CN103509815A - Preparation method of recombinant panda IL-2 immune adjuvant - Google Patents

Abstract

The invention discloses a preparation method of a recombinant panda IL-2 immune adjuvant. The recombinant panda IL-2 immune adjuvant is a recombinant panda IL-2 (gpIL-2) immune adjuvant, which has a prominent effect and a convenient extraction process, can be directly applied to production and clinical application, improves the immune effect of canine distemper vaccine strain and other conventional vaccines, controls the occurrence and epidemic of diseases, and protects the health of panda. Canine distemper is usually known as "the fatal infectious disease", has become the first fulminating infectious disease that threatens the species number and life safety of pandas, and badly affects the healthy development of pandas. The recombinant panda IL-2 (gpIL-2) immune adjuvant can prominently strengthen the immune effect of canine distemper vaccine strain, and has an important meaning on preventing and treating of canine distemper.

Description

A kind of preparation method of the giant panda IL-2 immunological adjuvant of recombinating

Technical field

The present invention relates to biological technical field, in particular a kind of preparation method of restructuring giant panda IL-2 (gpIL-2) immunological adjuvant.

Background technology

Giant panda is described as " national treasure " of China, be world-class rare wild animal, yet it is endangered, and except artificial origin is to the destruction of Giant Panda Habitat, disease death becomes one of current Giant Panda Population key reason in imminent danger.In 40 various diseases of suffering from giant panda, canine distemper is the primary deadly infectious disease that threatens Giant Panda Population quantity and life security.Vaccine immunity is the major measure of prevention and control CDV at present, but the vaccine of current use is not very good, exist Immune efficiency low with poor stability two hang-ups.Therefore, under safe dose vaccine inoculation, improve the immunizing power of giant panda body, become the focus of current vaccine development research.Cytokine is to be produced by immune effector cell and other relevant cells, has various biological activity, in the generation of immunne response with in regulating, has vital role.

Interleukin II (IL-2) is the important member in interleukin-family, and its report that is used as immunological adjuvant enhancing immune effect of vaccine is of common occurrence.

IL-2 is SCIF, and its most important effect is inducer T lymphocyte propagation (entering the S phase from the GO phase) and differentiation.T cell is subject to can secrete IL-2 after multiple stimulation, and IL-2 acts on again T cell self, induces and self breeds, breaks up and performance function, and this effect is called " Autocrine ".IL-2 can strengthen the killing activity of T cell, in vitro with IL-4, the IL-5 generation of inducing cytotoxic T cell (TC) jointly together with IL-6, and its activity is strengthened greatly, extends its vegetative period; In vivo IL-2 also can enhancement antigen the TC of induction active, even can assist antigen and haptens directly in nude mouse induction produce TC.After in the TC input body being produced by IL-2 induction, can produce obvious antitumor action.

IL-2 can induce natural killer cell (NK cell) propagation, strengthens the activity of NK cell, killer cell (LAK cell) and the tumor infiltrating lymphocyte (til cell) of induction Lymphokine, and stimulate them to produce cytokine.NK cell surface has IL-2R chain, and IL-2 stimulates NK cell activation and propagation, and can promote NK emiocytosis IFN-γ, increases it and expresses IL-2R subunit etc.The IL-2 of high density can also induce in vitro the LAK cell proliferation of NK cell derived, T cell derived and promote its activity.

IL-2 all has certain promoter action to the growth of B cell and differentiation.Can impel the IL-2R of B cell expressing high-affinity, wherein α chain is by B cell mitogen abduction delivering, and β chain is by IL-4 or IL-6 abduction delivering.IL-2 is except supporting B Growth of Cells, secretion that can also Promote immunity sphaeroprotein (IgM/IgG), thus the synthetic IgM that accelerates of induction J chain is towards IgG secretion 2 conversions.IL-2 can strengthen the expression of the IL-2 acceptor (IL-2R) that activation (infection, autoimmune disease, tumour etc.) B cell and leukemia cell produce, and stimulate its propagation and generation of immunoglobulin (Ig): in addition, IL-2 can also stimulate less prominent shape neurogliocyte to produce marrow basic protein, the propagation of this kind of cell is to amphicheirality and regulates.

In the cytokine of humans and animals, IL-2 is the cytokine of making at first molecular structure, acceptor composition and biological function clear, and IL-2 research occupy the forefront of cytokine always, and its application is also to study the most widely.In animal medicine, recombinant il-2 demonstrates wide application prospect at the aspects such as immunoprophylaxis, immunotherapy and structure a new generation gene engineering vaccine of some Animal diseases.

IL-2, because of immune level that can enhancing body, improves the resistance against diseases of body, thereby for the immunotherapy of bacillary, viral and parasitic diseases.In addition, IL-2 also can affect the metabolism of medicine, makes the metabolism time lengthening of medicine, and increase action time, thereby improves curative effect of medication.That first is the cytokine medicine that united States food and drug administration (FDA) approval is used for the treatment of cancer.Zhang Guangqin is sick and suis polyinfection by pharmacological agent goatpoxes such as IL-2 combination antibiotics, result shows that recombinant interleukin 2 is the active drug for the treatment of goatpox and suis polyinfection, can significantly improve the curative ratio of goatpox and suis polyinfection, and shorten its course for the treatment of.Qin Cuili etc., by the therapeutic test to 40 routine colibacillosis rabbits with interleukin II, cure 38, and dead 2, curative ratio is 95%, shows that interleukin II has significant curative effect to rabbit E.coli disease.Li Xiangrui is the treatment for Streptococcus suis by recombinant il-2, has obtained good effect.Aspect parasite, the reports such as Lillehoj, at the day before yesterday or the metainfective first day of E.imeriatenella or E.acervulina infected chicken, by the cytokine that ConA activation chicken lymphocyte produces, process chicken, can obviously reduce the output of coccidium infection ovum gallinaceum capsule.

Summary of the invention

The present invention aims to provide a kind of effect and significantly and again extracts easy; and can directly apply to restructuring giant panda IL-2 (gpIL-2) immunological adjuvant of production and clinical application; improve the immune effect of canine distemper vaccine and other conventional vaccines; the generation of control disease and popular, the health of protection giant panda.

Technical scheme of the present invention is as follows:

The recombinate preparation method of giant panda IL-2 immunological adjuvant, comprises the following steps:

A1, giant panda IL-2 full gene cloning;

A11 giant panda peripheral blood lymphocyte vitro culture;

Under aseptic condition, take healthy giant panda anticoagulation 2ml, be placed in EDTA-Na2 valve tube, then add inward the dilution of equivalent D-Hanks liquid; Slowly add in the giant panda peripheral blood of 4ml lymphocyte separation medium after dilution the centrifugal 20min of 2000r/min; Carefully white corpuscle is moved in another test tube, add equivalent RPMI1640 liquid, the centrifugal 15min of 2000r/min, removes supernatant, and throw out adds 4mlRPMl1640 liquid again, washs 2 times, removes supernatant; Precipitation lymphocyte suspends with the RPMI1640 liquid containing 10 μ g/mL ConA, 10% calf serum, 100 μ g/mL penicillin and 100 μ g/mL Streptomycin sulphates, gets 10 μ L cell suspension countings, and cell suspension is diluted to 5 * 10 ⁶individual/mL, is sub-packed in Tissue Culture Flask CO ₂in incubator, cultivate 24h for 37 ℃;

The total RNA of A12 extracts;

A13cDNA's is synthetic;

The total RNA that more than walks extracting is template, according to Prime Script ^tMthe operation steps of RT reagent kit reverse transcription test kit is carried out reverse transcription;

The design of A14 primer is with synthetic;

According to the giant panda IL-2cDNA sequence of having reported in GenBank, use the primer of two pairs of full genes of amplification IL-2 of primer-design software Oligo6.0 design, concrete sequence is as shown in the table:

The pcr amplification of A15 goal gene;

Take giant panda genome cDNA as template, and the IL-2P1/P2 of take carries out pcr amplification as primer; Reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 40s, 58 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations;

The TA clone of A16 goal gene PCR product;

The recovery of A161 object fragment;

A162 object fragment is connected with cloning vector;

The product that upper step is reclaimed is connected with pMD18-T Simple Vector, and 16 ℃ of connections are spent the night;

The preparation of A163DH5 α competent cell;

A164 connects the conversion of product;

The preliminary evaluation of A17 recombinant plasmid;

The prokaryotic expression of A2, giant panda interleukin-22, the preparation of polyclonal antibody;

A21 design of primers is with synthetic;

According to IL-2 Gene in Ailuropoda Melanoleuca sequencing result, use Oligo7.0 design pair of primers: IL-2P1/P2; The cDNA base sequence of amplification coding IL-2 mature peptide;

The pcr amplification of A22 object fragment;

Using recombinant plasmid pMD18-T-IL2 as template, add synthetic Auele Specific Primer IL-2P1/P2, carry out pcr amplification; Reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 40s, 60.8 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations;

The TA clone of A23 goal gene;

The TA clone of A231IL-2;

The preliminary evaluation of A24 recombinant plasmid;

The structure of A25 prokaryotic expression carrier;

The cultivation of A26 recombinant strains and expression;

The optimization of A27 prokaryotic expression;

The solubility of A28 recombinant expression protein detects.

Canine distemper have the title of " crushing transmissible disease ", has become the primary deadly infectious disease that threatens Giant Panda Population quantity and life security, has had a strong impact on the sound development of giant panda.Therefore the present invention's giant panda IL-2 (gpIL-2) immunological adjuvant of recombinating can significantly strengthen canine distemper vaccine immune effect, significant to the control of canine distemper.

Accompanying drawing explanation

Fig. 1 is the clone of IL-2 gene, A:M.DL2000Marker; 1.IL-2 gene amplification product;

Fig. 2 is that pMD18-T-IL-2 double digestion is identified collection of illustrative plates, M:DNA molecular mass standard, and 1:pMD-IL-2 enzyme is cut product;

Fig. 3 is that the enzyme of cloning vector is cut evaluation, A:M.DL2000Marker; 1.pMD-2 enzyme is cut product;

Fig. 4 is that the enzyme of recombinant expression plasmid is cut evaluation, A:M.DNA Marker III; 1.pET-32-2 double digestion product;

Fig. 5 is that the SDS-PAGE of recombinant protein analyzes, M. albumen marker; 1. empty carrier pET-32; 2. the supernatant after ultrasonic disruption; 3. the supernatant after the washing of 2M urea for inclusion body; 4. the supernatant after the washing of 4M urea for inclusion body; 5. the supernatant after the washing of 6M urea for inclusion body; 6. the supernatant after the washing of 8M urea for inclusion body;

Fig. 6 is that sero-fast fine jade expands Potency Analysis;

Fig. 7 is purifying and the immunoblotting assay of recombinant protein; A:M. albumen marker; 1. the anti-IL-2 of recombinant protein pET-32-IL-22. rabbit of purifying is primary antibodie; 3. negative control.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

One, giant panda IL-2 full gene cloning

Experimental technique

1.1 giant panda peripheral blood lymphocyte vitro culture

Under aseptic condition, take healthy giant panda anticoagulation 2ml, be placed in EDTA-Na ₂in valve tube, then add inward the dilution of equivalent D-Hanks liquid.Slowly add in the giant panda peripheral blood of 4ml lymphocyte separation medium after dilution the centrifugal 20min of 2000r/min.Carefully white corpuscle is moved in another test tube, add equivalent RPMI1640 liquid, the centrifugal 15min of 2000r/min, removes supernatant, and throw out adds 4mlRPMl1640 liquid again, washs 2 times, removes supernatant; Precipitation lymphocyte suspends with the RPMI1640 liquid containing 10 μ g/mL ConA, 10% calf serum, 100 μ g/mL penicillin and 100 μ g/mL Streptomycin sulphates, gets 10 μ L cell suspension countings, and cell suspension is diluted to 5 * 10 ⁶individual/mL, is sub-packed in Tissue Culture Flask CO ₂in incubator, cultivate 24h for 37 ℃.

1.2 total RNA extract

The RNA extraction test kit that total RNA extracts with reference to Promega company carries out, and concrete steps are as follows:

(1) the cell bottle of cultivating after 24h is taken out, with pasteur pipet piping and druming bottle bottom surface, then pour the centrifugal 5min of 4000r/min in centrifuge tube into, abandon supernatant.

(2) to the lysate that adds 300 μ L in centrifuge tube, under room temperature, act on 5min.

(3) then add the Blue buffer of 600 μ L, mix.Be positioned in the water of 70 ℃ and act on 2.5min.

(4) centrifugal 10min, is transferred to supernatant in other clean EP pipe, adds the ethanol of 200 μ L95%, after mixing, proceed in adsorption column, and effect 5min, the centrifugal 1min of 12000r/min, abandons the liquid in collection tube.

(5) in adsorption column, add washings 600 μ L washing 1 time, centrifugal, abandon the liquid in collection tube.

(6) add stop buffer 200 μ L after adding DNAase mixed solution 50 μ L effect 15min, the centrifugal 1min of 12000r/min, abandons the liquid in collection tube.

(7) add 600 μ L washings washing 1 time, abandon the liquid in collection tube.

(8) add again 250 μ L washings washings, abandon the liquid in collection tube, the unloaded centrifugal 3min of 12000r/min.

(9) the EP pipe belt of changing a cleaning is for collection tube, toward the nuclease free water that adds 80 μ L on adsorption column, the centrifugal 1min of 13000r/min, the liquid in collection EP pipe, be stored in-70 ℃ standby.

1.3cDNA's is synthetic

The total RNA that more than walks extracting is template, according to Prime Script ^tMthe operation steps of RT reagent kit reverse transcription test kit is carried out reverse transcription, and its reverse transcription system is:

Table 1

37℃15min，85℃5sec。CDNA after reverse transcription be stored in-20 ℃ standby.

The design of 1.4 primers is with synthetic

According to the giant panda IL-2cDNA sequence of having reported in GenBank, use the primer of two pairs of full genes of amplification IL-2 of primer-design software Oligo6.0 design, concrete sequence is as shown in the table:

Table 2

The pcr amplification of 1.5 goal gene

Take giant panda genome cDNA as template, and the IL-2P1/P2 of take carries out pcr amplification as primer, and amplification reaction system is as follows:

Table 3

Reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 40s, 58 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations.

Get 5 μ l PCR products and carry out electrophoresis detection amplification with 1% sepharose.

The TA clone of 1.6 goal gene PCR products

1.6.1 the recovery of object fragment

The specification sheets that the recovery of object fragment provides with reference to the DNA of Tian Gen company glue recovery test kit carries out, and recycling step is as follows:

(1) in adsorption column, add 500 μ L balance liquid BL, the centrifugal 1min of 12000r/min, abandons waste liquid in collection tube, then adsorption column is put back to collection tube;

(2) the DNA band that contains object fragment in sepharose is cut and blot surperficial damping fluid and put into centrifuge tube and take weight;

(3) add 3 times of volume sol solutionses in blob of viscose.55 ℃ of water-baths, during constantly gently spin upside down centrifuge tube so that blob of viscose fully dissolves, water-bath is until dissolve completely;

(4) when sol solutions is cooled to room temperature, added in the adsorption column that balance has been crossed, standing 2min, the centrifugal 1min of 12000r/min, outwells waste liquid in collection tube;

(5) add 700 μ l rinsing liquids (before use, first whether check has added dehydrated alcohol) in adsorption column, standing 2min, the centrifugal 1min of 8000r/min, outwells waste liquid in collection tube;

(6) add 500 μ L rinsing liquid PW in adsorption column, the centrifugal 1min of 12000r/min, outwells waste liquid in collection tube;

(7) the unloaded centrifugal 2min of 12000r/min removes rinsing liquid as far as possible.Place adsorption column in room temperature number minute, until alcohol-free taste;

(8) adsorption column is put into a clean centrifuge tube, the elution buffer of unsettled dropping 60 μ l in adsorption film mid-way, the standing 2min of room temperature.The centrifugal 2min of 12000r/min collects DNA solution;

1.6.2 object fragment is connected with cloning vector

The product that upper step is reclaimed is connected with pMD18-T Simple Vector, and linked system is as follows:

Table 4

Cumulative volume 10 μ L, mix rear 16 ℃ of connections and spend the night.

1.6.3DH5 the preparation of α competent cell

(1) from long, there is single bacterium colony of picking bacillus coli DH 5 alpha flat board, be inoculated in 5mL LB liquid nutrient medium, in 37 ℃ of 200r/min overnight incubation;

(2) get 1mL incubated overnight liquid, transfer in 10ml LB liquid nutrient medium, shaking culture 2h (OD600 approximately 0.6);

(3) by the aseptic 15mL centrifuge tube of transferring to of the bacterium liquid 10mL of above-mentioned gained, ice bath 30min; 4 ℃ of centrifugal 8min of 4000r/min;

(4) abandon supernatant, to the 0.1mol/L CaCl that adds 12mL precooling in centrifuge tube ₂resuspended, the centrifugal 10min of 4000r/min after ice bath 10min;

(5) repeat above-mentioned steps once;

(6) abandon supernatant, then add the 0.1mol/L CaCl of prior ice precooling in precipitation ₂1mL is resuspended and be packed as the every pipe of 0.15mL/, is placed in ice bath spend the night (12～24h).

1.6.4 connect the conversion of product

(1) by connecting product, join in the competent cell having prepared, fully mix ice bath 30min;

(2) 42 ℃ of heat-shocked 2min, then put into rapidly 0 ℃ of ice bath 5min;

(3) add LB liquid nutrient medium 800 μ L, 37 ℃ of shaking culture 45min;

(4) aseptic condition is got bacterium liquid 300 μ L, coats uniformly on previously prepared good acillin agar LB flat board, and 37 ℃ of constant temperature culture are spent the night, and occurs that transparent whites transforms bacterium colony.

1.7 the preliminary evaluation of recombinant plasmid

1.7.1 a small amount of of recombinant plasmid is extracted

Specification sheets with reference to the little extraction reagent kit of OMEGA plasmid carries out, and concrete operations are as follows:

(1) get 4mL bacterium liquid, the centrifugal 10min of 12000r/min collects thalline;

(2) abandon bacterium supernatant, add refrigeration alkaline lysis liquid I250 μ l, on whirlpool instrument, vibration mixes, and resuspended bacterium is transferred in 1.5ml EP pipe;

(3) add alkaline lysis liquid II250 μ I, slowly repeatedly put upside down for several times, until crin becomes filament;

(4) add Solution III350 μ L, put upside down gently and mix to white floss appearance;

After (5) 4 ℃ of centrifugal 10min of 12000r/min, supernatant liquor is moved in adsorption column to standing 2min;

(6) add 700 μ L rinsing liquids (before use, first whether check has added dehydrated alcohol), the centrifugal 1min of 12000r/min, abandons waste liquid in collection tube;

(7) repeat above-mentioned steps once after, the unloaded centrifugal 2min of 12000r/min;

(8) adsorption column is placed on clean EP pipe, in adsorption film mid-way, the elution buffer of unsettled dropping 60 μ l, places 3min under room temperature;

(9) 12000r/min4 ℃ of centrifugal 1min, collects elutriant, and-20 ℃ save backup.

1.7.2 recombinant plasmid double digestion is identified

3 recombinant plasmids that [1.7.1] extracting is obtained carry out enzyme with EcoR I, Sal I respectively and cut evaluation, and endonuclease reaction system is as follows:

Table 5

After 37 ℃ of 2h, enzyme is cut to product and carry out agarose gel electrophoresis, observations.

1.7.3 the mensuration of nucleotide sequence

Positive recombinant plasmid bacterial classification after identifying is preserved to the order-checking of Ye Songbao biotech firm.

2. results and analysis

The pcr amplification of 2.1IL-2 gene

Take giant panda genomic dna as template, add after Auele Specific Primer pcr amplification, through 0.8% agarose gel electrophoresis, result shows amplified fragments big or small conform to expection (seeing Fig. 1).

2.2IL-2 gene TA cloned plasmids is identified

Above-mentioned amplified production is connected to pMD18-T carrier, and recombinant plasmid is identified and is shown by EcoR I and Sal I double digestion, obtains expection big or small fragment (seeing Fig. 2), positive plasmid called after pMD18-T-IL-2.Through order-checking, show, the fragment of amplification is identical with gene reference sequences.

Two, the prokaryotic expression of giant panda interleukin-22 is, the preparation of polyclonal antibody

Experimental technique

1.1 design of primers are with synthetic

According to IL-2 Gene in Ailuropoda Melanoleuca sequencing result, use the cDNA base sequence of Oligo7.0 design pair of primers (IL-2P1/P2) amplification coding IL-2 mature peptide.Concrete primer situation is in Table 6 (restriction enzyme site represents with underscore).

Table 6 primer sequence

The pcr amplification of 1.2 object fragments

Using recombinant plasmid pMD18-T-IL2 as template, add synthetic Auele Specific Primer (IL-2P1/P2), carry out pcr amplification.PCR reaction system is as following table 7:

Table 7

Reaction conditions is: 95 ℃ of denaturation 5min95 ℃ of sex change 40s, and 60.8 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations.

The TA clone of 1.3 goal gene

1.3.1IL-2 TA clone

Pcr amplification product carries out electrophoresis detection on 1% agarose gel plate, adopts sky, Beijing root sepharose to reclaim test kit and reclaims purifying object fragment.According to object fragment amount after purifying, determine add-on 4.5uL, pMD18-T Simple Vector0.5uL, by above-mentioned TA connecting fluid transformed clone bacterium DH5 α, after activation culture, transformed bacteria coating is contained to the LB solid medium of Amp (50mg/L), be placed in 37 ℃ of incubators, just putting absorption 0.5h, be then inverted and cultivate 12～24h.

The preliminary evaluation of 1.4 recombinant plasmids

At the single bacterium colony of the dull and stereotyped picking of the LB solid medium containing Amp (50mg/L), be inoculated in 5mL containing the LB liquid nutrient medium of Amp (50mg/L), 37 ℃ of shaken overnight.The plasmid of upper step extracting is done to double digestion with restriction enzyme EcoR I and Sal I to be identified.Finally send precious biology to check order the correct plasmid of preliminary evaluation.

The structure of 1.5 prokaryotic expression carriers

The plasmid that T clone identification is correct carries out double digestion to T-IL2 and pET32a (+) carrier according to the system of cutting of the enzyme in table 8 simultaneously.

Table 8

By glue, reclaim test kit and carry out glue recovery, linked system is as table 9:

Table 9

Cumulative volume 10 μ L, spend the night in 16 ℃ of connections after mixing.Connecting fluid is transformed into DH5a competent cell, by recombinant plasmid is carried out to double digestion, by the correct prokaryotic expression plasmid Song Bao biotech firm order-checking of preliminary evaluation.

The cultivation of 1.6 recombinant strains and expression

Recombined pronucleus expression plasmid is transformed and expresses bacterium E.coli Rosetta (DE3), and extracting recombinant plasmid, carries out PCR evaluation and restriction enzyme EcoR I and Sal I double digestion and identifies.

Identifying that correct E.coli Rosetta (DE3) the inoculum 100 μ l containing recombinant expression plasmid are inoculated in 10mL containing the LB liquid nutrient medium of Amp (50mg/L), 37 ℃ of thermal agitation overnight incubation, inoculate in fresh LB liquid nutrient medium next day with the ratio of 1: 100, is cultured to OD ₆₀₀=0.6 while being left and right, adds IPTG, continues to cultivate 4h.Set up negative control simultaneously.Get the bacterium liquid 1mL after IPTG induction, the centrifugal 1min of 13000r/min, collects thalline resuspended with 50 μ l PBS, then adds 50 μ l2 * SDS sample-loading buffer 50 μ l, and sample carries out SDS-PAGE after treatment, observations after dyeing, decolouring.

The optimization of 1.7 prokaryotic expressions

By recombinant expressed bacterium in containing in the LB liquid nutrient medium of Amp (50mg/L), 37 ℃ of thermal agitation overnight incubation, with 1: 100 ratio, inoculate respectively containing the LB liquid nutrient medium of Amp (50mg/L) and cultivate, respectively IPTG concentration (0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.7mmol/L and 1.0mmol/L), induction time (2h, 3h, 4h, 5h, 6h) and inducing temperature (25 ℃, 30 ℃ and 37 ℃) are optimized.The sample of collecting under various conditions is processed, SDS-PAGE electrophoretic analysis.

The solubility of 1.8 recombinant expression proteins detects

Abduction delivering 100mL bacterium liquid, 12000r/min4 ℃ of centrifugal 10min collects thalline, resuspended with 20ml20mM Tris-Cl (pH8.0).Ultrasonic disruption thalline 5min under condition of ice bath, per minute interval 30s, then 4 ℃, the centrifugal 10min collecting precipitation of 12000r/min and supernatant, after processing, carry out SDS-PAGE electrophoresis, detecting expression product is that soluble form exists (in supernatant) or inclusion body form exists (in precipitation).

The preparation of 1.9 interleukin-22 polyclonal antibodies and titration thereof

1.9.1 the preparation of polyclonal antibody

The complete Fei Shi adjuvant of immunogen and equal-volume is mixed, fully emulsified, in family's rabbit neck part, back multiple spot subcutaneous injection.After 2 weeks, the incomplete Fei Shi adjuvant of recombinant protein and equal-volume is mixed mutually, carry out booster immunization.After weekly booster immunization once, and blood sampling detect to produce antibody titer weekly.Until antibody horizontal is raised to, meet the requirements, serum is collected in heart blood sampling, puts-20 ℃ and saves backup (immune programme for children is in Table 10).

Table 10 immunization program

1.9.2 saturated ammonium sulphate method is slightly carried IgG:

Concrete operation step with reference to reference (to fine horse. the expression of duck plague virus VP19c albumen, thin inner cellular localization and RNA disturb the preliminary study [D] that suppresses virus replication; Sichuan Agricultural University, 2011.) slightly put forward IgG method.

1.9.3 agar diffusion measuring antibody titer

(1) culture dish is washed to rear 75% alcohol flushing of using, after drying, be placed on horizontal stand standby;

(2) agarose (1%) that NaCl that will 0.9% dissolves spreads glue after melting in culture dish, and about 3mm is thick, solidifies with punch tool, to punch afterwards;

(3) centre hole adds 20ul antigen samples (comprising IL2 purifying protein), and around Kong Neimei hole adds 20ul antiserum(antisera) (antibody is made i.e. 1: 2,1: 4,1: 8,1: 16,1: 32 equal proportion of serial doubling dilution in advance);

(4) culture dish is placed in wet box, then in 37 ℃ of incubators, places 24h, observations.

The immunoblotting of 1.10 fusion expressed products (Western-Blot)

The recombinant protein IL-2 of purifying of take is antigen, usings the immunize rabbit serum slightly carried as primary antibodie, and two resist the goat anti-rabbit igg for HRP mark, colour developing DAB reagent.If rabbit negative serum is contrast, concrete operations referring to reference (to fine horse. the expression of duck plague virus VP19c albumen, thin inner cellular localization and RNA disturb the preliminary study [D] that suppresses virus replication; Sichuan Agricultural University, the isolation identification of 2011. woods China .PRRSV variants, the research [D] of differential diagnosis and novel gene vaccine; Sichuan Agricultural University, 2010.)

The purifying of 1.11 expression products and renaturation

1.11.1 the purifying of soluble proteins

According to the histidine-tagged fusion protein purification test kit of HisTrap FF specification sheets, carry out, concrete steps are as follows:

(1) collect after thalline, every gram of thalline (weight in wet base) adds 5-10ml binding buffer liquid to suspend, ultrasonic disruption thalline 5min under condition of ice bath, per minute interval 30s, 4 ℃, the centrifugal 10min of 10000r/min collects supernatant, and with the membrane filtration of 0.45um, removes cell debris and cake mass;

(2) syringe is filled up with distilled water, the chromatography column top plug of outwarding winding, is connected to syringe pillar, keeps junction to have liquid always, avoids introducing bubble;

(3) remove chromatography column bottom plug, with the deionized water of 3-5 times of volume, wash away the dehydrated alcohol containing in pillar, then use the binding buffer liquid balance pillar of at least 5 volumes, suggestion flow velocity is 1ml/min;

(4) with syringe, add pretreated sample, in order to obtain best effect, when loading, keep the speed of 0.2ml/min;

(5) with binding buffer liquid, wash at least 5-10 column volume, in the process of washing, keeping flow velocity is 1-2ml/min;

(6) use elution buffer wash-out, common 5 column volumes;

(5) after wash-out, use the binding buffer liquid washing of 3-5 times of column volume, then use the ethanol balance of 5 times of column volumes 20%, 4 ℃ are stored in 20% ethanol, so that next purifying.

1.11.2 the purifying of inclusion body protein

Abduction delivering 200mL bacterium liquid, the centrifugal 10min of 10000r/min, collects thalline, the resuspended liquid of use 40ml thalline (50 mM Tris-HCl, 5mM EDTA, 0.15mM NaCl, 1mg/ml N,O-Diacetylmuramidase, pH=8.0) resuspended.Ultrasonic disruption thalline 5min under condition of ice bath, per minute interval 30s, then 4 ℃, the centrifugal 10min collecting precipitation of 10000r/min.Add inclusion body washings (50mM Tris-HCl, 5mM EDTA, 0.15mM NaCl, 2M urea, pH=8.0), is used transfer pipet purge for several times, the centrifugal 10min of 10000r/min then, collecting precipitation.Repeated washing three times, precipitation is dissolved with solubilization of inclusion bodies liquid (50mM Tris-HCl, 8M urea, 0.1M DTT, pH8.0), and-20 ℃ save backup.

1.11.3 the renaturation of purifying protein

The processing of dialysis tubing: dialysis tubing is cut into the segment of 10cm, at 2%NaHCO ₃(1000ml) in, dialysis tubing is boiled to 10min, then with distilled water, clean up, being placed on concentration is to continue to boil 10min in 1mmol/L EDTA (1000ml), the inclusion body after cooling, above-mentioned 8mol/L urea being dissolved is transferred in dialysis tubing, put into dialysis renaturation liquid (50mM Tris-HCl is housed, 5mM EDTA, 150mM NaCl, 1.25mM GSH, with 0.25mM GSSG, 20% glycerine, pH=8.0, in renaturation basal liquid, add successively 6mol/L, 4mol/L, 2mol/L urea and PBS) beaker in, in 4 ℃ of refrigerators, dialyse, each concentration dialysis 12h, albumen after dialysis renaturation is used for doing determination of activity.

It is active that 1.12MTT method detects expression product

The disconnected neck of mouse is put to death, the aseptic spleen of getting is put into and is filled PBS ampulla, after peeling off white connective tissue, put back in ampulla, add 4-6ml HankS, with eye scissors, shred spleen, by copper mesh, filter and remove fragment of tissue, cell suspension is added in 15ml centrifuge tube, the centrifugal 10min of 2000r/min, supernatant discarded; Add 5ml to remove erythrocyte cracked liquid, room temperature is placed 10min, the centrifugal 10min of 2000r/min, supernatant discarded; Add 5ml HankS to wash and carry out cell count, after the centrifugal 10min of 2000r/min, with 10%1640 nutrient solutions, adjusting cell count is 5 * 10 ⁵individual ^[2-3], the lymphocyte suspension of preparation is joined to 96 orifice plate Zhong，Mei hole 100 μ L, then (final concentration is respectively 1,2,5,10 μ gmL to add 50 μ L recombinant protein samples ^-1), establish 6 repeating holes, and to establish physiological saline and ConA be yin, yang contrast, put 5%CO ₂, cultivate after 48h in 37 ℃ of incubators, abandon supernatant ，Mei hole and add 5mg/mLMTT10 μ L, continue to cultivate 4h, add 100 μ LDMSO effect 30min, enzyme linked immunosorbent detection A ₅₇₀value.

2. results and analysis

2.1 object fragment TA cloned plasmids double digestions are identified

The ripe fragment IL-2 of amplification is connected to pMD18-T carrier, recombinant plasmid is identified and is shown by EcoR I and Sal I double digestion, obtain expection big or small fragment (Fig. 3), sequencing result is in full accord with corresponding sequence, proves that the TA cloned plasmids of object fragment successfully constructs.Positive plasmid called after pMD-2.

The double digestion of 2.2 object fragment prokaryotic expression carriers is identified

Above-mentioned three fragments enzyme from recombinant cloning vector is cut, directed cloning is to prokaryotic expression carrier pET-32a (+), transform Rosetta (DE3), extracting plasmid double digestion is identified, enzyme is cut result and is conformed to expection size, the success of proof recombined pronucleus expression plasmid construction, by positive plasmid difference called after pET-32-2, electrophoresis result is shown in Fig. 4.

The abduction delivering of 2.3 goal gene, solubility detect the optimization with condition

The positive recombinant expressed bacterium of the Rosetta that picking contains recombined pronucleus expression plasmid (DE3), IPTG inducing culture 4h, after centrifugal collection thalline, ultrasonic disruption, cleer and peaceful precipitation in collection, precipitation is cleaned to (2M with the urea of different concns, 4M, 6M, 8M) collect the supernatant liquor under different concns, through processing, do SDS-PAGE electrophoresis detection, result shows, after recombinant bacterium induction, sample is equipped with obvious amalgamation and expression band in certain bits, and solubility detects finds that recombinant protein IL-2 is that soluble form exists (seeing Fig. 5).

By discovery that different IP TG concentration, different induction time and different inducing temperature are compared, interleukin-22 is at 30 ℃, and IPTG concentration is under 0.1mmol/L, to induce the amount of 5h maximum.

The mensuration that 2.4 polyclonal antibodies are tired

According to test method [2.9.1] immunity test rabbit, to collect and separation of serum, it is 1: 4 (Fig. 6) that the Chinese People's Anti-Japanese Military and Political College's panda IL-2 polyclonal antibody that records preparation by agar diffusion test is tired.

The purifying of 2.5 target proteins and immunoblotting assay

By methods such as histidine-tagged protein purification kit and inclusion body washings, obtain the recombinant protein (Fig. 7) of purifying.Through Western blot, analyze recombinant protein, result shows the panda IL-2 of Qi Junkeyu Chinese People's Anti-Japanese Military and Political College positive serum generation specific immune response (Fig. 7), proves that obtaining product is that goal gene is expressed, and has good reactionogenicity.

The bioactive detection of 2.6 expression product

The fusion rotein of purification renaturation of take is sample, and physiological saline and ConA, for contrast, carry out the detection of mouse lymphocyte multiplication-stimulating activity, and result is as shown in table 11:

Table 11 reclaims recombination fusion protein IL-2 to mouse lymph parent cell multiplication-stimulating activity detected result

Result shows, the fusion rotein IL-2 of purification renaturation all has to mouse lymphocyte the effect of significantly stimulating proliferation.When IL-2 concentration is 2 μ gmL ^-1time recombinant protein the most obvious to the cultivation effect of mouse lymph parent cell.

Three, restructuring giant panda interleukin II and fusion rotein thereof are measured the adjuvant effect of canine distemper virus gene engineering vaccine in mouse model

Experimental technique

1.1 Cytokine adjuvants exempt from front preparation

The recombinant cytokine albumen of purifying is after biologic activity detects, mixed mutually with mineral oil equal-volume, fully mix, be stored in 4 ℃ standby.Exempt from front that recombinant cytokine albumen and canine distemper recombinant vaccine equal-volume is mixedly mutually, fully mix.The final concentration of recombinant protein is foundation according to MTT experimental result, and the final concentration of IL-2 is respectively 2 μ gmL ^-1.

The grouping of 1.2 experiment mices and immunity

1.2.1 animal grouping

18 BALB/c mouse that are 18～25g by 4～6 week age, body weight are divided into 3 groups at random, 6/group, are respectively A. intramuscular injection CDV recombiant vaccine (0.2mL)+IL-2 (final concentration 2 μ gmL-1); B. intramuscular injection CDV recombiant vaccine (0.2mL)+PBS; With C.PBS blank group.

1.2.2 immunization method

By previous grouping situation vaccination, and inject at twice the recombinant cytokine albumen containing the emulsification of IL-2 every day, inject continuously 3 days.One exempts from booster immunization after 2 weeks, and immunization ways and is exempted from identical.

1.3 serum are processed

After exempting from respectively at head 7,14,21,28,35,42d is from tail vein blood sample collection, and the blood sample of collection is put in 37 ℃ of incubators after one hour, be placed in 4 ℃ and spend the night.Second day is by the centrifugal 10min of blood sample 8000rp/min, and sucking-off serum, preserves in-20 ℃ of refrigerator-freezers of 56 ℃ of deactivations postposition half an hour.To make conventional ELASA after the 6th week serum is also collected simultaneously, detect, detect and exempted from mouse and produce IL-4, IFN-γ level in the IgG level of anti-CDV and serum at different times.

1.4CDV antibody test

Concrete operation step carries out with reference to mouse canine distemper virus antibody (CDV-Ab) enzyme immunoassay (ELISA) specification sheets.

IFN-γ, IL-4 assay in 1.5 serum

Operation steps is carried out according to mouse IFN-γ (IFN-γ) ELISA detection kit, mouse interleukin 4 (IL-4) ELISA detection kit specification sheets.

(1) from the aluminium foil bag equilibrium at room temperature 20min, take out required lath, residue lath is put back to 4 ℃ with valve bag sealing;

(2) standard substance hole and sample aperture are set, standard substance hole respectively adds the standard substance 50 μ L of different concns;

(3) sample aperture first adds sample to be tested 10 μ L, then adds sample diluent 40 μ L; Blank well does not add;

(4) except blank well, standard substance hole and sample aperture Zhong Mei hole add the detection antibody 100 μ L of horseradish peroxidase (HRP) mark, with shrouding film, seal reacting hole, 37 ℃ of water-baths or thermostat container incubation 60min;

(5) after being got rid of, liquid in plate pats dry on thieving paper, the washings configuring toward Kong Zhongjia, and standing 1min, abandons washings, so repeats to wash plate 5 times (the also available plate machine washing plate of washing).

(6) every hole adds substrate A, each 50 μ L of B, and 37 ℃ of lucifuges are hatched 15min.

(7) every hole adds 50ul stop buffer to mix.At 450nm place, measure the OD value in each hole.

1.6 statistical analysis

Collect the OD Value Data that ELISA experiment measuring obtains, drawing standard curve (concentration of standard substance of take is X-coordinate, and the OD value measuring is ordinate zou), reference standard curve OD value is per sample by searching corresponding concentration.Use SPSS17.0 statistical software to carry out variance analysis to data, and check the significance of difference between each test group data mean value with paired sample t method of inspection, use Microsorft Excel Software on Drawing chart.

2. results and analysis

CDV antibody detection in 2.1 serum

Result demonstration, before immunity, all mice serum sample standard deviations are CDV feminine gender.1 week rear immune mouse of immunity starts to produce CDV specific antibody, but there is no notable difference (table 12) between each test group.To latter the 2nd week of immunity, the test group of immune CDV+IL2 was the highest compared with other group anti-body contg rising levels, reaches 97.679 ± 2.517ng/L, and CDV+PBS compares with control group, significant difference (P=0.046, < 0.05).This trend of high anti-body contg is continued until the 6th week after immunity, and is significantly higher than CDV+PBS control group (P=0.001 at the 4th week and the 6th week utmost point; 0.005, < 0.01).

CDV anti-body contg (ng/L) in table 12 immune serum

A and a represent CDV vaccine+IL-2 group and CDV vaccine+PBS in 0.01 level with 0.05 level on significant difference

IL-4 detected result in 2.2 serum

Detect the content of cytokine IL-4 in immune serum.Result demonstration, the level of each immune group IL-4 has been compared and has significantly been increased (table 13) with blank group PBS.Wherein, the IL-4 level of CDV vaccine+IL-2 immune group induction is all significantly higher than the 1st thoughtful 6 the IL-4 level (P < 0.05) that CDV vaccine+PBS induces, and at the 1st week, present extremely significantly (P=0.002, < 0.01).

The content of IL-4 (pg/mL) in table 13 immune serum

A and a represent CDV vaccine+IL-2 group and CDV vaccine+PBS in 0.01 level with 0.05 level on significant difference

IFN-γ detected result in 2.3 serum

Detect the content of cytokine IFN-γ in immune serum.Result shows, after immunity, the generation level of each immune group IFN-γ is than CDV vaccine+PBS group and blank group PBS all high (table 14).Wherein, the IFN-γ level that CDV vaccine+IL-2 stimulates mouse to produce is the most obvious, is all significantly higher than CDV vaccine+PBS group (P < 0.05).

The detection (pg/mL) of table 14 cytokine IFN-γ

A represents CDV vaccine+IL-2 group and CDV vaccine+PBS significant difference in 0.05 level

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (1)

1. the recombinate preparation method of giant panda IL-2 immunological adjuvant, is characterized in that, comprises the following steps:

A1, giant panda IL-2 full gene cloning;

A11 giant panda peripheral blood lymphocyte vitro culture;

Under aseptic condition, take healthy giant panda anticoagulation 2ml, be placed in EDTA-Na ₂in valve tube, then add inward the dilution of equivalent D-Hanks liquid; Slowly add in the giant panda peripheral blood of 4ml lymphocyte separation medium after dilution the centrifugal 20min of 2000r/min; Carefully white corpuscle is moved in another test tube, add equivalent RPMI1640 liquid, the centrifugal 15min of 2000r/min, removes supernatant, and throw out adds 4mlRPMl1640 liquid again, washs 2 times, removes supernatant; Precipitation lymphocyte suspends with the RPMI1640 liquid containing 10 μ g/mL ConA, 10% calf serum, 100 μ g/mL penicillin and 100 μ g/mL Streptomycin sulphates, gets 10 μ L cell suspension countings, and cell suspension is diluted to 5 * 10 ⁶individual/mL, is sub-packed in Tissue Culture Flask CO ₂in incubator, cultivate 24h for 37 ℃;

The total RNA of A12 extracts;

A13cDNA's is synthetic;

The total RNA that more than walks extracting is template, according to Prime Script ^tMthe operation steps of RT reagent kit reverse transcription test kit is carried out reverse transcription;

The design of A14 primer is with synthetic;

According to the giant panda IL-2cDNA sequence of having reported in GenBank, use the primer of two pairs of full genes of amplification IL-2 of primer-design software Oligo6.0 design, concrete sequence is as shown in the table:

The pcr amplification of A15 goal gene;

Take giant panda genome cDNA as template, and the IL-2P1/P2 of take carries out pcr amplification as primer; Reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 40s, 58 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations;

The TA clone of A16 goal gene PCR product;

The recovery of A161 object fragment;

A162 object fragment is connected with cloning vector;

The product that upper step is reclaimed is connected with pMD18-T Simple Vector, and 16 ℃ of connections are spent the night;

The preparation of A163DH5 α competent cell;

A164 connects the conversion of product;

The preliminary evaluation of A17 recombinant plasmid;

The prokaryotic expression of A2, giant panda interleukin-22, the preparation of polyclonal antibody;

A21 design of primers is with synthetic;

According to IL-2 Gene in Ailuropoda Melanoleuca sequencing result, use Oligo7.0 design pair of primers: IL-2P1/P2; The cDNA base sequence of amplification coding IL-2 mature peptide;

The pcr amplification of A22 object fragment;

Using recombinant plasmid pMD18-T-IL2 as template, add synthetic Auele Specific Primer IL-2P1/P2, carry out pcr amplification; Reaction conditions is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 40s, 60.8 ℃ of annealing 40s, 72 ℃ of renaturation 50s, this step is carried out 30 circulations; 72 ℃ are extended 10min; In 10 ℃ of preservations;

The TA clone of A23 goal gene;

The TA clone of A231IL-2;

The preliminary evaluation of A24 recombinant plasmid;

The structure of A25 prokaryotic expression carrier;

The cultivation of A26 recombinant strains and expression;

The optimization of A27 prokaryotic expression;

The solubility of A28 recombinant expression protein detects.

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