CN1184674A

CN1184674A - Pseudomonas active protein and method for producing same

Info

Publication number: CN1184674A
Application number: CN95119866A
Authority: CN
Inventors: 余国华
Original assignee: Individual
Current assignee: Individual
Priority date: 1995-12-07
Filing date: 1995-12-07
Publication date: 1998-06-17
Anticipated expiration: 2015-12-07
Also published as: CN1086588C

Abstract

The pseudomonas active protein is composed of corpuscle protein, endotoxic protein, mucus protein and etc. By adopting 11 pseudomonas i. e. pyocyaneus pseudomonas 1. 2. 3. 4. 5. 6 and 11 serotype standard strains and malt-philic pseudomonas, malodorous pseudomonas, alkaligenic pseudomonas etc. lyophilized standard strains to separate and extract corpuscle protein, toxoid of exotoxin and endotoxic protein, which possess of good immune active components (antigen components) to synthesize the pseudo monas active protein. Said product is used to prevent and cure the chronic pulmonaryinfection of pulmonary heart disease and infections due to pseudo monas.

Description

Pseudomonas active protein and production method thereof

Pseudomonas is a kind of low toxicity pathogenic bacterium that see that extensively are present in nature, often be called conditioned pathogen (Opportunisticor conditioned pathogen), susceptible person and its body's immunity to this bacterium lowly have substantial connection, as immunodeficiency, malignant tumor combined chemotherapy patient, use immunosuppressant, tissue transplantation, major operation, urinary system infection, large-area burns etc. and all can merge the infection of this bacterium, often cause septicemia and death.The pseudomonas sickness rate increased to some extent in recent years, because this bacterium has natural bacterial drug resistance, the considerable part bacterial strain produces beta-lactamase, to antibiotics resistance, therefore to the infected or can infectible pseudomonas susceptible person, application specific immunoprophylaxis and treatment replace chemotherapy or are subjected to very big attention now as the ancillary drug of antibiotic therapy, and making specific active immunity with bacterial extract especially is effective best method with prevention infection.

Pseudomonas is that the chronic cardiopulmonary disease pulmonary infection state of an illness increases the weight of and one of cause of death, and by its nosocomial infection that causes and ascendant trend is year by year arranged, mortality rate is up to 50%.In the age of antibiotic high speed development, the M ﹠ M of pseudomonas still is growing on and on, and illustrates that clinical treatment to primary disease still has certain difficulty.Respiratory system Pseudomonas aeruginosa sickness rate accounts for 30～50%, and other pseudomonass account for 40～70%, and antibiosis is have the adaptability of height and drug resistance is the main cause that primary disease infects the long and refractory of duration.Relevant pseudomonas medicine research, the medicine that can be used for treating at present comprises the nearly kind more than 300 of the antibiotic of biological extraction, chemosynthesis, structure of modification, these medicines all have certain curative effect, but pharmacological action can be subjected to multiple factor affecting, and the antibacterial action performance of medicine is restricted.

The object of the present invention is to provide the specificity control medicine of a kind of anti-pseudomonas, and can thoroughly solve pseudomonas active protein and production method thereof prevention of pulmonary heart disease pseudomonas and treatment.

Constituent of the present invention and each components in proportions (weight ratio) are: particulate protein 40%, endotoxin protein 20%, mucus protein 20%, toxoid 20%.Its production method is: take 20 hours liquid cultures of 11 kinds of different pseudomonas third generations respectively, 12000 rev/mins of 4 ℃ of continuous centrifugals are got precipitation, use 0.1M TrisHcl, pH7.2,1mM EDTA select three times, reuse aforesaid liquid dissolution precipitation thing adds freshly prepared lysozyme (antalzyme activity＞25000) in ice bath, shake up gently, under zero degree, act on 20～30 minutes, this bacteria suspension is put-30 ℃, multigelation three times, make the complete cracking of thalline, with the outstanding mill of colloid mill secondary, each 10 minutes, 4 ℃ centrifugal, 3000 rev/mins, 30 minutes, abandon precipitation (thalline residue), get supernatant, add 5% aluminium potassium sulfate, put 4 ℃ and spend the night, take out next day, 4 ℃ centrifugal, 5000 rev/mins, 30 minutes, get precipitation, be suspended from the middle room temperature dialysis of pH7.2 phosphate buffer (PBS) 24 hours, get dialysate---particulate protein.

With the supernatant of above-mentioned third generation culture after centrifugal, with the aseptic filtration of Cai Sishi funnel, get filtrate and add 5% potassium thiocyanate, the limit edged stirs, and room temperature left standstill 2 hours, transferred to pH3.0 with 1N hydrochloric acid, putting 4 ℃ spends the night, take out next day, and 4 ℃ centrifugal, 12000 rev/mins, get precipitation, the 95% dissolve with ethanol precipitate that adds 0.03N-NaOH, 35 ℃ leave standstill 16 hours (fatty acid in the endotoxin is removed, and make the endotoxin detoxification), transposition is in PBS pH6.8,4 ℃ of low temperature dialysis 24 hours (sample and dialysis solution ratio are 1: 50) change water three times continuously, and it is standby to put 4 ℃ of refrigerators---endotoxin protein (OEP).

Get 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals, abandon precipitation and get supernatant,, collect filtrate with the acetate fiber membrane filtration of 0.3 μ m, put freezer and be refrigerated to 1～2 ℃, add potassium thiocyanate (first wiring solution-forming), the limit edged stirs, transfer to pH3.0 with 1N hydrochloric acid, put 4 ℃ of iceboxs and spend the night 12000 rev/mins, 4 ℃ of continuous centrifugals, abandon supernatant, get precipitation, add pre-cooling 95% ethanol, abundant dissolution precipitation thing, 4 ℃ centrifugal 3000 rev/mins, abandon precipitation and get supernatant, transfer to pH5.5 with 0.1N NaOH, 4 ℃ centrifugal, 3000 rev/mins, 30 minutes, abandon supernatant; Get precipitation, put PBS pH6.8,4 ℃ of dialysis 24 hours (sample and PBS ratio are 1: 100)---mucus proteins.

Get 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals, abandon precipitation and get supernatant, add the aseptic filtration of filter paper plate, collect filtrate with the Cai Shi funnel, add 5% potassium thiocyanate, the limit edged stirs, and transfers to pH3.0 with 1N hydrochloric acid, putting 4 ℃ of iceboxs spends the night, take out, 12000 rev/mins, 4 ℃ of continuous centrifugals, 95% ethanol that adds pre-cooling, fully the dissolution precipitation thing transfers to pH7.8 with 0.1N NaOH, and 4 ℃ centrifugal, 5000 rev/mins 30 minutes, abandon supernatant, get precipitation, place 0.1M TrisHCl buffer (pH8.0) dialysis that contains 4% formalin and 0.3M L-lysine, in 37 ℃ of dialysis detoxification in 4 days, again in pH8.0, in the 0.1M TrisHCl buffer, 24 hours (changing liquid three times) of 4 ℃ of dialysis, to remove formalin, get dialysate---ectotoxic toxoid.

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

Get 20 liters of 20 hours liquid cultures of 11 kinds of different pseudomonas third generations respectively, 12000 rev/mins of 4 ℃ of continuous centrifugals are got precipitation, use 0.1M TrisHcl, pH7.2,1mM EDTA select three times, reuse aforesaid liquid 1000ml dissolution precipitation thing, in ice bath, add freshly prepared lysozyme 800～1000mg (antalzyme activity＞25000), shake up gently, under zero degree, act on 20～30 minutes, this bacteria suspension is put-30 ℃, multigelation three times makes the complete cracking of thalline, with the outstanding mill of colloid mill secondary, each 10 minutes, 4 ℃ are centrifugal, and 3000 rev/mins, 30 minutes, abandon precipitation (thalline residue), get supernatant, add 5% aluminium potassium sulfate 100ml, put 4 ℃ and spend the night, take out next day, 4 ℃ are centrifugal, and 5000 rev/mins, 30 minutes, get precipitation, being suspended from the middle room temperature dialysis of pH7.2 phosphate buffer (PBS) 24 hours, getting dialysate---particulate protein gets particulate protein 1200mg.

With the supernatant of above-mentioned third generation culture after centrifugal, with the aseptic filtration of Cai Sishi funnel, get filtrate and add 5% potassium thiocyanate 2000ml, the limit edged stirs, and room temperature left standstill 2 hours, transfer to pH3.0 with 1N hydrochloric acid, put 4 ℃ and spend the night, take out next day, and 4 ℃ centrifugal, 12000 rev/mins, get precipitation, add the 95% ethanol 600ml dissolution precipitation thing of 0.03N-NaOH, 35 ℃ leave standstill and (fatty acid in the endotoxin were removed in 16 hours, and make the endotoxin detoxification), transposition is in PBS pH6.8, and 4 ℃ of low temperature dialysis 24 hours (sample and dialysis solution ratio are 1: 50) change water three times continuously, it is standby to put 4 ℃ of refrigerators---and endotoxin protein (OEP) gets endotoxin protein 760mg.

Get 20 liters of 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals, abandon precipitation and get supernatant,, collect filtrate with the acetate fiber membrane filtration of 0.3um, put freezer and be refrigerated to 1～2 ℃, add potassium thiocyanate (first wiring solution-forming), the limit edged stirs, transfer to pH3.0 with 1N hydrochloric acid, put 4 ℃ of iceboxs and spend the night 12000 rev/mins, 4 ℃ of continuous centrifugals, abandon supernatant, get precipitation, add pre-cooling 95% ethanol 1000ml, abundant dissolution precipitation thing, 4 ℃ centrifugal 3000 rev/mins, abandon precipitation and get supernatant, transfer to pH5.5 with 0.1N NaOH, 4 ℃ centrifugal, 3000 rev/mins, 30 minutes, abandon supernatant, get precipitation, put PBS pH6.8,4 ℃ of dialysis 24 hours (sample and PBS ratio are 1: 100)---mucus proteins get mucus protein 1030mg.

Get 20 liters of 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals are abandoned precipitation and are got supernatant, add the aseptic filtration of filter paper plate with the Cai Shi funnel, collect filtrate, add 5% potassium thiocyanate 1850ml, the limit edged stirs, transfer to pH3.0 with 1N hydrochloric acid, put 4 ℃ of iceboxs and spend the night, take out 12000 rev/mins, 4 ℃ of continuous centrifugals, the 95% ethanol 1000ml that adds pre-cooling, fully the dissolution precipitation thing transfers to pH7.8 with 0.1N NaOH, 4 ℃ centrifugal, 5000 rev/mins 30 minutes, abandon supernatant, get precipitation, place 0.1MTrisHCl buffer (pH8.0) dialysis that contains 4% formalin and 0.3M L-lysine, in 37 ℃ of dialysis detoxification in 4 days, again in pH8.0, in the 0.1M TrisHCl buffer, 24 hours (changing liquid three times) of 4 ℃ of dialysis, to remove formalin, get dialysate---ectotoxic toxoid gets ectotoxic toxoid 812mg.

The pseudomonas active protein portfolio ratio that this method makes: particulate protein 0.4gm, endotoxin protein 0.2gm, mucus protein 0.2gm, toxoid 0.2gm.The merging back is diluted to 1mg/ml with PBS 1000ml (pH6.8) or 2mg/ml supplies subcutaneous or intramuscular injection.

The invention is not restricted to an above-mentioned embodiment.

Use this product and prevent and treat pulmonary heart disease pulmonary and various pseudomonas infection, the Active immunity effect is very desirable, demonstrates efficient and specificity, and the activated protein good stability is taken repeatedly and had no drug resistance, and is safe in utilization, has no side effect.

Claims

1. pseudomonas active protein, the constituent and each components in proportions (weight ratio) that it is characterized by it are: particulate protein 40%, endotoxin protein 20%, mucus protein 20%, toxoid 20%.

2. the production method of a pseudomonas active protein is characterized by:

A. take 20 hours liquids of 11 kinds of different pseudomonas third generations respectively, 12000 rev/mins of 4 ℃ of continuous centrifugals are got precipitation, use 0.1M TrisHcl, pH7.2,1mM EDTA select three times, reuse aforesaid liquid dissolution precipitation thing adds freshly prepared lysozyme (antalzyme activity＞25000) in ice bath, shake up gently, under zero degree, act on 20～30 minutes, this bacteria suspension is put-30 ℃, multigelation three times, make the complete cracking of thalline, with the outstanding mill of colloid mill secondary, each 10 minutes, 4 ℃ centrifugal, 3000 rev/mins, 30 minutes, abandon precipitation (thalline residue), get supernatant, add 5% aluminium potassium sulfate, put 4 ℃ and spend the night, take out next day, 4 ℃ centrifugal, 5000 rev/mins, 30 minutes, get precipitation, be suspended from the middle room temperature dialysis of pH7.2 phosphate buffer (PBS) 24 hours, get dialysate---particulate protein;

B. use the supernatant of above-mentioned third generation culture after centrifugal, with the aseptic filtration of Cai Sishi funnel, get filtrate and add 5% potassium thiocyanate, the limit edged stirs, and room temperature left standstill 2 hours, transferred to pH3.0 with 1N hydrochloric acid, putting 4 ℃ spends the night, take out next day, and 4 ℃ centrifugal, 12000 rev/mins, get precipitation, the 95% dissolve with ethanol precipitate that adds 0.03N-NaOH, 35 ℃ leave standstill 16 hours (fatty acid in the endotoxin is removed, and make the endotoxin detoxification), transposition is in PBS pH6.8,4 ℃ of low temperature dialysis 24 hours (sample and dialysis solution ratio are 1: 50) change water three times continuously, and it is standby to put 4 ℃ of refrigerators---endotoxin protein (OEP);

C. get 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals, abandon precipitation and get supernatant,, collect filtrate with the acetate fiber membrane filtration of 0.3um, put freezer and be refrigerated to 1～2 ℃, add potassium thiocyanate (first wiring solution-forming), the limit edged stirs, transfer to pH3.0 with 1N hydrochloric acid, put 4 ℃ of iceboxs and spend the night, 12000 rev/mins, 4 ℃ of continuous centrifugals, abandon supernatant, get precipitation, add pre-cooling 95% ethanol, fully the dissolution precipitation thing, 4 ℃ centrifugal 3000 rev/mins, abandon precipitation and get supernatant, transfer to pH5.5 with 0.1N NaOH, 4 ℃ centrifugal, 3000 rev/mins, 30 minutes, abandon supernatant, get precipitation, put PBS pH6.8,4 ℃ of dialysis 24 hours (sample and PBS ratio are 1: 100)---mucus proteins;

D. get 24 hours liquid cultures of the pseudomonas third generation, 15000 rev/mins, 4 ℃ of continuous centrifugals, abandon precipitation and get supernatant, add the aseptic filtration of filter paper plate, collect filtrate with the Cai Shi funnel, add 5% potassium thiocyanate, the limit edged stirs, and transfers to pH3.0 with 1N hydrochloric acid, putting 4 ℃ of iceboxs spends the night, take out, 12000 rev/mins, 4 ℃ of continuous centrifugals, 95% ethanol that adds pre-cooling, fully the dissolution precipitation thing transfers to pH7.8 with 0.1N NaOH, and 4 ℃ centrifugal, 5000 rev/mins 30 minutes, abandon supernatant, get precipitation, place 0.1M TrisHCl buffer (pH8.0) dialysis that contains 4% formalin and 0.3M L-lysine, in 37 ℃ of dialysis detoxification in 4 days, again in pH8.0, in the 0.1M TrisHCl buffer, 24 hours (changing liquid three times) of 4 ℃ of dialysis, to remove formalin, get dialysate---ectotoxic toxoid.