CN104524565B - A kind of new Der p1 nano vaccines for the treatment of lung cancer and its production and use - Google Patents

A kind of new Der p1 nano vaccines for the treatment of lung cancer and its production and use Download PDF

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CN104524565B
CN104524565B CN201410812804.1A CN201410812804A CN104524565B CN 104524565 B CN104524565 B CN 104524565B CN 201410812804 A CN201410812804 A CN 201410812804A CN 104524565 B CN104524565 B CN 104524565B
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左建宏
刘志刚
肖小军
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University of South China
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Abstract

A kind of new Der p1 nano vaccines for the treatment of lung cancer and its production and use, its method is：First clone and express in Escherichia coli using technique for gene engineering and prepare Der p1 albumen, then the biodegradable PGLA nano-particles of application prepare PLGA dust mites as lapping and strain former Der p1 ﹙ PLGA Der p1 ﹚ nano vaccines, product as of the present invention.A kind of Der p1 nano vaccines of disclosed new treatment lung cancer, have good anti-cancer function, have significant therapeutic effect to lung cancer, both effect harmless to the human body, moreover it is possible to increase body immunity, and preparation technology is simple.

Description

A kind of new Der p1 nano vaccines for the treatment of lung cancer and its production and use
Technical field
The present invention relates to new generation vaccine invention field, specifically a kind of Der p1 nano vaccines of new treatment lung cancer And its production and use.
Background technology
Lung cancer has become the first place of all cancer morbidities in global range, and its five-year survival rate is about 15%, about Bone tumour occurs for 30-40% patients with lung cancer, once Bone tumour occurs, its median survival time only has 7 months.The U.S. 2012 Lung cancer has more than 1,630,000 new cases and 570,000 deaths.It is occurred frequently greatly that lung cancer becomes China first also above liver cancer simultaneously Cancer, and the incidence of disease and death rate growth are the rapidest, are increased every year with 13% speed.At present, lung cancer occupies cancer morbidity Seniority among brothers and sisters（Men and women integrates）First place, wherein ranked first in male's incidence of disease, before also being come all the time in the female cancer incidence of disease Three.Expect 2025, newly-increased cases of lung cancer will be more than 1,000,000 every year for China.Rejuvenation trend is also presented in patients with lung cancer, Especially in recent years, the patients with lung cancer of less than 45 years old occurs more.Therefore, lung cancer, which has had a strong impact on, threatens our people's Life and health.
Treatment method mainly has the methods of operative treatment, chemotherapy, radiotherapy at present.Make a general survey of current therapy, operative treatment master It is adapted to early metaphase（I ~ II phases）Lung cancer and tumour are confined to the IIIb phase lung cancer of the partial selective in side thoracic cavity, and radiotherapy is same Belong to local treatment, in terms of oncobiology angle, cancer is systemic disease, the characteristic of transfer and diffusion be present；Conventional puts The therapeutic modality such as treatment and chemotherapy, also damages organism normal cell and immune system while tumour cell is killed, and reduces immune Power, toxic side effect are big.Therefore, it is important and necessary to carry out the research and development of new cancer therapy drug, it is good to anticancer effect and to normal to find Tissue poisons medicine active demand really that is small, not damaging immunity of organism.Show by our inventor's early-stage Studies, dermatophagoides pteronyssinus Treatment of the Der p1 vaccines to lung cancer has obvious action, and can strengthen body immunity, has anti-cancer function.
The present invention clones and expressed Prepare restructuring Der p1 albumen using technique for gene engineering in Escherichia coli, overcomes The difficulty largely extracted from mite body.Dust mite recombinant allergen Der p1 vaccines are prepared using PLGA nanometers adjuvant simultaneously, are used for Treat lung cancer.It is of the invention that there is not been reported both at home and abroad.
The content of the invention
An object of the present invention is to overcome the above-mentioned deficiency of prior art, and provides a kind of Der of new treatment lung cancer P1 nano vaccines.
Realizing the technical scheme of foregoing invention purpose of the present invention is：A kind of Der p1 nano vaccines of new treatment lung cancer, It is prepared using following steps：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in containing In the LB of 50 μ g/ml kanamycins, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and stayed overnight, by 1 ~ 4% inoculum concentration by genetic engineering bacterium bacterium Fall to be inoculated in LB/kam nutrient solutions, 200 ~ 300rpm shake bacterium to OD600 be 0.5 ~ 0.7 when, add IPTG make its final concentration of 0.1 ~ 0.4mM, after inducing 4 ~ 6 hours, 2000 ~ 4000g centrifuges 5 ~ 15min at 2 ~ 6 DEG C, removes supernatant, precipitates and washed with 1xPBS, Bacterium is resuspended in 40ml 1xPBS/5mM EDTA, it is mould to 0.4 ~ 0.6mg/ml of final concentration, DTT to final concentration 5 to add bacteriolyze ~ 15mM, PMSF vibrate 20 to 0.8 ~ 1.2mmol/L of final concentration, Triton X-100 to final concentration 0.8 ~ 1.2% in room temperature ~ 40 minutes, genetic engineering bacterium culture lysate, -80 DEG C of preservations are made；
（2）Lysate is placed in 40W ultrasounds 5 ~ 15 times under ice bath, 20 ~ 40s of ultrasound, stops 20 ~ 40s, until thalline does not glue and is Only, 10000 ~ 12000g3 ~ 5 DEG C centrifuge 10 ~ 20min, remove supernatant, obtain genetic engineering bacterium inclusion body；
（3）Under ice bath, it is resuspended and is precipitated with 100ml inclusion bodys washing lotion 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifuge 10 ~ 20min, remove supernatant, then be resuspended successively with inclusion body washing lotion 2 and inclusion body washing lotion 3, ice bath magnetic force Stirring, centrifugation remove supernatant, finally precipitation are dissolved in 20ml solubilization of inclusion bodies liquid, and 10000 ~ 12000g centrifugation 30min, it is heavy to abandon Form sediment, obtain inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Step 2, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA to be dissolved in dichloromethane, add the restructuring room that the concentration that 100 μ L have been prepared is 100mg/ml Dust mite strains former Der p1, and DL 1min, 40w, the min of ultrasonic emulsification 1 form colostric fluid, add 2ml concentration be 2% it is poly- Vinyl alcohol (PVA), then the min of ultrasonic emulsification 1 ~ 2, double emulsion is formed, then double emulsion is transferred in 50ml distilled water, room Temperature 3 ~ 5h of lower stirring, makes organic solvent fully volatilize, 10000rpm/ min centrifugation 10min, particulate is collected by centrifugation, then steamed with double Water washing, into the PLGA- dust mite allergen Der p1 nano vaccines of fine-particle powder shape, 4 DEG C preserve last vacuum freeze drying.
Preferably, the step 1（1）In genetic engineering bacterium colony inoculation amount be 2%；Bacterium is shaken to final concentration of 0.2mM； PMSF to final concentration of 1mmol/L；Triton X-100 to final concentration of 1%.
Preferably, the step 1（3）In inclusion body washing lotion 1 be 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA；Inclusion body washing lotion 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100；Inclusion body washing lotion 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea；Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L Urea.
Preferably, the PLAG in the step 2 is LA:GA=65:35, Mw=40000-75000.
The second object of the present invention is to overcome the above-mentioned deficiency of prior art, and provides a kind of Der of new treatment lung cancer The preparation method of p1 nano vaccines.
Realizing the technical scheme of foregoing invention purpose of the present invention is：A kind of Der p1 nano vaccines of new treatment lung cancer Preparation method, its step are as follows：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in containing In the LB of 50 μ g/ml kanamycins, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and stayed overnight, by 1 ~ 4% inoculum concentration by genetic engineering bacterium bacterium Fall to be inoculated in LB/kam nutrient solutions, 200 ~ 300rpm shake bacterium to OD600 be 0.5 ~ 0.7 when, add IPTG make its final concentration of 0.1 ~ 0.4mM, after inducing 4 ~ 6 hours, 2000 ~ 4000g centrifuges 5 ~ 15min at 2 ~ 6 DEG C, removes supernatant, precipitates and washed with 1xPBS, Bacterium is resuspended in 40ml 1xPBS/5mM EDTA, it is mould to 0.4 ~ 0.6mg/ml of final concentration, DTT to final concentration 5 to add bacteriolyze ~ 15mM, PMSF vibrate 20 to 0.8 ~ 1.2mmol/L of final concentration, Triton X-100 to final concentration 0.8 ~ 1.2% in room temperature ~ 40 minutes, genetic engineering bacterium culture lysate, -80 DEG C of preservations are made；
（2）Lysate is placed in 40W ultrasounds 5 ~ 15 times under ice bath, 20 ~ 40s of ultrasound, stops 20 ~ 40s, until thalline does not glue and is Only, 10000 ~ 12000g3 ~ 5 DEG C centrifuge 10 ~ 20min, remove supernatant, obtain genetic engineering bacterium inclusion body；
（3）Under ice bath, it is resuspended and is precipitated with 100ml inclusion bodys washing lotion 1, and magnetic agitation 20 ~ 40min, 10000 ~ 12000g3 ~ 5 DEG C centrifuge 10 ~ 20min, remove supernatant, then be resuspended successively with inclusion body washing lotion 2 and inclusion body washing lotion 3, ice bath magnetic force Stirring, centrifugation remove supernatant, finally precipitation are dissolved in 20ml solubilization of inclusion bodies liquid, and 10000 ~ 12000g centrifugation 30min, it is heavy to abandon Form sediment, obtain inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Step 2, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA to be dissolved in dichloromethane, add the restructuring room that the concentration that 100 μ L have been prepared is 100mg/ml Dust mite strains former Der p1, and DL 1min, 40w, the min of ultrasonic emulsification 1 form colostric fluid, add 2ml concentration be 2% it is poly- Vinyl alcohol (PVA), then the min of ultrasonic emulsification 1 ~ 2, double emulsion is formed, then double emulsion is transferred in 50ml distilled water, room Temperature 3 ~ 5h of lower stirring, makes organic solvent fully volatilize, 10000rpm/ min centrifugation 10min, particulate is collected by centrifugation, then steamed with double Water washing, into the PLGA- dust mite allergen Der p1 nano vaccines of fine-particle powder shape, 4 DEG C preserve last vacuum freeze drying.
Preferably, the step 1（1）In genetic engineering bacterium colony inoculation amount be 2%；Bacterium is shaken to final concentration of 0.2mM； PMSF to final concentration of 1mmol/L；Triton X-100 to final concentration of 1%.
Preferably, the step 1（3）In inclusion body washing lotion 1 be 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA；Inclusion body washing lotion 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100；Inclusion body washing lotion 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea；Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L Urea.
Preferably, the PLAG in the step 2 is LA:GA=65:35, Mw=40000-75000.
The third object of the present invention is to overcome the above-mentioned deficiency of prior art, and provides a kind of Der of new treatment lung cancer The purposes of p1 nano vaccines.
Realizing the technical scheme of foregoing invention purpose of the present invention is：A kind of Der p1 nano vaccines of new treatment lung cancer, For treating lung cancer.
The present invention has the following advantages that compared with prior art：A kind of Der p1 nanometers of new treatment lung cancer of the present invention Vaccine, there is good anti-cancer function, have significant therapeutic effect to lung cancer, both effect harmless to the human body, moreover it is possible to increase Body immunity, and preparation technology is simple.
Below by way of specific embodiment, the invention will be further described.
Embodiment
Embodiment 1：
A kind of Der p1 nano vaccines of new treatment lung cancer, it is prepared using following steps：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in In LB of the 20ml containing 50 μ g/ml kanamycins, 35 DEG C, 160rpm shakes bacterium and stayed overnight, by 1% inoculum concentration by genetic engineering bacterium bacterium colony Be inoculated in 1L LB/kam nutrient solutions, 200rpm shake bacterium to OD600 be 0.5 when, add IPTG make its final concentration of 0.1mM, lure After leading 4 hours, 2000g centrifuges 5min at 2 DEG C, removes supernatant, precipitates and washed one time with 1xPBS, bacterium is resuspended in into 40ml In 1xPBS/5mM EDTA, it is mould to final concentration 0.4mg/ml, DTT to final concentration 5mM, PMSF to final concentration to add bacteriolyze 0.8mmol/L, Triton X-100 vibrate 20 minutes to final concentration 0.8% in room temperature, and genetic engineering bacterium culture is made Lysate, -80 DEG C of preservations；
（2）Lysate is placed in 40W ultrasounds 5 times under ice bath, ultrasonic 20s, stops 20s, untill thalline is not viscous, 10000g3 DEG C of centrifugation 10min, removes supernatant, obtains genetic engineering bacterium inclusion body；
（3）Under ice bath, with 100ml inclusion bodys washing lotion 1（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA）It is resuspended precipitation, and magnetic agitation 20min, 10000g3 DEG C of centrifugation 10min removes supernatant, then successively with forgiving Body washing lotion 2（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100）And inclusion body Washing lotion 3（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea）It is resuspended, ice bath magnetic force Stirring, centrifugation remove supernatant, precipitation finally are dissolved in into 20ml solubilization of inclusion bodies liquid（50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L urea）In, 10000g centrifugation 30min, precipitation is abandoned, obtains inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Step 2, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) it is dissolved in 1ml dichloromethane, add 100 μ The restructuring dermatophagoides pteronyssinus that the concentration that L has been prepared is 100mg/ml strains former Der p1, DL 1min40w, the min shapes of ultrasonic emulsification 1 Into colostric fluid, the polyvinyl alcohol (PVA) that 2ml concentration is 2%, then ultrasonic emulsification 1min are added, forms double emulsion, then will be multiple Emulsion is transferred in 50ml distilled water, stirs 3h at room temperature, organic solvent is fully volatilized, 10000rpm/ min centrifugations 10min, particulate is collected by centrifugation, then is washed 3 times with distilled water, PLGA- dust mite of the last vacuum freeze drying into fine-particle powder shape Allergen Der p1 nano vaccines, 4 DEG C of preservations.
Embodiment 2
A kind of Der p1 nano vaccines of new treatment lung cancer, it is prepared using following steps：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in In LB of the 20ml containing 50 μ g/ml kanamycins, 37 DEG C, 240rpm shakes bacterium and stayed overnight, by 4% inoculum concentration by genetic engineering bacterium bacterium colony Be inoculated in 1L LB/kam nutrient solutions, 300rpm shake bacterium to OD600 be 0.7 when, add IPTG make its final concentration of 0.4mM, lure After leading 6 hours, 4000g centrifuges 15min at 6 DEG C, removes supernatant, precipitates and washed one time with 1xPBS, bacterium is resuspended in into 40ml In 1xPBS/5mM EDTA, it is mould to final concentration 0.6mg/ml, DTT to final concentration 15mM, PMSF to final concentration to add bacteriolyze 1.2mmol/L, Triton X-100 vibrate 40 minutes to final concentration 1.2% in room temperature, and genetic engineering bacterium culture is made Lysate, -80 DEG C of preservations；
（2）Lysate is placed in 40W ultrasounds 15 times under ice bath, ultrasonic 40s, stops 40s, untill thalline is not viscous, 12000g5 DEG C of centrifugation 20min, removes supernatant, obtains genetic engineering bacterium inclusion body；
（3）Under ice bath, with 100ml inclusion bodys washing lotion 1（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA）It is resuspended precipitation, and magnetic agitation 40min, 12000g5 DEG C of centrifugation 20min removes supernatant, then successively with forgiving Body washing lotion 2（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100）And inclusion body Washing lotion 3（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea）It is resuspended, ice bath magnetic force Stirring, centrifugation remove supernatant, precipitation finally are dissolved in into 20ml solubilization of inclusion bodies liquid（50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L urea）In, 12000g centrifugation 30min, precipitation is abandoned, obtains inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Second step, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) it is dissolved in 1ml dichloromethane, add 100 μ The restructuring dermatophagoides pteronyssinus that the concentration that L has been prepared is 100mg/ml strains former Der p1, DL 1min40w, the min shapes of ultrasonic emulsification 1 Into colostric fluid, the polyvinyl alcohol (PVA) that 2ml concentration is 2%, then the min of ultrasonic emulsification 2 are added, forms double emulsion, then will Double emulsion is transferred in 50ml distilled water, stirs 5h at room temperature, organic solvent is fully volatilized, 10000rpm/ min centrifugations 10min, particulate is collected by centrifugation, then is washed 3 times with distilled water, PLGA- dust mite of the last vacuum freeze drying into fine-particle powder shape Allergen Der p1 nano vaccines, 4 DEG C of preservations.
Embodiment 3：
A kind of preparation method of the Der p1 nano vaccines of new treatment lung cancer, its step are as follows：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in In LB of the 20ml containing 50 μ g/ml kanamycins, 35 DEG C, 160rpm shakes bacterium and stayed overnight, by 1% inoculum concentration by genetic engineering bacterium bacterium colony Be inoculated in 1L LB/kam nutrient solutions, 200rpm shake bacterium to OD600 be 0.5 when, add IPTG make its final concentration of 0.1mM, lure After leading 4 hours, 2000g centrifuges 5min at 2 DEG C, removes supernatant, precipitates and washed one time with 1xPBS, bacterium is resuspended in into 40ml In 1xPBS/5mM EDTA, it is mould to final concentration 0.4mg/ml, DTT to final concentration 5mM, PMSF to final concentration to add bacteriolyze 0.8mmol/L, Triton X-100 vibrate 20 minutes to final concentration 0.8% in room temperature, and genetic engineering bacterium culture is made Lysate, -80 DEG C of preservations；
（2）Lysate is placed in 40W ultrasounds 5 times under ice bath, ultrasonic 20s, stops 20s, untill thalline is not viscous, 10000g3 DEG C of centrifugation 10min, removes supernatant, obtains genetic engineering bacterium inclusion body；
（3）Under ice bath, with 100ml inclusion bodys washing lotion 1（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA）It is resuspended precipitation, and magnetic agitation 20min, 10000g3 DEG C of centrifugation 10min removes supernatant, then successively with forgiving Body washing lotion 2（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100）And inclusion body Washing lotion 3（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea）It is resuspended, ice bath magnetic force Stirring, centrifugation remove supernatant, precipitation finally are dissolved in into 20ml solubilization of inclusion bodies liquid（50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L urea）In, 10000g centrifugation 30min, precipitation is abandoned, obtains inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Step 2, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) it is dissolved in 1ml dichloromethane, add 100 μ The restructuring dermatophagoides pteronyssinus that the concentration that L has been prepared is 100mg/ml strains former Der p1, DL 1min40w, the min shapes of ultrasonic emulsification 1 Into colostric fluid, the polyvinyl alcohol (PVA) that 2ml concentration is 2%, then ultrasonic emulsification 1min are added, forms double emulsion, then will be multiple Emulsion is transferred in 50ml distilled water, stirs 3h at room temperature, organic solvent is fully volatilized, 10000rpm/ min centrifugations 10min, particulate is collected by centrifugation, then is washed 3 times with distilled water, PLGA- dust mite of the last vacuum freeze drying into fine-particle powder shape Allergen Der p1 nano vaccines, 4 DEG C of preservations.
Embodiment 4
A kind of preparation method of the Der p1 nano vaccines of new treatment lung cancer, its step are as follows：
Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
（1）Picking it is single containing recombinant expression plasmid restructuring pET-24a (+) Der p1 genetic engineering bacterium colony inoculation in In LB of the 20ml containing 50 μ g/ml kanamycins, 37 DEG C, 240rpm shakes bacterium and stayed overnight, by 4% inoculum concentration by genetic engineering bacterium bacterium colony Be inoculated in 1L LB/kam nutrient solutions, 300rpm shake bacterium to OD600 be 0.7 when, add IPTG make its final concentration of 0.4mM, lure After leading 6 hours, 4000g centrifuges 15min at 6 DEG C, removes supernatant, precipitates and washed one time with 1xPBS, bacterium is resuspended in into 40ml In 1xPBS/5mM EDTA, it is mould to final concentration 0.6mg/ml, DTT to final concentration 15mM, PMSF to final concentration to add bacteriolyze 1.2mmol/L, Triton X-100 vibrate 40 minutes to final concentration 1.2% in room temperature, and genetic engineering bacterium culture is made Lysate, -80 DEG C of preservations；
（2）Lysate is placed in 40W ultrasounds 15 times under ice bath, ultrasonic 40s, stops 40s, untill thalline is not viscous, 12000g5 DEG C of centrifugation 20min, removes supernatant, obtains genetic engineering bacterium inclusion body；
（3）Under ice bath, with 100ml inclusion bodys washing lotion 1（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA）It is resuspended precipitation, and magnetic agitation 40min, 12000g5 DEG C of centrifugation 20min removes supernatant, then successively with forgiving Body washing lotion 2（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2%Triton X-100）And inclusion body Washing lotion 3（50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/L urea）It is resuspended, ice bath magnetic force Stirring, centrifugation remove supernatant, precipitation finally are dissolved in into 20ml solubilization of inclusion bodies liquid（50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L urea）In, 12000g centrifugation 30min, precipitation is abandoned, obtains inclusion body；
Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+It is affine that metal chelate chromatography carries out restructuring Der p1 Chromatographic purifying, dermatophagoides pteronyssinus allergen Der p1 must be recombinated；
Second step, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
Claim 50mg PLGA (LA:GA=65:35, Mw=40000-75000) it is dissolved in 1ml dichloromethane, add 100 μ The restructuring dermatophagoides pteronyssinus that the concentration that L has been prepared is 100mg/ml strains former Der p1, DL 1min40w, the min shapes of ultrasonic emulsification 1 Into colostric fluid, the polyvinyl alcohol (PVA) that 2ml concentration is 2%, then the min of ultrasonic emulsification 2 are added, forms double emulsion, then will Double emulsion is transferred in 50ml distilled water, stirs 5h at room temperature, organic solvent is fully volatilized, 10000rpm/ min centrifugations 10min, particulate is collected by centrifugation, then is washed 3 times with distilled water, PLGA- dust mite of the last vacuum freeze drying into fine-particle powder shape Allergen Der p1 nano vaccines, 4 DEG C of preservations.
Drugloading rate, entrapment efficiency determination and the nanometer of PLGA-Der p1 nano vaccines characterize observation：
With protein concentration in BCA protein quantification determination method PLGA-Der p1 nano vaccines, the lyophilized PLGA- of 5mg are weighed For Der p1 nanometer powders in 2ml NaOH containing the 0.05mol/L and 1%SDS aqueous solution, 37 DEG C of constant temperature oscillation instrument vibrations 24 are small When, then with BCA protein quantification kit measurement protein concentrations.Nano vaccine is calculated according to PLGA-Der p1 protein concentrations Drugloading rate and envelop rate：
Drugloading rate（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 to be scanned Electronic Speculum lyophilized PLGA-Der p1 nanometer powders（SEM） Observation.Conduction is adhesive on sample stage first, then is gently uniformly sprinkling upon nanometer powder on conducting resinl, is then blown with rubber pipette bulb Remove nonadherent particle, then metal coating（Metal spraying）, the particle diameter and form of electron microscopy observation nanoparticle can be gone up after metal spraying.As a result such as Under：
After nano vaccine is lyophilized the quality of nanoparticle is referred to as 63.8mg；
Der p1 albumen 0.524mg in 5mg nano vaccines is measured with BCA protein quantification determination methods；
Vaccine drugloading rate（LC）=（0.524/5）*100%=10.5%（That is the μ g of load medicine 105 of 1mg dry powder nano vaccine）；
Envelop rate（EE）=[（0.105*63.8）/10]*100%=67.0%；
Observation Der p1 nano vaccine grain particles are satisfactory under ESEM, size is homogeneous, good dispersion, and nano particle is straight Footpath is in the range of 200nm ~ 500nm.
Utilize the control experiment of Der p1 nano vaccines immunization therapy lung cancer of the present invention：
（1）The foundation of the transplantable tumor animal model of lung cancer
The transplantable tumor animal model of lung cancer is established in C57Bl/6 mouse using LLC lung cancer cell lines：In vitro culture LLC cells Cell, is inoculated in that the C57Bl/6 mouse armpits of 8 week old or so are subcutaneous, and the number of inoculating cell is by system respectively：3x106Individual cell, Mouse is divided into control group and treatment group at random after inoculation, establishes the mouse transplantable tumor of LLC cell lines.
（2）Study influence of the Der p1 nano vaccines to Lung Cancer xenograft
Using the transplantable tumor animal model of foundation as research object, after mouse subcutaneous injection cell 10 days, treatment group is opened Begin to treat using Der p1 nano vaccines, injected with Der p1 nano vaccines（100μg）Immune 10 times, every minor tick 1 day is right According to group without vaccine injection.The weight of transplantable tumor is measured, compares the difference between control group and treatment group, is shown in Table 1.
The control group of table 1. and treatment group's mice-transplanted tumor weight compare（g）
Control 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, a kind of Der p1 nano vaccines of new treatment lung cancer of the present invention can to the inhibiting rate of tumour growth Up to 67.53%.
Note：Tumour inhibiting rate=（Control group average tumor weight-treatment group's average tumor weight）/ control group average tumor weight Amount * 100%.
（3）Analyze influence of the Der p1 nano vaccines to lung cancer Th1/Th2 states, it was demonstrated that Der p1 nano vaccines can pass through Th1/Th2 is reversed to have the function that immunization therapy lung cancer.After analyzing the treatment of Der p1 nano vaccines, detect outside above-mentioned experimental animal In all blood and spleen cell cultures liquid in Th1/Th2 cell associated cytokines change.
1st, blood sample：It is all from above-mentioned experimental animal.
2nd, the detection of Th1/Th2 cell associated cytokines：IFN-γ is determined using ELISA method and IL-4 is horizontal.
Test result indicates that：Der p1 nano vaccines can effectively suppress the growth diffusion of lung neoplasm, play good treatment Effect.

Claims (1)

  1. A kind of 1. application of Der p1 nano vaccines on treatment lung-cancer medicament is prepared, it is characterized in that the Der p1 nano vaccines Prepare with the following method：
    Step 1, with technique for gene engineering Prepare restructuring dermatophagoides pteronyssinus allergen Der p1：
    （1）Single pET-24a (+) the Der p1 containing recombinant expression plasmid restructuring of picking genetic engineering bacterium colony inoculation is in containing 50 μ In the LB of g/ml kanamycins, 35 ~ 37 DEG C, 160 ~ 240rpm shakes bacterium and stayed overnight, by 1 ~ 4% inoculum concentration by genetic engineering bacterium bacterium colony Be inoculated in LB/kam nutrient solutions, 200 ~ 300rpm shake bacterium to OD600 be 0.5 ~ 0.7 when, add IPTG make its final concentration of 0.1 ~ 0.4mM, after inducing 4 ~ 6 hours, 2000 ~ 4000g centrifuges 5 ~ 15min at 2 ~ 6 DEG C, removes supernatant, precipitates and washed with 1xPBS, will be thin Bacterium is resuspended in 40ml 1xPBS/5mM EDTA, addition lysozyme to 0.4 ~ 0.6mg/ml of final concentration, and DTT to final concentration 5 ~ 15mM, PMSF to 0.8 ~ 1.2mmol/L of final concentration, Triton X-100 to final concentration 0.8 ~ 1.2%, and in room temperature vibration 20 ~ 40 minutes, genetic engineering bacterium culture lysate, -80 DEG C of preservations are made；
    （2）Lysate is placed in 40W ultrasounds 5 ~ 15 times under ice bath, 20 ~ 40s of ultrasound, stops 20 ~ 40s, untill thalline is not viscous, 10000 ~ 12000g3 ~ 5 DEG C centrifuge 10 ~ 20min, remove supernatant, obtain genetic engineering bacterium inclusion body；
    （3）Under ice bath, it is resuspended and is precipitated with 100ml inclusion bodys washing lotion 1, and 20 ~ 40min of magnetic agitation, 10000 ~ 12000g3 ~ 5 DEG C 10 ~ 20min of centrifugation, removes supernatant, then be resuspended successively with inclusion body washing lotion 2 and inclusion body washing lotion 3, ice bath magnetic agitation, centrifuges Supernatant is removed, finally precipitation is dissolved in 20ml solubilization of inclusion bodies liquid, 10000 ~ 12000g centrifugation 30min, precipitation is abandoned, must forgive Body；
    Because recombinant protein N-end carries 6-His labels, therefore pass through Ni2+Metal chelate chromatography carries out recombinating Der p1 affinity chromatographys Purifying, must recombinate dermatophagoides pteronyssinus allergen Der p1；
    Step 2, prepare PLGA- dust mites and strain former Der p1 ﹙ PLGA-Der p1 ﹚ nano vaccines：
    Claim 50mg PLGA to be dissolved in dichloromethane, add the restructuring dermatophagoides pteronyssinus that the concentration that 100 μ L have been prepared is 100mg/ml Former Der p1 are strained, the min of DL 1min, 40w ultrasonic emulsification 1 forms colostric fluid, adds the polyvinyl alcohol that 2ml concentration is 2% (PVA) double emulsion, then the min of ultrasonic emulsification 1 ~ 2, is formed, then double emulsion is transferred in 50ml distilled water, stirred at room temperature 3 ~ 5h is mixed, organic solvent is fully volatilized, 10000rpm/ min centrifugation 10min, particulate is collected by centrifugation, then is washed with distilled water, Into the PLGA- dust mite allergen Der p1 nano vaccines of fine-particle powder shape, 4 DEG C preserve last vacuum freeze drying；
    The step 1（1）In genetic engineering bacterium colony inoculation amount be 2%；Bacterium is shaken to final concentration of 0.2mM；PMSF is dense to end Spend for 1mmol/L；Triton X-100 to final concentration of 1%；
    It is characterized in that：The step 1（3）In inclusion body washing lotion 1 be 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA；Inclusion body washing lotion 2 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 2% Triton X-100；Inclusion body washing lotion 3 is 50mmol/L PB PH8.0,100mmol/L Nacl, 5mmol/L EDTA, 4mol/ L urea；Solubilization of inclusion bodies liquid is 50mmol/L PB PH8.0,300mmol/L Nacl, 20mmol/L imidazoles, 6mol/L urea；
    Described PLGA in the step 2 is LA:GA=65:35, Mw=40000-75000.
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