CN104524565A - New Der p1 nano vaccine for treating lung cancer as well as preparation method and application thereof - Google Patents
New Der p1 nano vaccine for treating lung cancer as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a new Der p1 nano vaccine for treating lung cancer as well as a preparation method and application thereof. The preparation method of the new Der p1 nano vaccine for treating the lung cancer comprises the following steps: firstly cloning and expressing in escherichia coli by adopting a genetic engineering technology for preparing Der p1 protein, and then applying biodegradable PGLA (poly (lactic-glycolic acid)) nanoparticle as a coating material for preparing PLGA-dust mite strain source Der p1(PLGA-Der p1) nano vaccine, namely the new Der p1 nano vaccine for treating the lung cancer product. The new Der p1 nano vaccine disclosed by the invention has a good anticancer function, has an obvious treatment effect on the lung cancer, no toxic or side effect is produced to a human body, organism immunity ability can also be enhanced, and a preparation process is simple.
Description
Technical field
The present invention relates to new generation vaccine invention field, specifically a kind of Der p1 nano vaccine and its production and use for the treatment of pulmonary carcinoma newly.
Background technology
Pulmonary carcinoma has become the first place of all cancer morbidities in global range, and its five-year survival rate is approximately 15%, the patients with lung cancer generation Bone tumour of about 30-40%, once there is Bone tumour, its median survival time only has 7 months.U.S.'s pulmonary carcinoma in 2012 has more than 1,630,000 new cases and 570,000 deaths.Pulmonary carcinoma simultaneously also exceedes hepatocarcinoma becomes the first cancer occurred frequently of China, and sickness rate and mortality rate increase the rapidest, every year with 13% speed increment.At present, pulmonary carcinoma occupies the first place of cancer morbidity seniority among brothers and sisters (men and women is comprehensive), wherein ranked first in male's sickness rate, in female cancer sickness rate, also comes front three all the time.Expect 2025, the annual newly-increased cases of lung cancer of China will more than 1,000,000 examples.Patients with lung cancer also presents rejuvenation trend, and especially in recent years, the patients with lung cancer of less than 45 years old occurred more.Therefore, pulmonary carcinoma has had a strong impact on the life and health threatening our people.
Current Therapeutic Method mainly contains the methods such as operative treatment, chemotherapy, radiotherapy.Make a general survey of current therapy, operative treatment mainly applicable early metaphase (I ~ II phase) pulmonary carcinoma and tumor is confined to the IIIb phase pulmonary carcinoma of the partial selective in thoracic cavity, side, and radiotherapy belongs to topical therapeutic together, from oncobiology angle, cancer is systemic disease, there is the characteristic of metastasis and extension; The therapeutic modalities such as conventional radiation and chemotherapy, also damage organism normal cell and immune system while killing tumor cell, and reduce immunity, toxic and side effects is large.Therefore, the research and development carrying out new cancer therapy drug are important and necessary, find and poison little, not damage immunity of organism medicine urgent needs really to anticancer effective and normal tissue.Show through our inventor's early-stage Study, the treatment of dermatophagoides pteronyssinus Der p1 vaccine to pulmonary carcinoma has significant effect, and energy enhancing human body immunity ability, there is anti-cancer function.
The present invention adopts technique for gene engineering cloning and expressing preparation restructuring Der p1 albumen in escherichia coli, overcomes a large amount of difficulty extracted from demodicid mite body.Adopt PLGA nanometer adjuvant to prepare dirt demodicid mite recombinant allergen Der p1 vaccine simultaneously, be used for the treatment of pulmonary carcinoma.There is not been reported both at home and abroad in the present invention.
Summary of the invention
An object of the present invention is the above-mentioned deficiency overcoming prior art, and provides a kind of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly.
The technical scheme realizing foregoing invention object of the present invention is: a kind of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly, and it adopts following steps to be prepared from:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking is in the LB containing 50 μ g/ml kanamycin, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and spends the night, inoculum concentration by 1 ~ 4% by genetic engineering bacterium colony inoculation in LB/kam culture fluid, 200 ~ 300rpm shakes bacterium when being 0.5 ~ 0.7 to OD600, adding IPTG makes its final concentration be 0.1 ~ 0.4mM, induce after 4 ~ 6 hours, 2000 ~ 4000g is at 2 ~ 6 DEG C of centrifugal 5 ~ 15min, remove supernatant, precipitation 1xPBS washs, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4 ~ 0.6mg/ml, DTT is to final concentration 5 ~ 15mM, PMSF is to final concentration 0.8 ~ 1.2mmol/L, Triton X-100 is to final concentration 0.8 ~ 1.2%, and vibrate 20 ~ 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 ~ 15 times, ultrasonic 20 ~ 40s, to stop 20 ~ 40s, till thalline is not sticky, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, by the resuspended precipitation of 100ml inclusion body washing liquid 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, remove supernatant, use successively again inclusion body washing liquid 2 and inclusion body washing liquid 3 resuspended, ice bath magnetic agitation, centrifugally removes supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid, the centrifugal 30min of 10000 ~ 12000g, abandons precipitation, obtains inclusion body;
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
50mg PLGA is claimed to be dissolved in dichloromethane, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min, 40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1 ~ 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3 ~ 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, wash with distilled water again, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
Preferably, the genetic engineering bacterium colony inoculation amount in described step one (1) is 2%; Shaking bacterium to final concentration is 0.2mM; PMSF is 1mmol/L to final concentration; Triton X-100 is 1% to final concentration.
Preferably, the inclusion body washing liquid 1 in described step one (3) is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA; Inclusion body washing liquid 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100; Inclusion body washing liquid 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide; Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide.
Preferably, the PLAG in described step 2 is LA:GA=65:35, Mw=40000-75000.
Two of object of the present invention is the above-mentioned deficiencies overcoming prior art, and provides a kind of preparation method of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly.
The technical scheme realizing foregoing invention object of the present invention is: a kind of preparation method of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly, and its step is as follows:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking is in the LB containing 50 μ g/ml kanamycin, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and spends the night, inoculum concentration by 1 ~ 4% by genetic engineering bacterium colony inoculation in LB/kam culture fluid, 200 ~ 300rpm shakes bacterium when being 0.5 ~ 0.7 to OD600, adding IPTG makes its final concentration be 0.1 ~ 0.4mM, induce after 4 ~ 6 hours, 2000 ~ 4000g is at 2 ~ 6 DEG C of centrifugal 5 ~ 15min, remove supernatant, precipitation 1xPBS washs, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4 ~ 0.6mg/ml, DTT is to final concentration 5 ~ 15mM, PMSF is to final concentration 0.8 ~ 1.2mmol/L, Triton X-100 is to final concentration 0.8 ~ 1.2%, and vibrate 20 ~ 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 ~ 15 times, ultrasonic 20 ~ 40s, to stop 20 ~ 40s, till thalline is not sticky, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, by the resuspended precipitation of 100ml inclusion body washing liquid 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, remove supernatant, use successively again inclusion body washing liquid 2 and inclusion body washing liquid 3 resuspended, ice bath magnetic agitation, centrifugally removes supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid, the centrifugal 30min of 10000 ~ 12000g, abandons precipitation, obtains inclusion body;
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
50mg PLGA is claimed to be dissolved in dichloromethane, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min, 40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1 ~ 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3 ~ 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, wash with distilled water again, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
Preferably, the genetic engineering bacterium colony inoculation amount in described step one (1) is 2%; Shaking bacterium to final concentration is 0.2mM; PMSF is 1mmol/L to final concentration; Triton X-100 is 1% to final concentration.
Preferably, the inclusion body washing liquid 1 in described step one (3) is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA; Inclusion body washing liquid 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100; Inclusion body washing liquid 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide; Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide.
Preferably, the PLAG in described step 2 is LA:GA=65:35, Mw=40000-75000.
Three of object of the present invention is the above-mentioned deficiencies overcoming prior art, and provides a kind of purposes of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly.
The technical scheme realizing foregoing invention object of the present invention is: a kind of Der p1 nano vaccine for the treatment of pulmonary carcinoma newly, is used for the treatment of pulmonary carcinoma.
The present invention compared with prior art tool has the following advantages: the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly of the present invention, there is good anti-cancer function, have significant therapeutic effect to pulmonary carcinoma, both to the effect of human non-toxic's evil, body immunity can also be increased, and preparation technology is simple.
Below by way of specific embodiment, the invention will be further described.
Detailed description of the invention
Embodiment 1:
A Der p1 nano vaccine for treatment pulmonary carcinoma newly, it adopts following steps to be prepared from:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking contains in the LB of 50 μ g/ml kanamycin in 20ml, 35 DEG C, 160rpm shakes bacterium and spends the night, inoculum concentration by 1% by genetic engineering bacterium colony inoculation in 1L LB/kam culture fluid, 200rpm shakes bacterium when being 0.5 to OD600, adding IPTG makes its final concentration be 0.1mM, induce after 4 hours, 2000g is at 2 DEG C of centrifugal 5min, remove supernatant, precipitation 1xPBS washs one time, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4mg/ml, DTT is to final concentration 5mM, PMSF is to final concentration 0.8mmol/L, Triton X-100 is to final concentration 0.8%, and vibrate 20 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 times, ultrasonic 20s, to stop 20s, till thalline is not sticky, 10000g3 DEG C of centrifugal 10min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, with 100ml inclusion body washing liquid 1(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA) resuspended precipitation, and magnetic agitation 20min, 10000g3 DEG C of centrifugal 10min, remove supernatant, use inclusion body washing liquid 2(50mmol/L PB PH8.0 more successively, 100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100) and inclusion body washing liquid 3(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide) resuspended, ice bath magnetic agitation, centrifugally remove supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid (50mmol/L PB PH8.0, 300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide) in, the centrifugal 30min of 10000g, abandon precipitation, obtain inclusion body,
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) be dissolved in the dichloromethane of 1ml, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, 3 times are washed again with distilled water, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
Embodiment 2
A Der p1 nano vaccine for treatment pulmonary carcinoma newly, it adopts following steps to be prepared from:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking contains in the LB of 50 μ g/ml kanamycin in 20ml, 37 DEG C, 240rpm shakes bacterium and spends the night, inoculum concentration by 4% by genetic engineering bacterium colony inoculation in 1L LB/kam culture fluid, 300rpm shakes bacterium when being 0.7 to OD600, adding IPTG makes its final concentration be 0.4mM, induce after 6 hours, 4000g is at 6 DEG C of centrifugal 15min, remove supernatant, precipitation 1xPBS washs one time, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.6mg/ml, DTT is to final concentration 15mM, PMSF is to final concentration 1.2mmol/L, Triton X-100 is to final concentration 1.2%, and vibrate 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 15 times, ultrasonic 40s, to stop 40s, till thalline is not sticky, 12000g5 DEG C of centrifugal 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, with 100ml inclusion body washing liquid 1(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA) resuspended precipitation, and magnetic agitation 40min, 12000g5 DEG C of centrifugal 20min, remove supernatant, use inclusion body washing liquid 2(50mmol/L PB PH8.0 more successively, 100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100) and inclusion body washing liquid 3(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide) resuspended, ice bath magnetic agitation, centrifugally remove supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid (50mmol/L PB PH8.0, 300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide) in, the centrifugal 30min of 12000g, abandon precipitation, obtain inclusion body,
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Second step, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) be dissolved in the dichloromethane of 1ml, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, 3 times are washed again with distilled water, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
Embodiment 3:
A preparation method for the Der p1 nano vaccine for the treatment of pulmonary carcinoma newly, its step is as follows:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking contains in the LB of 50 μ g/ml kanamycin in 20ml, 35 DEG C, 160rpm shakes bacterium and spends the night, inoculum concentration by 1% by genetic engineering bacterium colony inoculation in 1L LB/kam culture fluid, 200rpm shakes bacterium when being 0.5 to OD600, adding IPTG makes its final concentration be 0.1mM, induce after 4 hours, 2000g is at 2 DEG C of centrifugal 5min, remove supernatant, precipitation 1xPBS washs one time, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4mg/ml, DTT is to final concentration 5mM, PMSF is to final concentration 0.8mmol/L, Triton X-100 is to final concentration 0.8%, and vibrate 20 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 times, ultrasonic 20s, to stop 20s, till thalline is not sticky, 10000g3 DEG C of centrifugal 10min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, with 100ml inclusion body washing liquid 1(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA) resuspended precipitation, and magnetic agitation 20min, 10000g3 DEG C of centrifugal 10min, remove supernatant, use inclusion body washing liquid 2(50mmol/L PB PH8.0 more successively, 100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100) and inclusion body washing liquid 3(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide) resuspended, ice bath magnetic agitation, centrifugally remove supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid (50mmol/L PB PH8.0, 300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide) in, the centrifugal 30min of 10000g, abandon precipitation, obtain inclusion body,
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) be dissolved in the dichloromethane of 1ml, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, 3 times are washed again with distilled water, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
Embodiment 4
A preparation method for the Der p1 nano vaccine for the treatment of pulmonary carcinoma newly, its step is as follows:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking contains in the LB of 50 μ g/ml kanamycin in 20ml, 37 DEG C, 240rpm shakes bacterium and spends the night, inoculum concentration by 4% by genetic engineering bacterium colony inoculation in 1L LB/kam culture fluid, 300rpm shakes bacterium when being 0.7 to OD600, adding IPTG makes its final concentration be 0.4mM, induce after 6 hours, 4000g is at 6 DEG C of centrifugal 15min, remove supernatant, precipitation 1xPBS washs one time, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.6mg/ml, DTT is to final concentration 15mM, PMSF is to final concentration 1.2mmol/L, Triton X-100 is to final concentration 1.2%, and vibrate 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 15 times, ultrasonic 40s, to stop 40s, till thalline is not sticky, 12000g5 DEG C of centrifugal 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, with 100ml inclusion body washing liquid 1(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA) resuspended precipitation, and magnetic agitation 40min, 12000g5 DEG C of centrifugal 20min, remove supernatant, use inclusion body washing liquid 2(50mmol/L PB PH8.0 more successively, 100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100) and inclusion body washing liquid 3(50mmol/L PB PH8.0, 100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide) resuspended, ice bath magnetic agitation, centrifugally remove supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid (50mmol/L PB PH8.0, 300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide) in, the centrifugal 30min of 12000g, abandon precipitation, obtain inclusion body,
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Second step, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) be dissolved in the dichloromethane of 1ml, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, 3 times are washed again with distilled water, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
The drug loading of PLGA-Der p1 nano vaccine, entrapment efficiency determination and nanometer characterize observes:
With protein concentration in BCA protein quantification algoscopy PLGA-Der p1 nano vaccine, the PLGA-Der p1 nanometer powder taking 5mg lyophilizing contains in 2ml in the aqueous solution of 0.05mol/L NaOH and 1%SDS, 37 DEG C of constant temperature oscillation instrument vibrate 24 hours, then use BCA protein quantification kit measurement protein concentration.Drug loading and the envelop rate of nano vaccine is calculated according to PLGA-Der p1 protein concentration:
Drug loading (LC)=(total protein-floating preteins)/total nanoparticle weight * 100%
Envelop rate (EE)=(total protein-floating preteins)/total protein * 100%
Inst. of Optoelectronics, Shenzhen Univ. is sent by the PLGA-Der p1 nanometer powder of lyophilizing to carry out scanning electron microscope (SEM) observation.First conducting resinl is bonded on sample stage, more gently nanometer powder is evenly sprinkling upon on conducting resinl, then blow away the particle do not adhered to rubber pipette bulb, then metal coating (metal spraying), particle diameter and the form of electron microscopy observation nanoparticle can be gone up after metal spraying.Result is as follows:
Claim the quality of nanoparticle is 63.8mg after nano vaccine lyophilizing;
Der p1 albumen 0.524mg in 5mg nano vaccine is recorded by BCA protein quantification algoscopy;
The medicine carrying 105 μ g of vaccine drug loading (LC)=(0.524/5) * 100%=10.5%(and 1mg dry powder nano vaccine);
Envelop rate (EE)=[(0.105*63.8)/10] * 100%=67.0%;
Under scanning electron microscope, observe that Der p1 nano vaccine grain granule is satisfactory, size is homogeneous, good dispersion, nano-particle diameter is within the scope of 200nm ~ 500nm.
Utilize the control experiment of Der p1 nano vaccine immunization therapy pulmonary carcinoma of the present invention:
(1) foundation of the transplanted tumor animal model of pulmonary carcinoma
Utilize LLC lung cancer cell line to set up the transplanted tumor animal model of pulmonary carcinoma C57Bl/6 Mus: In vitro culture LLC cell line, the C57Bl/6 Mus armpit be inoculated in by cell respectively about 8 week age is subcutaneous, and the number of inoculating cell is: 3x10
6individual cell, inoculation is random is afterwards divided into matched group and treatment group by mice, sets up the Mus transplanted tumor of LLC cell line.
(2) Der p1 nano vaccine is studied on the impact of Lung Cancer xenograft
With the transplanted tumor animal model set up for object of study, at mouse subcutaneous injection cell after 10 days, Der p1 nano vaccine is brought into use to treat to treatment group, with Der p1 nano vaccine injection (100 μ g) immunity 10 times, every minor tick 1 day, matched group does not carry out vaccine injection.Measure the weight of transplanted tumor, the difference between comparative control group and treatment group, in table 1.
table 1. matched group and treatment group mice-transplanted tumor weight ratio are comparatively (g)
| Matched group | Treatment group | |
| 1 | 2.92 | 1.45 |
| 2 | 3.90 | 0.13 |
| 3 | 2.07 | 1.35 |
| 4 | 2.02 | 1.63 |
| 5 | 3.78 | 0.21 |
| x±s | 2.938±0.9 | 0.954±0.72 |
As seen from the above table, the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly of the present invention can reach 67.53% to the suppression ratio of tumor growth.
Note: tumour inhibiting rate=(matched group average tumor weight-treatment group average tumor weight)/matched group average tumor weight * 100%.
(3) analyzing Der p1 nano vaccine to the impact of pulmonary carcinoma Th1/Th2 state, confirming that Der p1 nano vaccine reaches the effect of immunization therapy pulmonary carcinoma by reversing Th1/Th2.After analyzing the treatment of Der p1 nano vaccine, detect the change with Th1/Th2 cell associated cytokines in spleen cell cultures liquid in above-mentioned laboratory animal peripheral blood.
1, blood sample: all from above-mentioned laboratory animal.
2, the detection of Th1/Th2 cell associated cytokines: adopt ELISA method to measure IFN-γ and IL-4 level.
Experimental result shows: Der p1 nano vaccine can effectively suppress the growth of lung tumor to be spread, and plays good therapeutical effect.
Claims (9)
1. a Der p1 nano vaccine for new treatment pulmonary carcinoma, is characterized in that adopting following steps to be prepared from:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking is in the LB containing 50 μ g/ml kanamycin, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and spends the night, inoculum concentration by 1 ~ 4% by genetic engineering bacterium colony inoculation in LB/kam culture fluid, 200 ~ 300rpm shakes bacterium when being 0.5 ~ 0.7 to OD600, adding IPTG makes its final concentration be 0.1 ~ 0.4mM, induce after 4 ~ 6 hours, 2000 ~ 4000g is at 2 ~ 6 DEG C of centrifugal 5 ~ 15min, remove supernatant, precipitation 1xPBS washs, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4 ~ 0.6mg/ml, DTT is to final concentration 5 ~ 15mM, PMSF is to final concentration 0.8 ~ 1.2mmol/L, Triton X-100 is to final concentration 0.8 ~ 1.2%, and vibrate 20 ~ 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 ~ 15 times, ultrasonic 20 ~ 40s, to stop 20 ~ 40s, till thalline is not sticky, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, by the resuspended precipitation of 100ml inclusion body washing liquid 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, remove supernatant, use successively again inclusion body washing liquid 2 and inclusion body washing liquid 3 resuspended, ice bath magnetic agitation, centrifugally removes supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid, the centrifugal 30min of 10000 ~ 12000g, abandons precipitation, obtains inclusion body;
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
50mg PLGA is claimed to be dissolved in dichloromethane, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min, 40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1 ~ 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3 ~ 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, wash with distilled water again, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
2. the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 1, is characterized in that: the genetic engineering bacterium colony inoculation amount in described step one (1) is 2%; Shaking bacterium to final concentration is 0.2mM; PMSF is 1mmol/L to final concentration; Triton X-100 is 1% to final concentration.
3. the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 1, is characterized in that: the inclusion body washing liquid 1 in described step one (3) is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA; Inclusion body washing liquid 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100; Inclusion body washing liquid 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide; Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide.
4. the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 1, is characterized in that: the PLAG in described step 2 is LA:GA=65:35, Mw=40000-75000.
5. a preparation method for the Der p1 nano vaccine of new treatment pulmonary carcinoma, its feature is as follows:
Step one, prepare restructuring dermatophagoides pteronyssinus allergen Der p1 with technique for gene engineering:
(1) the single genetic engineering bacterium colony inoculation containing recombinant expression plasmid restructuring pET-24a (+) Der p1 of picking is in the LB containing 50 μ g/ml kanamycin, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and spends the night, inoculum concentration by 1 ~ 4% by genetic engineering bacterium colony inoculation in LB/kam culture fluid, 200 ~ 300rpm shakes bacterium when being 0.5 ~ 0.7 to OD600, adding IPTG makes its final concentration be 0.1 ~ 0.4mM, induce after 4 ~ 6 hours, 2000 ~ 4000g is at 2 ~ 6 DEG C of centrifugal 5 ~ 15min, remove supernatant, precipitation 1xPBS washs, antibacterial is resuspended in 40ml 1xPBS/5mM EDTA, add bacteriolyze mould to final concentration 0.4 ~ 0.6mg/ml, DTT is to final concentration 5 ~ 15mM, PMSF is to final concentration 0.8 ~ 1.2mmol/L, Triton X-100 is to final concentration 0.8 ~ 1.2%, and vibrate 20 ~ 40 minutes in room temperature, make genetic engineering bacterium culture lysate,-80 DEG C of preservations,
(2) lysate to be placed under ice bath 40W ultrasonic 5 ~ 15 times, ultrasonic 20 ~ 40s, to stop 20 ~ 40s, till thalline is not sticky, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, removes supernatant, obtains genetic engineering bacterium inclusion body;
(3) under ice bath, by the resuspended precipitation of 100ml inclusion body washing liquid 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifugal 10 ~ 20min, remove supernatant, use successively again inclusion body washing liquid 2 and inclusion body washing liquid 3 resuspended, ice bath magnetic agitation, centrifugally removes supernatant, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid, the centrifugal 30min of 10000 ~ 12000g, abandons precipitation, obtains inclusion body;
Because recombinant protein N-end band has 6-His label, therefore pass through Ni
2+metal chelate chromatography carries out restructuring Der p1 affinitive layer purification, and must recombinate dermatophagoides pteronyssinus allergen Der p1;
Step 2, preparation PLGA-dirt demodicid mite strains former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccine:
50mg PLGA is claimed to be dissolved in dichloromethane, adding concentration that 100 μ L have prepared is again that the restructuring dermatophagoides pteronyssinus of 100mg/ml strains former Der p1, DL 1min40w, ultrasonic emulsification 1 min forms colostric fluid, add the polyvinyl alcohol (PVA) that 2ml concentration is 2% again, ultrasonic emulsification 1 ~ 2 min again, form double emulsion, then double emulsion is transferred in the distilled water of 50ml, stirred at ambient temperature 3 ~ 5h, organic solvent is fully volatilized, the centrifugal 10min of 10000rpm/ min, collected by centrifugation microgranule, wash with distilled water again, last vacuum lyophilization becomes the PLGA-dust mite allergen Der p1 nano vaccine of fine-particle powder shape, 4 DEG C of preservations.
6. the preparation method of the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 5, is characterized in that: the genetic engineering bacterium colony inoculation amount in described step one (1) is 2%; Shaking bacterium to final concentration is 0.2mM; PMSF is 1mmol/L to final concentration; Triton X-100 is 1% to final concentration.
7. the preparation method of the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 5, is characterized in that: the inclusion body washing liquid 1 in described step one (3) is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA; Inclusion body washing liquid 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100; Inclusion body washing liquid 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L carbamide; Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L carbamide.
8. the preparation method of the Der p1 nano vaccine of a kind for the treatment of pulmonary carcinoma newly as claimed in claim 5, is characterized in that: the described PLAG in described step 2 is LA:GA=65:35, Mw=40000-75000.
9. a purposes for the Der p1 nano vaccine of new treatment pulmonary carcinoma, is characterized in that: be used for the treatment of pulmonary carcinoma.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110354098A (en) * | 2019-07-23 | 2019-10-22 | 中国农业大学 | The preparation and application of recombinant protein c FP-10 nano particle |
| CN110368488A (en) * | 2019-07-23 | 2019-10-25 | 中国农业大学 | Recombinate the preparation and application of argF protein nano particle |
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|---|---|---|---|---|
| CN1706494A (en) * | 2005-04-07 | 2005-12-14 | 深圳大学 | Method of preparing dust mite allergen vaccine with nano particle carrier |
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| CN1706494A (en) * | 2005-04-07 | 2005-12-14 | 深圳大学 | Method of preparing dust mite allergen vaccine with nano particle carrier |
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| A TODO BOM ET AL: "Allergic respiratory disease in the elderly", 《RESPIRATORY MEDICINE》 * |
| JOON LIN CHEW ET AL.: "Chitosan nanoparticles containing plasmid DNA encoding house dust mite allergen, Der p1 for oral vaccination in mice", 《VACCINE》 * |
| KRISZTINA SZALAI ET AL.: "Establishing an allergic eczema model employing recombinant house dust mite allergens Der p 1 and Der p 2 in BALB/c mice", 《EXPERIMENTAL DERMATOLOGY》 * |
| NACKSUNG KIM ET AL: "Protective effect of the DNA vaccine encoding the major house dust mite allergens on allergic inflammation in the murine model of house dust mite allergy", 《CLINICAL AND MOLECULAR ALLERGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110354098A (en) * | 2019-07-23 | 2019-10-22 | 中国农业大学 | The preparation and application of recombinant protein c FP-10 nano particle |
| CN110368488A (en) * | 2019-07-23 | 2019-10-25 | 中国农业大学 | Recombinate the preparation and application of argF protein nano particle |
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