CN103834687A - Preparation method of periodic Wuchereria malayi compound polyvalent protein vaccine - Google Patents

Abstract

The invention discloses a preparation method of a periodic Wuchereria malayi compound polyvalent protein vaccine. The preparation method comprises the steps of extraction of recombinant plasmids of the periodic Wuchereria malayi pcDNA3.1(+)-BmCPI/BmGAPDH, gene transfection, screening of positive clones, extraction and enlarge cultivation of the positive clones, cryopreservation of the positive clones, detection of the positive clones, extraction of cell total RNA (Ribose Nucleic Acid), molecular level detection of expression of the recombinant plasmids in cells, protein level detection of expression of the recombinant plasmids in cells, SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analysis of expression products, extraction and purification of periodic Wuchereria malayi recombinant protein, and the like. The preparation method is simple and convenient, and good in effect.

Description

The preparation method of the compound multivalence protein vaccine of Periodic Brugia

Technical field

The present invention relates to the preparation method of the compound multivalence protein vaccine of a kind of Periodic Brugia.

Background technology

The popular people's the health of not only giving of filaricide is brought serious harm, also becomes one of major reason of driving into poverty by medical crises.The main method for the treatment of filaricide is to adopt chemicals at present, though medicine can reduce infection rate and sickness rate, but due to frequent application, still can not get rid of filaria has potential drug-fast danger, add that repetition chemotherapy and long-term monitoring measure incur great expense and large quantities of professional and technical personnel.Therefore, assist or alternative method for finding control filaricide, the research of filaria vaccine becomes the problem that people pay close attention to day by day.

Although nucleic acid vaccine demonstrates huge potential in the control of infectious diseases and tumour.But in actual applications, limit its widespread use and further developed because DNA vaccination exists some problem demanding prompt solutions, be mainly that safety issue has limited the application on human body, can cause the activation of oncogene or the inactivation of cancer suppressor gene as plasmid DNA is incorporated in host genome; The long-term expression of antibody in body may cause host's immunological tolerance or autoimmune response etc.; And plasmid DNA surprisingly imports the normal function that can affect cell in non-target cell.Secondly, DNA vaccination is because of reasons such as transfection efficiency in body are not good enough, the immune response strength that nucleic acid vaccine excites relatively a little less than, especially in large animal and human body, be not enough to cause enough immune protective effects.

Protein vaccine is that the gene of certain exogenous antigen of coding is imported to recipient bacterium (as intestinal bacteria) or cell, makes its high efficient expression in recipient bacterium or cell, then a class subunit vaccine made from this biosynthetic gene expression product.The key of protein successful expression is to select suitable expression system.Prokaryotic expression system is the most ripe exogenous protein expression system, simple to operate, can great expression albumen, but lack the processing Modifying Capability of Eukaryotic gene expression regulation mechanism and protein, expression product can not spontaneously fold curling generation the protein of certain space structure and particular organisms function, often exist with inclusion body form, after sex change renaturation, expression product easily loses again immunocompetence.And mammalian cell expression system possesses complete protein assembly and modification system, the conformation of expression product approaches natural protein more.

Summary of the invention

The object of the present invention is to provide one easy to prepare, the preparation method of the compound multivalence protein vaccine of effective Periodic Brugia.

Technical solution of the present invention is:

A preparation method for the compound multivalence protein vaccine of Periodic Brugia, is characterized in that: comprise the following steps:

(1) Periodic Brugia pcDNA3.1(+) extracting of-BmCPI/BmGAPDH recombinant plasmid:

(1) join by getting 50ul after the bacterial classification room temperature thawing that contains recombinant plasmid the shaking table cultivation 12-16h that is placed in 37 ℃ of constant temperature, 250rpm in the 100ml LB solution that contains 50ul, 100mg/ml ampicillin, place afterwards 4 ℃ of refrigerators cooling;

(2) bacterium liquid is cultivated after 12-16h, uses ultraviolet spectrophotometer to measure its OD600 value, if OD600 numerical value between 0.6-0.8, shows the logarithmic phase of bacterium in vigorous growth, can be used for carrying out the extracting of plasmid;

(3) get bacterium after above-mentioned cultivation to 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation, abandons supernatant;

(4) every pipe adds 5ml solution I (suspension), re-suspended cell precipitation;

(5) every pipe adds 5ml solution II (lysate) afterwards, puts upside down centrifuge tube 4-6 time, and room temperature is placed 3-5 minute, makes the complete cracking of bacterium, and solution is transparent;

(6) every pipe adds 5ml solution III (in conjunction with liquid), puts upside down immediately centrifuge tube and mixes for 4-6 time, and visible white floss produces, the then centrifugal 10-20 minute of 5000rpm room temperature;

(7) supernatant is poured into or is drawn in plasmid purification column, centrifugal 2 minutes of 5000rpm room temperature, abandons collection liquid in pipe;

(8) in plasmid purification column, add 7ml solution IV (washings), centrifugal 2 minutes of 5000rpm room temperature, washes away impurity, abandons collection liquid in pipe, and centrifugal 2 minutes of 5000rpm room temperature more afterwards, removes residual liquid and trace ethanol is volatilized completely;

(9) plasmid purification column is placed on 50ml centrifuge tube, add 2ml solution V(elutriant) to pipe inner cylinder, place 2 minutes, then centrifugal 2 minutes of 5000rpm room temperature, adds 10ml solution VI(DNA purifying in conjunction with liquid in gained liquid) be added in former plasmid purification column after mixing;

(10) room temperature, after centrifugal 2 minutes, is abandoned collection liquid in pipe again;

(11) in plasmid purification column, add 15ml solution IV (washings), centrifugal 2 minutes of 5000rpm room temperature, a nearly step washes away after impurity, abandons collection liquid in pipe;

(12) centrifugal 2 minutes of 5000rpm room temperature, removes residual liquid and trace ethanol is volatilized completely;

(13) plasmid purification column is placed on 50ml centrifuge tube, adds 2ml solution V(elutriant) to pipe inner cylinder, place 2 minutes;

(14) centrifugal 2 minutes of 5000rpm room temperature, gained liquid is and turns the ultrapure plasmid of cell grade;

(15) get aqueous dna, with 400 times of distilled water dilutions, make blank with distilled water, on ultraviolet spectrophotometer, read the mensuration concentration of sample and the value of A260/A280, and be stored in-20 ℃ for gene transfection;

Described solution I is the suspension of a large amount of extraction agent boxes of plasmid; Solution II is the lysate of a large amount of extraction agent boxes of plasmid; Solution III is the combination liquid of a large amount of extraction agent boxes of plasmid; Solution IV is the washings of a large amount of extraction agent boxes of plasmid; Solution V is the elutriant of a large amount of extraction agent boxes of plasmid; Solution VI is that the DNA purifying of a large amount of extraction agent boxes of plasmid is in conjunction with liquid;

(2) recovery of Hela cell, cultivate and go down to posterity

(1) from-80 ℃ of refrigerators, take out the EP pipe of frozen Hela cell, and directly drop in 37 ℃ of thermostat water baths, shake frequently makes its thawing;

(2) in the time melting completely, from 37 ℃ of warm water, take out EP pipe, 1200 revs/min, centrifugal 3 minutes;

(3) abandon supernatant, by the DMEM perfect medium re-suspended cell precipitation that contains 10% volume calf serum and dual anti-(penicillin and Streptomycin sulphate), be inoculated in culturing bottle, be placed on 37 ℃, the standing cultivation of 5%CO2 cell culture incubator, change nutrient solution next day one time, continue to cultivate;

(4) at the bottom of Hela cell covers with culturing bottle 70%～80% when full, go down to posterity, outwell the nutrient solution in culturing bottle, by 2ml PBS damping fluid washed twice;

(5) add 0.25%W/V pancreatin lmL digestion lmin, in the time that starting to come off, outwells a bottle floor cells pancreatin, add 8mL to contain 10% foetal calf serum and dual anti-DMEM perfect medium, blow and beat a bottle floor cells with liquid-transfering gun and it is come off and mix, draw 4mL and continue to cultivate to another culturing bottle;

(3) gene transfection

(1) shift to an earlier date 24 hours inoculating cell 55 holes in 96 orifice plates, every kind of concentration is established 5 multiple holes, and inoculum size, for making within second day, grow up to 25% individual layer, is put 37 ℃ of overnight incubation in CO2 incubator;

(2) nutrient solution is changed into the substratum containing microbiotic G418, antibiotic concentration increases progressively by gradient: 0 μ g/ml, 100 μ g/ml, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml and 1000 μ g/ml;

(3) cultivate and be as the criterion with most necrocytosis concentration for 10-14 days, finally obtaining best screening concentration is 400 μ g/ml, when screening stably express clone, improves a rank than this concentration, uses the half of screening concentration while maintaining;

(4) transfection the day before yesterday, by 0.25%W/V trysinization for cell, counting bed board, making it is 80-90% at transfection cytogamy on same day degree, cell bed board 2mL containing foetal calf serum and dual anti-DMEM substratum in; In six orifice plates, carry out in the following manner transfection, a, b in contrast:

A.No DNA transfection control(DNA transfection blank)

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/BmGAPDH；

(5), for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 4ug～5ug DNA, preparation in batches when porous operation;

(6) for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 10ul Lipofect lipofectamine, after Lipofect dilution, the DNA with dilution in 5min mixes, soaking time is long can reduce activity, can prepare in batches;

(7) the Lipofect lipofectamine of the DNA of mixed diluting and dilution, at room temperature insulation 15min;

(8) transfection media washed cell before transfection starts in use 2ml/ hole, transfection media is replaced by the not DMEM substratum containing serum;

(9) mixture of dilution is joined in the middle of cell, wave and culture plate, mixes gently in 37 ℃, 5% CO2 and is incubated 24-48h;

(4) screening of positive colony

(1) transfection was gone down to posterity transfectional cell in the ratio of 1:10 after 24 hours in 6 orifice plates, cultivate again the G418 that the substratum in six orifice plates after 24h is replaced by the 400 μ g/ml concentration of determining in trial test and select substratum, then positive cell is screened by screening culture medium according to the cell of different growing states below:

A.No DNA transfection control(DNA transfection blank)

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/GAPDH

Add by the G418 screening culture medium preparing, when screening, improve a grade concentration than preliminary experiment;

(2), according to the color of substratum and Growth of Cells situation, within every 2～3 days, change primary screening substratum; When observing in a, b, c in the most of death of most cells or a cell when all dead, substratum G418 concentration is reduced by half and maintains screening as maintain base, treat that it increases gradually, now serum-concentration can add greatly 15%;

(3), in the time having seen under the microscope positive colony formation, prepare picking clone;

(5) extraction of positive colony and enlarged culturing

(1) observation of cell clone, makes marks to the positive colony that will separate in culture plate bottom surface with marker pen;

(2) remove substratum, rinse gently cell clone with PBS;

(3) cultivate ring with clone of aseptic tweezer gripping, dip sterilized Vaseline, Vaseline face sweeps away, and presses in culture plate on eugonic positive monoclonal, make Vaseline be evenly distributed on clone and cultivate ring bottom surface, clone's ring is in required cloning cluster;

(4) repeating step (3) on the six same holes of orifice plate, by 2 to 3 other required cloning cluster of snare;

(5) add 0.25% pancreatin and be full of in ring, after 20 seconds, discard pancreatin;

(6) cover six orifice plates, 37 ℃, 15 minutes;

(7) in each ring, add the substratum of 0.1ml to 0.2ml;

(8) successively each clone is blown and beaten with cell dispersion with suction pipe, and cell suspension is moved into respectively in the hole of 24 orifice plates, each cell clone should be equipped with suction pipe or rifle head separately;

(9) rinse clone's ring with the substratum of 0.1ml to 0.2ml again, then these substratum are moved into respectively in corresponding 24 orifice plates;

(10) substratum in culture hole is added to 0.5ml, build, continue to cultivate;

(11) in the time that clone cell covers with 24 orifice plate, get final product in had digestive transfer culture to six orifice plate, add 2ml substratum and continue enlarged culturing;

In (12) six orifice plates, cell is passaged to culturing bottle continuation cultivation after covering with, until frozen;

(6) positive colony is frozen

After positive colony cell (pcDNA3.1 (+), pcDNA3.1 (+)-BmCPI/BmGAPDH) growth conditions in screening culture medium is stable, start them to carry out frozen:

(1) choose the cell of logarithmic phase, change a subculture in frozen the day before yesterday;

(2) outwell substratum, PBS rinses after cell clone gently, with the trypsin digestion cell of 0.25%W/V, the cell harvesting having digested is centrifugal in centrifuge tube, outwells substratum and pancreatin;

(3) add frozen storing liquid, the ultimate density of the cell in frozen storing liquid is 5 × 10 ⁶, with liquid-transfering gun piping and druming, be then distributed in cell cryopreservation tube and seal, put into-80 ℃ of refrigerators frozen; Described frozen storing liquid comprises the foetal calf serum of the frozen substratum of the DMSO of 10% volume fraction and 10% volume fraction;

(7) detect positive colony, extracting cell total rna, molecular level detects the expression of recombinant plasmid in cell:

In (1) six orifice plate, cell is abandoned supernatant, and after PBS cleans, every hole adds 1ml Trizol reagent, with moving in new 1.5ml Ep pipe after liquid-transfering gun piping and druming dissolved cell;

(2) 15-30 ℃ of incubation 5min, guarantees the thorough separation of nucleoprotein complex;

(3) add 0.1ml chloroform, the 15s that fully vibrates, 15-30 ℃ of incubation 3min, the 2-8 ℃ of centrifugal 15min of 12000rpm;

(4) supernatant liquid is transferred in another Ep pipe, adds 0.3ml Virahol, 15-30 ℃ of incubation 10min; The 2-8 ℃ of centrifugal 10min of 12000rpm;

(5) abandon supernatant, use 0.5ml75%DEPC-alcohol flushing to precipitate once, the 2-8 ℃ of centrifugal 15min of 12000rpm.

(6) dry 5-10min, adds 20 μ l DEPC-DDW, and 55-60 ℃ of incubation 10min is the RNA of extraction, be stored in-80 ℃ for subsequent use;

(7) synthetic cDNA the first chain of reverse transcription adds following reaction mixture in the centrifuge tube of the nuclease free of ice bath:

(1-5ug) total RNA;

2ul?oligo（dT）15；

2ul?dNTP（2.5mM?each）；

Mend RNase-free ddH2O and be settled to 14.5ul;

(8) 70 ℃ heating 5min after at cooled on ice 2min; After brief centrifugal collection reaction solution, add following component:

4ul5 × First-Strand buffer (containing DTT); 0.5ul RNasin.

(9) add the TIANScript M_MLV of 1ul, 200U, mix with pipettor gently.

(10) 42 ℃ of temperature temperature 50min;

(11) 95 ℃ of heating 5min termination reactions, put and carry out subsequent experimental or the freezing preservation of-20 degree on ice.

(12) pcr amplification gene; Calculate primer Tm value, P1 Tm=62.02, P2 Tm=64.94, in 0.5m1 Eppendorf pipe, according to the form below is set up the PCR reaction system of 25 μ l:

Table 1 PCR reaction system

Reaction system, in modulation on ice, mixes, centrifugal rearmounted PCR instrument, and after 94 ℃ of denaturation 3min, 94 ℃ of sex change 45 seconds;

57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling termination reactions; After amplification, product is identified through 1.0% agarose gel electrophoresis;

(8) protein level detects the expression of recombinant plasmid in cell

Albumen in collecting cell, the institute of crack protein all carries out on ice in steps:

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add the ratio of 200 μ l～250 μ l to add lysate according to the 6 every holes of orifice plate, several with rifle piping and druming under, lysate is fully contacted with cell;

(3) after cracking, the centrifugal 15min of 15000rpm, gets supernatant, puts in 1.5ml sterilizing centrifuge tube;

(4) in the EP pipe of new collecting cell albumen, add equivalent loading buffer, 100 ℃ are boiled 5-10min, and-20 ℃ save backup;

(9) expression product carries out SDS-PAGE analysis

(1) join glue: resolving gel concentration is 12%, concentrated gum concentration is 5%.According to the form below adds successively each reagent and mixes, glue.Each sample adds volume as following table:

Table 2 separation gel component preparation (12%)

The concentrated glue component preparation of table 3 (5%)

Electrophoresis sheet glass is fixed on clip, records separation gel in sheet glass interlayer, with the Virahol moulding of binding, level leaves standstill, polymerized at room temperature 1h immediately; Remove separation gel upper strata Virahol, sop up unnecessary liquid with filter paper, evenly add concentrated glue, insert immediately comb, polymerized at room temperature 45min;

(2) loading: extract comb after concentrated gelling is poly-, the double glazing unit that contains glue is placed in to electrophoresis chamber, add electrophoretic buffer, by protein molecular weight standard object of reference and sample loading successively;

(3) electrophoresis: sample side joint negative pole, concentrated glue voltage 80V, separation gel voltage 100V; Need electrophoresis 4～6h, until tetrabromophenol sulfonphthalein reaches separation gel bottom;

(4) coomassie brilliant blue staining: peel gel, be placed in coomassie brilliant blue staining liquid, shake on shaking table, approximately 1～2h of vibration dyeing;

(5) gel is transferred in the plate that fills destainer, on shaking table, shaken, every 15min changes a not good liquor, repeats after 2～3 times, colourless to background, the observations of taking pictures;

(10) extraction and the purifying of periodicity Wuchereria malayi recombinant protein

1. the serum-free culture of positive colony cell

(1) all positive colony cells are first cultivated with screening culture medium and maintain base;

(2) after at the bottom of cell monolayer covers with bottle, go down to posterity in the ratio of 1:2 to 1:4, change afterwards 5% serum-concentration mixed culture medium into;

Within (3) two days, after three days, repetitive operation (2), and progressively reduce successively serum-concentration in substratum, uses serum free medium instead after three to four times entirely;

(4) use serum free medium cultured cells now can start suspension growth and produce big and small cell mass;

(5) generation of cell mass produces puzzlement to suspension culture, obtains 5U/ml heparin sodium can solve the problem of cell conglomeration by preliminary experiment, therefore, uses instead containing heparin sodium substratum and by cell piping and druming evenly, make the complete suspension growth of cell when change liquid next time;

(6) after cultivating, retain cells and supernatant, treat that next step carries out purifying to the target protein in cell and cells and supernatant;

2. thick leach protein

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add lysate according to the ratio that adds 400 μ l～500 μ l in each Tissue Culture Flask, several with rifle piping and druming under;

(3) add after the resuspended precipitation of RIPA cell pyrolysis liquid, be placed on ice, with 40% peak power output, 1 time/10s ultrasonic degradation cell;

(4) leave standstill on ice after 30min, 4 ℃, the centrifugal 30min of 12000rmp, collects supernatant liquor; With the protein concn in BCA colorimetric method for determining supernatant liquor;

3. the purifying of target protein in serum-free cell culture supernatant

(1) use the strainer of 0.45um to filter the supernatant 20ml of serum free medium;

(2) cells and supernatant being filled into and serum free medium and papoid 0.5ml are stirred 24 hours at 4 ℃;

(3) preparation of packed column: 5ml retains the syringe of metal needle, and in bottom nylon hair beyond the Great Wall;

(4) balance pillar: with the dcq buffer liquid washing pillar of 5-10 times of pillar volume, the volume of the damping fluid that estimation nylon hair adsorbs is as pillar volume;

(5), the mixed solution upper prop in (2), cross post;

(6) cross post with the dcq buffer liquid of 5-10 times of column volume;

(7) the flow velocity eluted product with 50 times of column volumes per hour with elution buffer;

(8) sample collection under wash-out is placed in after packing in-20 ℃ in neutralization buffer.

The present invention is easy to prepare, effective.

Below in conjunction with embodiment, the invention will be further described.

Embodiment

A preparation method for the compound multivalence protein vaccine of Periodic Brugia, comprises the following steps:

(1) Periodic Brugia pcDNA3.1(+) extracting of-BmCPI/BmGAPDH recombinant plasmid:

(1) join by getting 50ul after the bacterial classification room temperature thawing that contains recombinant plasmid the shaking table cultivation 12-16h that is placed in 37 ℃ of constant temperature, 250rpm in the 100ml LB solution that contains 50ul, 100mg/ml ampicillin, place afterwards 4 ℃ of refrigerators cooling;

(2) bacterium liquid is cultivated after 12-16h, uses ultraviolet spectrophotometer to measure its OD600 value, if OD600 numerical value between 0.6-0.8, shows the logarithmic phase of bacterium in vigorous growth, can be used for carrying out the extracting of plasmid;

(3) get bacterium after above-mentioned cultivation to 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation, abandons supernatant;

(4) every pipe adds 5ml solution I (suspension), re-suspended cell precipitation;

(5) every pipe adds 5ml solution II (lysate) afterwards, puts upside down centrifuge tube 4-6 time, and room temperature is placed 3-5 minute, makes the complete cracking of bacterium, and solution is transparent;

(6) every pipe adds 5ml solution III (in conjunction with liquid), puts upside down immediately centrifuge tube and mixes for 4-6 time, and visible white floss produces, then the centrifugal 10-20 of 5000rpm room temperature minute (example 10,15,20 minutes);

(7) supernatant is poured into or is drawn in plasmid purification column, centrifugal 2 minutes of 5000rpm room temperature, abandons collection liquid in pipe;

(8) in plasmid purification column, add 7ml solution IV (washings), centrifugal 2 minutes of 5000rpm room temperature, washes away impurity, abandons collection liquid in pipe, and centrifugal 2 minutes of 5000rpm room temperature more afterwards, removes residual liquid and trace ethanol is volatilized completely;

(9) plasmid purification column is placed on 50ml centrifuge tube, add 2ml solution V(elutriant) to pipe inner cylinder, place 2 minutes, then centrifugal 2 minutes of 5000rpm room temperature, adds 10ml solution VI(DNA purifying in conjunction with liquid in gained liquid) be added in former plasmid purification column after mixing;

(10) room temperature, after centrifugal 2 minutes, is abandoned collection liquid in pipe again;

(11) in plasmid purification column, add 15ml solution IV (washings), centrifugal 2 minutes of 5000rpm room temperature, a nearly step washes away after impurity, abandons collection liquid in pipe;

(12) centrifugal 2 minutes of 5000rpm room temperature, removes residual liquid and trace ethanol is volatilized completely;

(13) plasmid purification column is placed on 50ml centrifuge tube, adds 2ml solution V(elutriant) to pipe inner cylinder, place 2 minutes;

(14) centrifugal 2 minutes of 5000rpm room temperature, gained liquid is and turns the ultrapure plasmid of cell grade;

(15) get aqueous dna, with 400 times of distilled water dilutions, make blank with distilled water, on ultraviolet spectrophotometer, read the mensuration concentration of sample and the value of A260/A280, and be stored in-20 ℃ for gene transfection;

Described solution I is the suspension of a large amount of extraction agent boxes of plasmid; Solution II is the lysate of a large amount of extraction agent boxes of plasmid; Solution III is the combination liquid of a large amount of extraction agent boxes of plasmid; Solution IV is the washings of a large amount of extraction agent boxes of plasmid; Solution V is the elutriant of a large amount of extraction agent boxes of plasmid; Solution VI is that the DNA purifying of a large amount of extraction agent boxes of plasmid is in conjunction with liquid;

(2) recovery of Hela cell, cultivate and go down to posterity

(1) from-80 ℃ of refrigerators, take out the EP pipe of frozen Hela cell, and directly drop in 37 ℃ of thermostat water baths, shake frequently makes its thawing;

(2) in the time melting completely, from 37 ℃ of warm water, take out EP pipe, 1200 revs/min, centrifugal 3 minutes;

(3) abandon supernatant, by the DMEM perfect medium re-suspended cell precipitation that contains 10% volume calf serum and dual anti-(penicillin and Streptomycin sulphate), be inoculated in culturing bottle, be placed on 37 ℃, the standing cultivation of 5%CO2 cell culture incubator, change nutrient solution next day one time, continue to cultivate;

(4) at the bottom of Hela cell covers with culturing bottle 70%～80% when full, go down to posterity, outwell the nutrient solution in culturing bottle, by 2ml PBS damping fluid washed twice;

(5) add 0.25%W/V pancreatin lmL digestion lmin, in the time that starting to come off, outwells a bottle floor cells pancreatin, add 8mL to contain 10% foetal calf serum and dual anti-DMEM perfect medium, blow and beat a bottle floor cells with liquid-transfering gun and it is come off and mix, draw 4mL and continue to cultivate to another culturing bottle;

(3) gene transfection

(1) shift to an earlier date 24 hours inoculating cell 55 holes in 96 orifice plates, every kind of concentration is established 5 multiple holes, and inoculum size, for making within second day, grow up to 25% individual layer, is put 37 ℃ of overnight incubation in CO2 incubator;

(2) nutrient solution is changed into the substratum containing microbiotic G418, antibiotic concentration increases progressively by gradient: 0 μ g/ml, 100 μ g/ml, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml and 1000 μ g/ml;

(3) cultivate and be as the criterion with most necrocytosis concentration for 10-14 days, finally obtaining best screening concentration is 400 μ g/ml, when screening stably express clone, improves a rank than this concentration, uses the half of screening concentration while maintaining;

(4) transfection the day before yesterday, by 0.25%W/V trysinization for cell, counting bed board, making it is 80-90% at transfection cytogamy on same day degree, cell bed board 2mL containing foetal calf serum and dual anti-DMEM substratum in; In six orifice plates, carry out in the following manner transfection, a, b in contrast:

A.No DNA transfection control(DNA transfection blank)

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/BmGAPDH；

(5), for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 4ug～5ug DNA, preparation in batches when porous operation;

(6) for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 10ul Lipofect lipofectamine, after Lipofect dilution, the DNA with dilution in 5min mixes, soaking time is long can reduce activity, can prepare in batches;

(7) the Lipofect lipofectamine of the DNA of mixed diluting and dilution, at room temperature insulation 15min;

(8) transfection media washed cell before transfection starts in use 2ml/ hole, transfection media is replaced by the not DMEM substratum containing serum;

(9) mixture of dilution is joined in the middle of cell, wave and culture plate, mixes gently in 37 ℃, 5% CO2 and is incubated 24-48h;

(4) screening of positive colony

(1) transfection was gone down to posterity transfectional cell in the ratio of 1:10 after 24 hours in 6 orifice plates, cultivate again the G418 that the substratum in six orifice plates after 24h is replaced by the 400 μ g/ml concentration of determining in trial test and select substratum, then positive cell is screened by screening culture medium according to the cell of different growing states below:

A.No DNA transfection control(DNA transfection blank)

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/GAPDH

Add by the G418 screening culture medium preparing, when screening, improve a grade concentration than preliminary experiment;

(2), according to the color of substratum and Growth of Cells situation, within every 2～3 days, change primary screening substratum; When observing in a, b, c in the most of death of most cells or a cell when all dead, substratum G418 concentration is reduced by half and maintains screening as maintain base, treat that it increases gradually, now serum-concentration can add greatly 15%;

(3), in the time having seen under the microscope positive colony formation, prepare picking clone;

(5) extraction of positive colony and enlarged culturing

(1) observation of cell clone, makes marks to the positive colony that will separate in culture plate bottom surface with marker pen;

(2) remove substratum, rinse gently cell clone with PBS;

(3) cultivate ring with clone of aseptic tweezer gripping, dip sterilized Vaseline, Vaseline face sweeps away, and presses in culture plate on eugonic positive monoclonal, make Vaseline be evenly distributed on clone and cultivate ring bottom surface, clone's ring is in required cloning cluster;

(4) repeating step (3) on the six same holes of orifice plate, by 2 to 3 other required cloning cluster of snare;

(5) add 0.25% pancreatin and be full of in ring, after 20 seconds, discard pancreatin;

(6) cover six orifice plates, 37 ℃, 15 minutes;

(7) in each ring, add the substratum of 0.1ml to 0.2ml;

(8) successively each clone is blown and beaten with cell dispersion with suction pipe, and cell suspension is moved into respectively in the hole of 24 orifice plates, each cell clone should be equipped with suction pipe or rifle head separately;

(9) rinse clone's ring with the substratum of 0.1ml to 0.2ml again, then these substratum are moved into respectively in corresponding 24 orifice plates;

(10) substratum in culture hole is added to 0.5ml, build, continue to cultivate;

(11) in the time that clone cell covers with 24 orifice plate, get final product in had digestive transfer culture to six orifice plate, add 2ml substratum and continue enlarged culturing;

In (12) six orifice plates, cell is passaged to culturing bottle continuation cultivation after covering with, until frozen;

(6) positive colony is frozen

After positive colony cell (pcDNA3.1 (+), pcDNA3.1 (+)-BmCPI/BmGAPDH) growth conditions in screening culture medium is stable, start them to carry out frozen:

(1) choose the cell of logarithmic phase, change a subculture in frozen the day before yesterday;

(2) outwell substratum, PBS rinses after cell clone gently, with the trypsin digestion cell of 0.25%W/V, the cell harvesting having digested is centrifugal in centrifuge tube, outwells substratum and pancreatin;

(3) add frozen storing liquid, the ultimate density of the cell in frozen storing liquid is 5 × 10 ⁶, with liquid-transfering gun piping and druming, be then distributed in cell cryopreservation tube and seal, put into-80 ℃ of refrigerators frozen; Described frozen storing liquid comprises the foetal calf serum of the frozen substratum of the DMSO of 10% volume fraction and 10% volume fraction;

(7) detect positive colony, extracting cell total rna, molecular level detects the expression of recombinant plasmid in cell:

In (1) six orifice plate, cell is abandoned supernatant, and after PBS cleans, every hole adds 1ml Trizol reagent, with moving in new 1.5ml Ep pipe after liquid-transfering gun piping and druming dissolved cell;

(2) 15-30 ℃ of incubation 5min, guarantees the thorough separation of nucleoprotein complex;

(3) add 0.1ml chloroform, the 15s that fully vibrates, 15-30 ℃ of incubation 3min, the 2-8 ℃ of centrifugal 15min of 12000rpm;

(4) supernatant liquid is transferred in another Ep pipe, adds 0.3ml Virahol, 15-30 ℃ of incubation 10min; The 2-8 ℃ of centrifugal 10min of 12000rpm;

(5) abandon supernatant, use 0.5ml75%DEPC-alcohol flushing to precipitate once, the 2-8 ℃ of centrifugal 15min of 12000rpm.

(6) dry 5-10min, adds 20 μ l DEPC-DDW, and 55-60 ℃ of incubation 10min is the RNA of extraction, be stored in-80 ℃ for subsequent use;

(7) synthetic cDNA the first chain of reverse transcription adds following reaction mixture in the centrifuge tube of the nuclease free of ice bath:

(1-5ug) total RNA;

2ul?oligo（dT）15；

2ul?dNTP（2.5mM?each）；

Mend RNase-free ddH2O and be settled to 14.5ul;

(8) 70 ℃ heating 5min after at cooled on ice 2min; After brief centrifugal collection reaction solution, add following component:

4ul5 × First-Strand buffer (containing DTT); 0.5ul RNasin.

(9) add the TIANScript M_MLV of 1ul, 200U, mix with pipettor gently.

(10) 42 ℃ of temperature temperature 50min;

(11) 95 ℃ of heating 5min termination reactions, put and carry out subsequent experimental or the freezing preservation of-20 degree on ice.

(12) pcr amplification gene; Calculate primer Tm value, P1 Tm=62.02, P2 Tm=64.94, in 0.5m1 Eppendorf pipe, according to the form below is set up the PCR reaction system of 25 μ l:

Table 1 PCR reaction system

Reaction system, in modulation on ice, mixes, centrifugal rearmounted PCR instrument, and after 94 ℃ of denaturation 3min, 94 ℃ of sex change 45 seconds;

57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling termination reactions; After amplification, product is identified through 1.0% agarose gel electrophoresis;

(8) protein level detects the expression of recombinant plasmid in cell

Albumen in collecting cell, the institute of crack protein all carries out on ice in steps:

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add the ratio of 200 μ l～250 μ l to add lysate according to the 6 every holes of orifice plate, several with rifle piping and druming under, lysate is fully contacted with cell;

(3) after cracking, the centrifugal 15min of 15000rpm, gets supernatant, puts in 1.5ml sterilizing centrifuge tube;

(4) in the EP pipe of new collecting cell albumen, add equivalent loading buffer, 100 ℃ are boiled 5-10min, and-20 ℃ save backup;

(9) expression product carries out SDS-PAGE analysis

(1) join glue: resolving gel concentration is 12%, concentrated gum concentration is 5%.According to the form below adds successively each reagent and mixes, glue.Each sample adds volume as following table:

Table 2 separation gel component preparation (12%)

The concentrated glue component preparation of table 3 (5%)

Electrophoresis sheet glass is fixed on clip, records separation gel in sheet glass interlayer, with the Virahol moulding of binding, level leaves standstill, polymerized at room temperature 1h immediately; Remove separation gel upper strata Virahol, sop up unnecessary liquid with filter paper, evenly add concentrated glue, insert immediately comb, polymerized at room temperature 45min;

(2) loading: extract comb after concentrated gelling is poly-, the double glazing unit that contains glue is placed in to electrophoresis chamber, add electrophoretic buffer, by protein molecular weight standard object of reference and sample loading successively;

(3) electrophoresis: sample side joint negative pole, concentrated glue voltage 80V, separation gel voltage 100V; Need electrophoresis 4～6h, until tetrabromophenol sulfonphthalein reaches separation gel bottom;

(4) coomassie brilliant blue staining: peel gel, be placed in coomassie brilliant blue staining liquid, shake on shaking table, approximately 1～2h of vibration dyeing;

(5) gel is transferred in the plate that fills destainer, on shaking table, shaken, every 15min changes a not good liquor, repeats after 2～3 times, colourless to background, the observations of taking pictures;

(10) extraction and the purifying of periodicity Wuchereria malayi recombinant protein

1. the serum-free culture of positive colony cell

(1) all positive colony cells are first cultivated with screening culture medium and maintain base;

(2) after at the bottom of cell monolayer covers with bottle, go down to posterity in the ratio of 1:2 to 1:4, change afterwards 5% serum-concentration mixed culture medium into;

Within (3) two days, after three days, repetitive operation (2), and progressively reduce successively serum-concentration in substratum, uses serum free medium instead after three to four times entirely;

(4) use serum free medium cultured cells now can start suspension growth and produce big and small cell mass;

(5) generation of cell mass produces puzzlement to suspension culture, obtains 5U/ml heparin sodium can solve the problem of cell conglomeration by preliminary experiment, therefore, uses instead containing heparin sodium substratum and by cell piping and druming evenly, make the complete suspension growth of cell when change liquid next time;

(6) after cultivating, retain cells and supernatant, treat that next step carries out purifying to the target protein in cell and cells and supernatant;

2. thick leach protein

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add lysate according to the ratio that adds 400 μ l～500 μ l in each Tissue Culture Flask, several with rifle piping and druming under;

(3) add after the resuspended precipitation of RIPA cell pyrolysis liquid, be placed on ice, with 40% peak power output, 1 time/10s ultrasonic degradation cell;

(4) leave standstill on ice after 30min, 4 ℃, the centrifugal 30min of 12000rmp, collects supernatant liquor; With the protein concn in BCA colorimetric method for determining supernatant liquor;

3. the purifying of target protein in serum-free cell culture supernatant

(1) use the strainer of 0.45um to filter the supernatant 20ml of serum free medium;

(2) cells and supernatant being filled into and serum free medium and papoid 0.5ml are stirred 24 hours at 4 ℃;

(3) preparation of packed column: 5ml retains the syringe of metal needle, and in bottom nylon hair beyond the Great Wall;

(4) balance pillar: with the dcq buffer liquid washing pillar of 5-10 times of pillar volume, the volume of the damping fluid that estimation nylon hair adsorbs is as pillar volume;

(5), the mixed solution upper prop in (2), cross post;

(6) cross post with the dcq buffer liquid of 5-10 times of column volume;

(7) the flow velocity eluted product with 50 times of column volumes per hour with elution buffer;

(8) sample collection under wash-out is placed in after packing in-20 ℃ in neutralization buffer.

4. Western-blotting detects the albumen of collecting

(1) SDS-PAGE(carries out SDS-PAGE analysis referring to experiment one 1.2.7.2.2 by expression product)

(2) transfer groove transferring protein is to pvdf membrane:

1) assembling transfer printing interlayer: pvdf membrane-filter paper-Scotch-Brite pad-anode that negative electrode-Scotch-Brite pad-filter paper-gel-balance is crossed in transfer liquid in advance;

2) transfer printing interlayer is put into transfer groove, add transfer liquid, by correct polar orientation, transfer groove is put into electroporation, connect power supply;

3) under cooling conditions, 350mA electricity turns 2.5 hours;

4) powered-down, takes out transfer film, carries out protein immunodetection.

(3) protein immunodetection

1) transfer film is put into confining liquid (containing the TBST of 5% skim-milk) room temperature 2 hours;

2) transfer film is put into the primary antibodie (the mice serum 1:160 dilution that mouse immune gathered after 6 weeks) containing the TBST dilution of 5% skim-milk, room temperature 1 hour, 4 ℃ are spent the night;

3) TBST washes film 3 × 10 minutes;

4) transfer film is put into two anti-(goat anti-mouse igg-HRP1:2000 dilutions) with the TBST dilution of 5% skim-milk, room temperature 2 hours;

5) TBST washes film 3 × 10 minutes;

6) add Luminol Ecl Reagent(same volume to mix A and B liquid), 0.125mL/cm2, room temperature 1 minute, in darkroom, transfer film, to the exposure of X sensitive film, develops and photographic fixing.

The immunization of (11) Periodic Brugia BmCPI/BmGAPDH recombinant protein vaccine

(1) immunological adjuvant CpG ODN prepare in novel adjuvant take non-methylated cytosine(Cyt) and guanylic acid as the oligodeoxynucleotide (CpG ODN) of core the most representative, CpG ODN is synthetic by Shanghai Ying Jun biotech firm, and synthetic sequence is as follows:

CpG ODN18265 '-TCCATGACGTTCCTGACGTT-3 ', and full chain thiophosphoric acid backbone modification, strengthen its nuclease resistance.Be mixed with 1mg/mL with physiological saline solution.

(2) the immunization laboratory animal of vaccine grouping, by the sequence of weighing in of 45 experiment mices, divides 4 groups according to table of random number method, and 10 every group, A group: PBS control group; B group: empty carrier pcDNA3.1 (+)/CpG group; C group: BmCPI/BmGAPDH recombinant protein/CpG group); D group: BmCPI/BmGAPDH recombinant plasmid and recombinant protein combined immunization group, immunization protocol is 0,2,4 week.

(3) inoculation position pre-treatment recombinant protein vaccine is subcutaneous multi-point injection.LmL bupivacaine hydrochloride inj is diluted in 14mL physiological saline, obtains 0.5mg/mL diluent.24h before each inoculation, in inoculation position injection, 50 μ l carry out pre-treatment.

(4) immunization A group: (100 μ l) for PBS group, B group: empty plasmid group (pcDNA3.1 (+)/CpG (100 μ g/30 μ g)), C group: (100 μ g/30 μ g) for BmCPI/BmGAPDH recombinant protein/CpG group; D group: first 2 times each every injection recombinant plasmid 100 μ g and immunological adjuvants, inject for the last time recombinant protein 100 μ g and immunological adjuvants.Immunity 3 times altogether, 2 weeks, each immune interval.

(12) sample collection

(1) extraction of immune mouse injection site muscle, after last immunity 6 weeks, gets the left back leg tibialis anterior muscle of 1 mouse ,-70 ℃ of preservations for every group.

(2) collect mice serum respectively at after last immunity 2,4,6 weeks by eyeball excise method, get 3 mouse for every group:

1) left hand is caught mouse, and head inclination is downward, and holds neck with forefinger and thumb and make eyeball evagination.Due to the pressure of neck is made to cerebrovascular extravasated blood, can make to feel temporary transient forfeiture.

2) right hand is held aseptic elbow tweezer, extracts fast the eyeball of evagination, rapidly drop of blood is entered in aseptic 1.5ml Ep pipe, and (approximately 500 μ are l) time, and left hand loosens, with the hemostasis of aseptic dry cotton ball compressing eye socket to reach aequum.

3) room temperature is placed 1h, after solidifying, puts at 4 ℃, spend the night, and serum to be separated out, centrifugal 10 minutes of 4 ℃ of 4000rpm.Under aseptic condition, sucking-off serum, packing (0.05～0.02mL), posts label, is stored in-70 ℃.

(13) ELISA method detects mice serum antibody titer

Preliminary experiment is determined serum dilution and antigen concentration

(1) antigen coated: antigen diluent is become to 2 μ g/mL with antigen coated damping fluid, arranging under the prerequisite of blank and negative control, in Sptting plate, every hole adds 100 μ L antigens.

(2) sealing: every hole adds confining liquid 200 μ L, 4 ℃ of sealings are spent the night or 37 ℃ of sealing 2h, and PBST washes 3 times.

(3) react with mouse resisting anteserum: mouse resisting anteserum is first diluted to different concns with confining liquid: 1:20,1:40,1:80,1:160,1:320, negative control group is done same than the dilution of row, each sample is all established three multiple holes, the every hole of experimental group adds 100 μ L and is diluted to the antiserum(antisera) of different concns, hatches 2h for 37 ℃, PBST washing 3 times.

(4) react with second antibody: do square formation with the goat anti-mouse igg of confining liquid dilution horseradish peroxidase-labeled with 1:500,1:1000,1:1500,1:2000 dilution proportion and test, every hole adds 100 μ L and is diluted to the second antibody of different concns, hatch 1.5h for 37 ℃, PBST washing 3 times;

(5) colour developing: add immediately freshly prepared substrate solution, every hole 100 μ L.37 ℃ of lucifuge reaction 30min left and right.

(6) termination reaction: in the time that positive control occurs that obvious colour-change or negative control slightly develop the color, add stop buffer 50 μ L termination reactions.

(7) result judgement: enzyme mark detector is measured every hole in the light absorption value A at 490nm place value, blank zeroing, approach 1.0 with positive serum OD value, negative serum OD value <0.1, P/N value maximum (P/N=(testing sample hole OD value-blank well OD value)/(negative sample hole OD value-blank well OD value)) is ELISA best effort concentration.

It is 1:160 that serum dilution is chosen in this experiment, and second antibody extent of dilution is: 1:2000.

(14) immune mouse spleen cell collection (operating on super clean bench):

(1) after last immunity, the whole cervical vertebra dislocations of mouse in 6 weeks are lethal.

(2) get aseptic 10ml screw socket pipe, add 3ml lymphocyte separation medium, place 37 ℃ of preheating 2h.

(3) cervical vertebra is dislocated lethal mouse is put 2～3min in 75% ethanolic soln, sterilization skin.

(4) routine disinfection small white mouse belly, left veutro is upward; Left mouse skin of abdomen is mentioned gently with surgical forceps, cut about 1cm rimmer knife mouth with operating scissors; With hand, skin of abdomen is torn, exposed peritonaeum, red color visible strip spleen; Mention mouse peritoneum with aseptic operation tweezer, cut off to expose completely abdominal cavity with aseptic operation cutter; Careful separation goes out spleen.

(5) spleen is placed in to the little plate of containing appropriate aseptic Hank ' s liquid, cleans spleen.

(6) aseptic 100 order copper sieves put into another contain the plate of appropriate aseptic Hank ' s liquid, spleen is placed on it, grind gently with aseptic glass stick, until copper reticular tissue of remaining spleen only on sieving.

(7) the 10ml screw socket pipe of preheating is tilted 45 °, aseptic dropper pastes tube wall about suspension in plate 6ml is slowly transferred to (lymphocyte separation medium: cell suspension=1:2) in pipe, and attention can not be broken through the interface of lymph parting liquid.

(8) the centrifugal 2000rpm × 20min of room temperature, suspension is divided into 4 layers.

(9) get the 2nd layer of splenocyte suspension, move in the 50ml centrifuge tube containing 10mlHank ' s liquid the centrifugal 1000rpm × 10min of room temperature.

(10) discard supernatant liquid, add 10mlHank ' s liquid, piping and druming mixes, the centrifugal 1000rpm × 10min of room temperature.

(11) discard supernatant liquid, add 10mlHank ' s liquid, piping and druming mixes, and gets 10 μ l and adds in tally, counting.

(12) the centrifugal 1000rpm × 10min of room temperature, discards supernatant liquid, adds 5ml perfect medium, and piping and druming mixes.

(15) lymphocyte transformation test (mtt assay):

(1) use Trypan Blue living cell counting to answer more than 95% lymphocyte suspension, adjusting cell concn is 2 × 10 ⁶individual/m1.

(2) add cell suspension 100 μ l in the 96 every holes of well culture plate, 3 multiple holes are established in every hole, wherein in 1 hole, add 5 μ l ConA, put the CO containing 5% ₂37 ℃ of cultivation 72h of incubator.

(3) cultivate and finish front 4h, in every porocyte, add MTT10 μ l, on vibrator, mix 1min, put in incubator and continue to cultivate.

(4) after 4h, take out culture plate, every hole adds 100 μ l DMSO, mixes after 1min on vibrator, leaves standstill 20min, and purple crystal is dissolved completely.

(5) use enzyme-linked immunosorbent assay instrument to detect the OD value of each porocyte in 570nm place.

(stimulation index SI=adds concanavalin A (ConA) stimulates A respectively to organize lymphocyte stimulation indices ₅₇₀mean value/do not add ConA stimulate A ₅₇₀mean value).

The mensuration of (16) INF-γ:

(1) in advance 20min takes out test kits from 4 ℃, with balance to room temperature.

(2) by DDW dilution (1:20) for concentrated cleaning solution.

(3) with standard substance diluent proportional diluted standard substance.

1) establish standard substance 10 holes;

2) first, second hole (50ul): standard substance 100ul+ standard substance diluent 50ul, mix, each hole is respectively

Take out 100ul to the three, the 4th hole;

3) the 3rd, the 4th hole (50ul): the standard substance diluent 50ul in the each hole of 100ul+ of respectively getting from first, second hole, mix, first abandon respectively 50ul mixed solution, respectively get 50ul and be added to the 5th, the 6th hole;

4) the 5th, the 6th hole (50ul): the standard substance diluent in the each hole of 50ul+ of respectively getting from the 3rd, the 4th hole

50ul, mixes, respectively gets that 50ul is added to the 7th, octal;

5) the 7th, octal (50ul): the standard substance diluent 50ul in the each hole of 50ul+ of respectively getting from the 5th, the 6th hole, mixes, respectively get 50ul and be added to the 9th, the tenth hole;

6) the 9th, the tenth hole (50ul): the standard substance diluent 50ul in the each hole of 50ul+ of respectively getting from the 7th, octal, mixes, respectively get 50ul and discard.

(after dilution, each hole application of sample amount is 50ul, 2. 3. 4. 5. 6. concentration be respectively: 1200ng/L, 800ng/L, 400ng/L, 200ng/L, 100ng/L)

(4) to the sealing bag of room temperature, take out lath from balance, except blank well, respectively by sample diluting liquid 40ul and testing sample 10 sample ul, seal reacting hole, 37 ℃ of incubator incubation 30min with shrouding gummed paper.(5) wash plate, get rid of liquid in most hole, washings is filled it up with in every hole, after standing 30s, gets rid of most liquid, on thieving paper, pats dry, and so repeats 5 times.

(6) except blank well, every hole adds enzyme marking reagent 50 μ l, seals reacting hole, 37 ℃ of incubator incubation 30min with shrouding gummed paper.

(7) wash plate 5 times, operation is with (5).

(8) except blank well, every hole first adds developer A50 μ l, then adds developer B50 μ l, and light shaking mixes, 37 ℃ of lucifuge colour developing 15min.

(9) stop, every hole adds stop buffer 50 μ l, termination reaction (the now blue vertical yellow that turns).

(10) measure: with blank well zeroing, 450nm wavelength is sequentially measured the absorbancy (OD value) in each hole.(11) calculate OD value: the OD value of each standard substance and sample deducts the OD value in zero hole.

(12) manual drawing typical curve: make X-coordinate with standard substance concentration, OD value is made ordinate zou, connects the coordinate point of each standard substance with sweep.

(13) can on typical curve, find concentration by the OD value of sample,

The mensuration (method is the same) of (17) IL-4.

2.2.4 data analysis

Application SPSS16 statistics software carries out statistical study to sampled data, adopts paired t-test.

The detected result that table 4 mouse antibodies is tired

Table 5 mouse lymphocyte proliferative response test-results (mtt assay ± s, A ₅₇₀value)

Note: ^*with ^#comparing difference has statistical significance, P<0.05.

Cytokine IFN-γ level (± s, pg/ml) in the different immunity of table 6 time mice serum

Note: ^*with ^#comparing difference has statistical significance, P<0.05.

Cytokine IL-4 level (± s, pg/ml) in the different immunity of table 7 time mice serum

Note: ^*with ^#comparing difference has statistical significance, P<0.05.

Recombinant plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH stable transfection is entered to Hela cell, after the operation of lysis and extraction albumen, institute's leach protein is carried out to the analysis of SDS-PAGE electrophoresis detection, there is a protein expression band near being presented at the position of about 54KD left and right in film making result, matches with the molecular weight theoretical value of the expressed target protein of expection complex gene.Institute's leach protein is carried out to Western-blotting analysis, and film making result is presented near the protein expression band that occurs in position of about 54KD left and right, matches with the molecular weight theoretical value of the expressed albumen of expecting.After the purifying of target protein in thick leach protein and serum-free cell culture supernatant, by subcutaneous recombinant protein vaccine multi-point injection BALB/c mouse immunization.Detect humoral immunization and the Study On Cellular Immune of its vaccine by MTT, ELISA method, the result of tiring that detects mouse immune 4 weeks and 6 weeks rear Serum Antibodies by ELISA indirect method shows: compound recombinant protein/CpG group and compound recombinant plasmid/compound recombinant protein/CpG organize tiring of immune mouse 4 weeks and 6 weeks rear Serum Antibodies, all apparently higher than two control groups.Lymphocyte transformation test result shows: immune group mouse spleen lymphocyte is external after ConA stimulates, and its lymphocyte stimulation indices raises gradually with the prolongation of immunity time, apparently higher than control group (P<0.05); Immune cell factor detected result shows: IFN-γ level in 2,4,6 weeks two groups of immune serums after immunity, compare with blank plasmid control group with PBS control group that all there were significant differences (P<0.05), immune group mouse the 4th week and more obviously (P<0.05) of the horizontal ascensional range of serum I FN-γ after 6 weeks.Latter 4 weeks of immunity, compound recombinant protein/CpG group mice serum IL-4 level, higher than PBS control group and blank plasmid group, starts there is statistical significance (P<0.05); Latter 6 weeks of immunity, immune group mice serum IL-4 level rises more obvious, there is statistical significance (P<0.05) higher than control group, the compound multivalence protein vaccine of above the results show Periodic Brugia can produce specific immune response by inducing mouse, obtains promising result.

Claims (3)

1. a preparation method for the compound multivalence protein vaccine of Periodic Brugia, is characterized in that: comprise the following steps:

(1) Periodic Brugia pcDNA3.1(+) extracting of-BmCPI/BmGAPDH recombinant plasmid:

(1) join by getting 50ul after the bacterial classification room temperature thawing that contains recombinant plasmid the shaking table cultivation 12-16h that is placed in 37 ℃ of constant temperature, 250rpm in the 100ml LB solution that contains 50ul, 100mg/ml ampicillin, place afterwards 4 ℃ of refrigerators cooling;

(2) bacterium liquid is cultivated after 12-16h, uses ultraviolet spectrophotometer to measure its OD600 value, and OD600 numerical value, between 0.6-0.8, shows the logarithmic phase of bacterium in vigorous growth, can be used for carrying out the extracting of plasmid;

(3) get bacterium after above-mentioned cultivation to 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation, abandons supernatant;

(4) every pipe adds 5ml solution I, re-suspended cell precipitation;

(5) every pipe adds 5ml solution II afterwards, puts upside down centrifuge tube 4-6 time, and room temperature is placed 3-5 minute, makes the complete cracking of bacterium, and solution is transparent;

(6) every pipe adds 5ml solution III, puts upside down immediately centrifuge tube and mixes for 4-6 time, and visible white floss produces, the then centrifugal 10-20 minute of 5000rpm room temperature;

(7) supernatant is poured into or is drawn in plasmid purification column, centrifugal 2 minutes of 5000rpm room temperature, abandons collection liquid in pipe;

(8) in plasmid purification column, add 7ml solution IV, centrifugal 2 minutes of 5000rpm room temperature, washes away impurity, abandons collection liquid in pipe, and centrifugal 2 minutes of 5000rpm room temperature more afterwards, removes residual liquid and trace ethanol is volatilized completely;

(9) plasmid purification column is placed on 50ml centrifuge tube, adds 2ml solution V to pipe inner cylinder, place 2 minutes, then centrifugal 2 minutes of 5000rpm room temperature, is added to former plasmid purification column in after adding 10ml solution VI to mix in gained liquid;

(10) room temperature, after centrifugal 2 minutes, is abandoned collection liquid in pipe again;

(11) in plasmid purification column, add 15ml solution IV, centrifugal 2 minutes of 5000rpm room temperature, a nearly step washes away after impurity, abandons collection liquid in pipe;

(12) centrifugal 2 minutes of 5000rpm room temperature, removes residual liquid and trace ethanol is volatilized completely;

(13) plasmid purification column is placed on 50ml centrifuge tube, adds 2ml solution V to pipe inner cylinder, place 2 minutes;

(14) centrifugal 2 minutes of 5000rpm room temperature, gained liquid is and turns the ultrapure plasmid of cell grade;

(15) get aqueous dna, with 400 times of distilled water dilutions, make blank with distilled water, on ultraviolet spectrophotometer, read the mensuration concentration of sample and the value of A260/A280, and be stored in-20 ℃ for gene transfection;

Described solution I is the suspension of a large amount of extraction agent boxes of plasmid; Solution II is the lysate of a large amount of extraction agent boxes of plasmid; Solution III is the combination liquid of a large amount of extraction agent boxes of plasmid; Solution IV is the washings of a large amount of extraction agent boxes of plasmid; Solution V is the elutriant of a large amount of extraction agent boxes of plasmid; Solution VI is that the DNA purifying of a large amount of extraction agent boxes of plasmid is in conjunction with liquid;

(2) recovery of Hela cell, cultivate and go down to posterity

(1) from-80 ℃ of refrigerators, take out the EP pipe of frozen Hela cell, and directly drop in 37 ℃ of thermostat water baths, shake frequently makes its thawing;

(2) in the time melting completely, from 37 ℃ of warm water, take out EP pipe, 1200 revs/min, centrifugal 3 minutes;

(3) abandon supernatant, by the DMEM perfect medium re-suspended cell precipitation that contains 10% volume calf serum and dual anti-(penicillin and Streptomycin sulphate), be inoculated in culturing bottle, be placed on 37 ℃, the standing cultivation of 5%CO2 cell culture incubator, change nutrient solution next day one time, continue to cultivate;

(4) at the bottom of Hela cell covers with culturing bottle 70%～80% when full, go down to posterity, outwell the nutrient solution in culturing bottle, by 2ml PBS damping fluid washed twice;

(5) add 0.25%W/V pancreatin lmL digestion lmin, in the time that starting to come off, outwells a bottle floor cells pancreatin, add 8mL to contain 10% foetal calf serum and dual anti-DMEM perfect medium, blow and beat a bottle floor cells with liquid-transfering gun and it is come off and mix, draw 4mL and continue to cultivate to another culturing bottle;

(3) gene transfection

(1) shift to an earlier date 24 hours inoculating cell 55 holes in 96 orifice plates, every kind of concentration is established 5 multiple holes, and inoculum size, for making within second day, grow up to 25% individual layer, is put 37 ℃ of overnight incubation in CO2 incubator;

(2) nutrient solution is changed into the substratum containing microbiotic G418, antibiotic concentration increases progressively by gradient: 0 μ g/ml, 100 μ g/ml, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml and 1000 μ g/ml;

(3) cultivate and be as the criterion with most necrocytosis concentration for 10-14 days, finally obtaining best screening concentration is 400 μ g/ml, when screening stably express clone, improves a rank than this concentration, uses the half of screening concentration while maintaining;

(4) transfection the day before yesterday, by 0.25%W/V trysinization for cell, counting bed board, making it is 80-90% at transfection cytogamy on same day degree, cell bed board 2mL containing foetal calf serum and dual anti-DMEM substratum in; In six orifice plates, carry out in the following manner transfection, a, b in contrast:

A.DNA transfection blank

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/BmGAPDH；

(5), for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 4ug～5ug DNA, preparation in batches when porous operation;

(6) for every porocyte, use 250ul not containing foetal calf serum and dual anti-DMEM substratum dilution 10ul Lipofect lipofectamine, after Lipofect dilution, the DNA with dilution in 5min mixes, soaking time is long can reduce activity, can prepare in batches;

(7) the Lipofect lipofectamine of the DNA of mixed diluting and dilution, at room temperature insulation 15min;

(8) transfection media washed cell before transfection starts in use 2ml/ hole, transfection media is replaced by the not DMEM substratum containing serum;

(9) mixture of dilution is joined in the middle of cell, wave and culture plate, mixes gently in 37 ℃, 5% CO2 and is incubated 24-48h;

(4) screening of positive colony

(1) transfection was gone down to posterity transfectional cell in the ratio of 1:10 after 24 hours in 6 orifice plates, cultivate again the G418 that the substratum in six orifice plates after 24h is replaced by the 400 μ g/ml concentration of determining in trial test and select substratum, then positive cell is screened by screening culture medium according to the cell of different growing states below:

A.DNA transfection blank

b．pcDNA3.1(+)

c．pcDNA3.1(+)-BmCPI/GAPDH

Add by the G418 screening culture medium preparing, when screening, improve a grade concentration than preliminary experiment;

(2), according to the color of substratum and Growth of Cells situation, within every 2～3 days, change primary screening substratum; When observing in a, b, c in the most of death of most cells or a cell when all dead, substratum G418 concentration is reduced by half and maintains screening as maintain base, treat that it increases gradually, now serum-concentration can add greatly 15%;

(3), in the time having seen under the microscope positive colony formation, prepare picking clone;

(5) extraction of positive colony and enlarged culturing

(1) observation of cell clone, makes marks to the positive colony that will separate in culture plate bottom surface with marker pen;

(2) remove substratum, rinse gently cell clone with PBS;

(3) cultivate ring with clone of aseptic tweezer gripping, dip sterilized Vaseline, Vaseline face sweeps away, and presses in culture plate on eugonic positive monoclonal, make Vaseline be evenly distributed on clone and cultivate ring bottom surface, clone's ring is in required cloning cluster;

(4) repeating step (3) on the six same holes of orifice plate, by 2 to 3 other required cloning cluster of snare;

(5) add 0.25% pancreatin and be full of in ring, after 20 seconds, discard pancreatin;

(6) cover six orifice plates, 37 ℃, 15 minutes;

(7) in each ring, add the substratum of 0.1ml to 0.2ml;

(8) successively each clone is blown and beaten with cell dispersion with suction pipe, and cell suspension is moved into respectively in the hole of 24 orifice plates, each cell clone should be equipped with suction pipe or rifle head separately;

(9) rinse clone's ring with the substratum of 0.1ml to 0.2ml again, then these substratum are moved into respectively in corresponding 24 orifice plates;

(10) substratum in culture hole is added to 0.5ml, build, continue to cultivate;

(11) in the time that clone cell covers with 24 orifice plate, get final product in had digestive transfer culture to six orifice plate, add 2ml substratum and continue enlarged culturing;

In (12) six orifice plates, cell is passaged to culturing bottle continuation cultivation after covering with, until frozen;

(6) positive colony is frozen

After positive colony cell (pcDNA3.1 (+), pcDNA3.1 (+)-BmCPI/BmGAPDH) growth conditions in screening culture medium is stable, start them to carry out frozen:

(1) choose the cell of logarithmic phase, change a subculture in frozen the day before yesterday;

(2) outwell substratum, PBS rinses after cell clone gently, with the trypsin digestion cell of 0.25%W/V, the cell harvesting having digested is centrifugal in centrifuge tube, outwells substratum and pancreatin;

(3) add frozen storing liquid, the ultimate density of the cell in frozen storing liquid is 5 × 10 ⁶, with liquid-transfering gun piping and druming, be then distributed in cell cryopreservation tube and seal, put into-80 ℃ of refrigerators frozen; Described frozen storing liquid comprises the foetal calf serum of the frozen substratum of the DMSO of 10% volume fraction and 10% volume fraction;

(7) detect positive colony, extracting cell total rna, molecular level detects the expression of recombinant plasmid in cell:

In (1) six orifice plate, cell is abandoned supernatant, and after PBS cleans, every hole adds 1ml Trizol reagent, with moving in new 1.5ml Ep pipe after liquid-transfering gun piping and druming dissolved cell;

(2) 15-30 ℃ of incubation 5min, guarantees the thorough separation of nucleoprotein complex;

(3) add 0.1ml chloroform, the 15s that fully vibrates, 15-30 ℃ of incubation 3min, the 2-8 ℃ of centrifugal 15min of 12000rpm;

(4) supernatant liquid is transferred in another Ep pipe, adds 0.3ml Virahol, 15-30 ℃ of incubation 10min; The 2-8 ℃ of centrifugal 10min of 12000rpm;

(5) abandon supernatant, use 0.5ml75%DEPC-alcohol flushing to precipitate once, the 2-8 ℃ of centrifugal 15min of 12000rpm.

(6) dry 5-10min, adds 20 μ l DEPC-DDW, and 55-60 ℃ of incubation 10min is the RNA of extraction, be stored in-80 ℃ for subsequent use;

(7) synthetic cDNA the first chain of reverse transcription adds following reaction mixture in the centrifuge tube of the nuclease free of ice bath:

(1-5ug) total RNA;

2ul?oligo（dT）15；

2ul?dNTP（2.5mM?each）；

Mend RNase-free ddH2O and be settled to 14.5ul;

(8) 70 ℃ heating 5min after at cooled on ice 2min; After brief centrifugal collection reaction solution, add following component:

4ul5 × First-Strand buffer (containing DTT); 0.5ul RNasin.

(9) add the TIANScript M_MLV of 1ul, 200U, mix with pipettor gently.

(10) 42 ℃ of temperature temperature 50min;

(11) 95 ℃ of heating 5min termination reactions, put and carry out subsequent experimental or the freezing preservation of-20 degree on ice.

(12) pcr amplification gene; Calculate primer Tm value, P1 Tm=62.02, P2 Tm=64.94, in 0.5m1 Eppendorf pipe, according to the form below is set up the PCR reaction system of 25 μ l:

PCR reaction system

Reaction system, in modulation on ice, mixes, centrifugal rearmounted PCR instrument, and after 94 ℃ of denaturation 3min, 94 ℃ of sex change 45 seconds;

57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling termination reactions; After amplification, product is identified through 1.0% agarose gel electrophoresis;

(8) protein level detects the expression of recombinant plasmid in cell

Albumen in collecting cell, the institute of crack protein all carries out on ice in steps:

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add the ratio of 200 μ l～250 μ l to add lysate according to the 6 every holes of orifice plate, several with rifle piping and druming under, lysate is fully contacted with cell;

(3) after cracking, the centrifugal 15min of 15000rpm, gets supernatant, puts in 1.5ml sterilizing centrifuge tube;

(4) in the EP pipe of new collecting cell albumen, add equivalent loading buffer, 100 ℃ are boiled 5-10min, and-20 ℃ save backup;

(9) expression product carries out SDS-PAGE analysis

(1) join glue: resolving gel concentration is 12%, concentrated gum concentration is 5%.According to the form below adds successively each reagent and mixes, glue.Each sample adds volume as following table:

Separation gel component preparation (12%)

Concentrated glue component preparation (5%)

Electrophoresis sheet glass is fixed on clip, records separation gel in sheet glass interlayer, with the Virahol moulding of binding, level leaves standstill, polymerized at room temperature 1h immediately; Remove separation gel upper strata Virahol, sop up unnecessary liquid with filter paper, evenly add concentrated glue, insert immediately comb, polymerized at room temperature 45min;

(2) loading: extract comb after concentrated gelling is poly-, the double glazing unit that contains glue is placed in to electrophoresis chamber, add electrophoretic buffer, by protein molecular weight standard object of reference and sample loading successively;

(3) electrophoresis: sample side joint negative pole, concentrated glue voltage 80V, separation gel voltage 100V; Need electrophoresis 4～6h, until tetrabromophenol sulfonphthalein reaches separation gel bottom;

(4) coomassie brilliant blue staining: peel gel, be placed in coomassie brilliant blue staining liquid, shake on shaking table, approximately 1～2h of vibration dyeing;

(5) gel is transferred in the plate that fills destainer, on shaking table, shaken, every 15min changes a not good liquor, repeats after 2～3 times, colourless to background, the observations of taking pictures;

(10) extraction and the purifying of periodicity Wuchereria malayi recombinant protein

1. the serum-free culture of positive colony cell

(1) all positive colony cells are first cultivated with screening culture medium and maintain base;

(2) after at the bottom of cell monolayer covers with bottle, go down to posterity in the ratio of 1:2 to 1:4, change afterwards 5% serum-concentration mixed culture medium into;

Within (3) two days, after three days, repetitive operation (2), and progressively reduce successively serum-concentration in substratum, uses serum free medium instead after three to four times entirely;

(4) use serum free medium cultured cells now can start suspension growth and produce big and small cell mass;

(5) generation of cell mass produces puzzlement to suspension culture, obtains 5U/ml heparin sodium can solve the problem of cell conglomeration by preliminary experiment, therefore, uses instead containing heparin sodium substratum and by cell piping and druming evenly, make the complete suspension growth of cell when change liquid next time;

(6) after cultivating, retain cells and supernatant, treat that next step carries out purifying to the target protein in cell and cells and supernatant;

2. thick leach protein

(1) from-20 ℃, RIPA lysate is taken out and melted, mix; Get lysate, within several minutes before use, add PMSF, the ultimate density that makes PMSF is 1mM;

(2) remove nutrient solution, wash one time with serum-free medium, add lysate according to the ratio that adds 400 μ l～500 μ l in each Tissue Culture Flask, several with rifle piping and druming under;

(3) add after the resuspended precipitation of RIPA cell pyrolysis liquid, be placed on ice, with 40% peak power output, 1 time/10s ultrasonic degradation cell;

(4) leave standstill on ice after 30min, 4 ℃, the centrifugal 30min of 12000rmp, collects supernatant liquor; With the protein concn in BCA colorimetric method for determining supernatant liquor;

3. the purifying of target protein in serum-free cell culture supernatant

(1) use the strainer of 0.45um to filter the supernatant 20ml of serum free medium;

(2) cells and supernatant being filled into and serum free medium and papoid 0.5ml are stirred 24 hours at 4 ℃;

(3) preparation of packed column: 5ml retains the syringe of metal needle, and in bottom nylon hair beyond the Great Wall;

(4) balance pillar: with the dcq buffer liquid washing pillar of 5-10 times of pillar volume, the volume of the damping fluid that estimation nylon hair adsorbs is as pillar volume;

(5), the mixed solution upper prop in (2), cross post;

(6) cross post with the dcq buffer liquid of 5-10 times of column volume;

(7) the flow velocity eluted product with 50 times of column volumes per hour with elution buffer;

(8) sample collection under wash-out is placed in after packing in-20 ℃ in neutralization buffer.

2. the preparation method of the compound multivalence protein vaccine of Periodic Brugia according to claim 1, is characterized in that: centrifugal 15 minutes of 5000rpm room temperature in step () (6).

3. the preparation method of the compound multivalence protein vaccine of Periodic Brugia according to claim 1, is characterized in that: centrifugal 20 minutes of 5000rpm room temperature in step () (6).