that negative control OD value is lower, as the suitableeest antigen coated concentration, measure the best effort concentration of HRP mark goat anti-mouse igg antibody again.The best effort concentration of determining is the VacA recombinant protein: every hole 100 μ l (0.3ug), HRP mark goat anti-mouse igg antibody: 1: 5000.
Serum antibody titer detects:
Antigen coated: every hole 100 μ l (0.3ug) bag of VacA antigen of getting purifying is by the polystyrene reactant plate, and 4 ℃ are spent the night in the moisture releasing box.Abandon coating buffer, dried with washings washing 3 times and button, add 4 ℃ of sealing 2h in the rearmounted wet box of 200 μ l confining liquids, wash then 3 times and detain and do.
Detect antibody: add cells and supernatant to be checked, establish positive control, negative control and blank simultaneously.Negative control adds corresponding cell culture fluid, every hole 100 μ l; Positive control adds immune mouse serum (dilution in 1: 1000), 100 μ l/ holes; The blank hole adds PBS, and control wells is all established multiple hole.Put in the wet box 37 ℃ of incubations 1 hour.
Adding two resists: every hole adds 1: 5000 HRP mark goat anti-mouse igg antibody of 100 μ l, puts 37 ℃ of 30min in the wet box, washs 3 times and pats dry.
Colour developing: every hole adds 100 μ l substrate solutions, after 37 ℃ of lucifuges develop the color 15-20min in the wet box, measures the 450nm OD of place value and gets final product result of determination.
The result judges: with the zeroing of blank hole, as treat gaging hole OD value 2.1 times more than or equal to negative control hole, promptly be judged to be the positive; As treat gaging hole OD value 1.5 times less than negative control hole, promptly be judged to be feminine gender; As between 1.5 and 2.1, then be probable positive.
(3) result
Mice serum antibody production is measured in behind booster immunization 4 days, and 2 mice serum antibody titers were respectively 1: 320 and 1: 640, and selecting tires is that 1: 640 mouse is used for MONOCLONAL ANTIBODIES SPECIFIC FOR.
The preparation of embodiment 2 myeloma cell's suspensions
Merge first two weeks recovery myeloma cell (SP2/0), treat that the cell growth is stable after, be that 20 μ g/ml guanozola complete culture solutions cultivated for two weeks with final concentration, screening xanthoglobulin one guanine phosphoribosyl transferase deficient cell.In merging preceding 48 hours with cultivations of going down to posterity of SP2/0 cell, choose that growth is vigorous, form well, be in the cell of logarithmic phase.Before fusion, renew bright nutrient solution once, blow down gently, be collected in the 20ml centrifuge tube with suction pipe; 1000rpm 5-8 minute, cell is resuspended in the 50ml serum-free medium, and the adjustment cell count is 1-5 * 10
5/ ml is standby.Satisfy cytoactive more than 95%.
Embodiment 3 preparation feeder cell suspensions
Get non-immune mouse, dial and remove the eyeball bloodletting, separation of serum is made negative control.After the execution, get the 5ml serum-free medium and inject the abdominal cavity, aseptic absorption peritoneal macrophage suspension is with serum-free medium centrifuge washing once (1000rpm, 5 minutes); Be resuspended to the HAT nutrient solution that contains xanthoglobulin (H), thymidine (T), amino dish purine (A) and (adjust cell concn 2 * 10
5/ ml is inoculated in 96 well culture plates, and every hole 0.1ml contains 2 * 10
5Individual cell is cultivated under 37 ℃, 5%CO2 and saturated humidity condition.
Embodiment 4 preparation splenocyte suspensions
The eyeball bloodletting is extractd with immune mouse in behind the mouse booster immunization the 4th day, and separation of serum is used for positive control; Put to death mouse, spleen is won in aseptic exposure abdominal cavity, sterilization back, removes fat and reticular tissue, with serum-free medium flushing three times.The spleen immigration is filled in the culture dish of 5ml serum-free medium, place 200 order stainless steel sifts online, grind spleen gently with the syringe nook closing member, and, collect splenocyte suspension with the serum-free medium flushing, with 1000rpm, 5 minutes, abandoning supernatant was resuspended in the serum-free RPMI-1640, centrifuge washing once is suspended in the incomplete nutrient solution.Every mouse obtains 2.0 * 10 approximately
8Individual splenocyte, after the blue dyeing of 1% phenol not cytochrome account for 97%, adjusting cell count is 1.0 * 10
7/ ml.
Embodiment 5 PEG fused cells
The splenocyte and the myeloma cell of preparation are pressed 1: 6 mixed, in point end plastic centrifuge tube, be mixed into pasty state, with 1000rpm, 5 minutes, abandon supernatant, flick the centrifuge tube bottom with forefinger, make the cell precipitation agglomerate loose slightly.Centrifuge tube is placed 37 ℃ of beakers that are filled with water of temperature in advance, slowly inject 1ml50%PEG solution, left standstill for 90 seconds, add the 30ml serum-free mediums of 37 ℃ of pre-temperature immediately, make the PEG dilution and the termination cytogamy.With 800rpm, 8 minutes, abandon supernatant, be resuspended in the 40ml HAT nutrient solution.
Embodiment 6 indirect elisa method screening positive clone methods are with embodiment 1 (2)
The cloning screening of embodiment 7 antibody positive cells
Cell with in the curved suction pipe piping and druming antibody positive cell cultures plate hole is suspended in the complete culture solution.With blood counting chamber counting, adjust cell concn to 100,50,10/ml with the RPMI-1640 that contains 20% foetal calf serum.With the hybridoma suspension inoculation in containing 2 * 10
4In the culture plate of feeder cell, every hole adds 100 μ l, makes every hole contain 10,5,1 cells respectively.Under 37 ℃, 5%CO2 and saturated humidity condition, leave standstill cultivation, observe clone's formation situation and record in microscopically.1 all later half amounts are changed liquid, change liquid once in later every 2-3 days.Cloning was cultivated the back the 14th day, and it is big that cell colony grows to 1/4 hole, detected with the ELISA method to have or not the VacA antibody-secreting.Duplicate detection was once again in the 16th day.The 20th day, to get twice ELISA and detect all positive and microscopically is viewed as monoclonal hole and carries out cloning second time and cultivate, method all is same as first cloning cultivation.It is that the cultivation of cloning is for the third time carried out in monoclonal hole that the cultivation of cloning for the second time back selected the antibody-secreting positive and microscopically to observe in 5-7 days again, and 3 subclones, till clone's secreting specificity antibody of 100%.
Embodiment 8 enlarged culturing
Positive hole with 100% clone's secreting specificity antibody, examine under a microscope that to be cultured to about 2/3 hole of cell colony big, transitional cell to 24 well culture plate, treat that it grows to 2/3 hole when big, be transferred to 96 well culture plates, treat the cell well-grown, and grow to 3/4 hole when big, transitional cell is to the 5ml culturing bottle, at the bottom of cell covers with bottle 3/4 the time, and the cultivation of going down to posterity again.
Getting the strain of positive colony porocyte adopts limiting dilution assay to carry out 4 time cloningizations, four strain cells are wherein arranged along with clone's increased frequency, antibody positive cell growth hole increases to 100% from 60%, its excretory monoclonal antibody can with recombinant protein VacA specific combination, show the hybridoma cell strain that has obtained secreting anti-HpVacA monoclonal antibody.Choose cellular form and carry out enlarged culturing and frozen in cell growth hole preferably, and called after ZYA, ZYB, ZYC and ZYD (ZY represents first letter of operator and project leader's surname ", poplar " phonetic).
A large amount of preparations of embodiment 9 VacA monoclonal antibodies
(1) ascites preparation
Enlarged culturing positive hybridoma cell: by 10
5Individual cell/ml is from 96 well culture plates, 24 well culture plates, 6 well culture plates, 10ml culturing bottle, progressively enlarged culturing.Collected hybridoma: 1000rpm centrifugal 10 minutes, and collected hybridoma, and wash 1-2 time with serum-free medium, the flush away foreign protein of trying one's best, it is 10 that counting is made into concentration then
6The cell suspension of/ml.With 10
6An individual cell/mouse, under the aseptic condition abdominal injection before one week the abdominal cavity inoculate aseptic medical liquid paraffin 0.5ml/ BALB/C mice (especially the female mouse of multiparity is better) only.Hybridoma is with the breeding in a large number in mouse peritoneal of ascitic tumor form.Mouse web portion swells after 10-15 days, and ascites appears in intraperitoneal, and can collect ascites this moment, and centrifugal 10 minutes of 3000rpm gets supernatant.
(2) saturated ammonium sulphate salting-out process preliminary purification antibody
To collect the 4 times of dilutions of PBS of good ascites with 50% saturated ammonium sulphate with 0.02mol/L, PH7.2.Dropwise slowly add ammonium sulfate while shaking, make its concentration reach 50% saturation ratio, leave standstill then more than the 2h, make it form throw out.With 12000rpm, 15min abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Hocket 2-3 time with 50% saturated ammonium sulphate and 33% saturated ammonium sulphate grading extraction antibody (method is the same), at last precipitation is dissolved in the PBS (phosphate buffered saline buffer) of the 0.01M PH7.2 of small volume.Solute places dialysis tubing after treatment, with 0.01MPBS in 4 ℃ of dialysed overnight get final product the monoclonal antibody of purifying.
Embodiment 10 CHARACTERISTICS IDENTIFICATION
Other evaluation of the immunoglobulin class of monoclonal antibody, subclass and light chain type is a critical index of confirming McAb, to the purifying of McAb with use and have important directive significance.Antibody because of molecular weight size, carried charge, solubleness and with the different different types and the subclass of being divided into of antigenic action.This laboratory animal immunity totally four times, the immunity time is longer relatively, and wherein the antibody of three strains generation is IgG, and another strain is respectively IgM.The different antibodies type has different effector functions with subclass, and with regard to mouse IgG, IgG2a, IgG2b, IgG3 be the energy activating complement all, on the contrary IgG1.Aspect enhancing phagocytic cell phagocytic function, IgG1 is the strongest, secondly is IgG3, IgG2a, IgG2b.In the present invention, we adopt the Hybridoma Cell Culture supernatant is sample, and avoids using the ascites that contains monoclonal antibody, because also contain all kinds of Ig of mouse self in ascites, can influence qualification result.
Embodiment 11 ELISA methods detect stomach mucous membrane sample HpVacA antigen
1. techniqueflow:
Treated stomach mucous membrane → as antigen coated enzyme plate (18 hours, 4 ℃) → plate (3 times)+confining liquid washed (4 hours, 4 ℃) → monoclonal antibody of plate (3 times)+preparation → hatched (40 minutes washed, 37 ℃) → plate (3 times)+enzyme labelled antibody (horseradish peroxidase-labeled) → hatched (30 minutes washed, 37 ℃) wash plate (3 times)+substrate solution → hatched (20 minutes, 37 ℃)+stop buffer (10 minutes, lucifuge) → colour developing
2. gordian technique:
(1) processing of stomach mucous membrane sample
Stomach mucous membrane sample liquid nitrogen grinding, broken tissue, low-temperature centrifugation are got supernatant liquor.
(2) best effort concentration
Utilize the square formation volumetry to determine that best effort concentration is: gastric mucosa sample bag was by concentration 1: 2, and the monoclonal antibody extension rate is 1: 10, and the extent of dilution of ELIAS secondary antibody is 1: 2500.
(3) dividing value
50 parts of no Hp the infected's gastric mucosa detected result OD
450nmMean value is 0.25, and standard variance is 0.072, so the yin and yang attribute threshold value is 0.466, in order to use conveniently threshold value is decided to be 0.5.
Claims (7)
1, a kind of hybridoma cell line ZYA2, ZYB5, ZYC8, ZYD4 that can secrete helicobacter pylori recombinant VacA monoclonal antibody continually and steadily, they are made by following steps: the helicobacter pylori VacA recombinant protein that adopts the genetically engineered abduction delivering to obtain is the antigen immune BALB/c mouse, the splenocyte of immune mouse, active SP2/0 murine myeloma cell greater than 95% are carried out cytogamy, and colony screening is the hybridoma cell strain of secrete monoclonal antibody continually and steadily.
2, hybridoma cell line ZYA2, ZYB5, ZYC8, the ZYD4 that can secrete helicobacter pylori recombinant VacA monoclonal antibody continually and steadily as claimed in claim 1, wherein said immune mouse spleen cell carries out cytogamy with active SP2/0 murine myeloma cell greater than 95% in 10: 1 ratios, the molecular weight of used promotor PEG is 4000 during cytogamy, concentration is 50%, and cytogamy is placed in 96 orifice plates and cultivates.
3, hybridoma cell line ZYA2, ZYB5, ZYC8, the ZYD4 that can secrete helicobacter pylori recombinant VacA monoclonal antibody continually and steadily as claimed in claim 1 merges the antibody that the back began to detect in the culture supernatant in 7-10 days in hybridoma in its preparation process.
4, a kind of helicobacter pylori recombinant VacA monoclonal antibody is characterized in that the described hybridoma of claim 1 is obtained the proteic monoclonal antibody of anti-helicobacter pylori recombinant VacA with inducing method in the homology mouse peritoneal inoculum.
5, helicobacter pylori recombinant VacA monoclonal antibody as claimed in claim 4, the wherein dosage 1 * 10 of abdominal cavity inoculation hybridoma
7Milliliter, volume is 0.5 milliliter.
6, the application of a kind of helicobacter pylori recombinant VacA monoclonal antibody in the ELISA test kit that the positive strain of preparation diagnosis VacA is infected.
7, the application of a kind of helicobacter pylori recombinant VacA monoclonal antibody in the reagent that the positive strain of preparation treatment VacA is infected.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116120408A (en) * | 2022-12-09 | 2023-05-16 | 扬州大学 | B cell epitope target antigen carrying helicobacter pylori virulence factor, expression vector and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116120408A (en) * | 2022-12-09 | 2023-05-16 | 扬州大学 | B cell epitope target antigen carrying helicobacter pylori virulence factor, expression vector and application thereof |
| CN116120408B (en) * | 2022-12-09 | 2023-10-20 | 扬州大学 | B cell epitope target antigen carrying helicobacter pylori virulence factor, expression vector and application thereof |
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