CN104056280A - Preparation method of pasteurella multocida ptfa gene chitosan nano DNA vaccine - Google Patents
Preparation method of pasteurella multocida ptfa gene chitosan nano DNA vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method of a pasteurella multocida ptfa gene chitosan nano DNA vaccine and relates to a preparation method of a vaccine; according to a genomic sequence of pasteurella multocida, two primers for amplifying a ptfa gene, and the pasteurella multocida ptfa gene is amplified through a polymerase chain reaction (PCR); the pasteurella multocida ptfa gene and pcDNA3.1 (+) plasmid are enzymatically digested through restriction endonuclease (i) Bam (/i) HI and (i) Xho (/i) II, and a pasteurella multocida ptfa recombinant plasmid is constructed; such materials as sodium sulfate, anhydrous sodium acetate, sodium lauryl sulfate and calcium chloride are sequentially added and are uniformly mixed to obtain the pasteurella multocida ptfa gene chitosan nano DNA vaccine. The vaccine prepared by the method disclosed by the invention is free of live pathogenic bacteria, and can avoid pasteurella multocida diseases due to reasons thereof after being applied to animals; an immune protective rate reaches up to 85%; moreover, the vaccine, which is a nanoparticle containing recombinant plasmid, is easy to store and transport.
Description
Technical field
The present invention relates to a kind of DNA vaccination, specifically the preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine.
Background technology
Fowl pasteurella multocida disease, is a kind of contact septic infectious disease of the poultry such as the main infringement chicken that caused by fowl pasteurella multocida, turkey, duck, goose and wild fowl class, take generate heat, diarrhoea, dyspnea is feature, morbus acutissimus example is rapidly dead.Being one of main bacteria disease having a strong impact on China's aviculture development, is also a kind of poultry disease that there is no at present good prevention method and cause heavy economic losses.
At present China is for preventing the commercialization vaccine of fowl pasteurella multocida disease to mainly contain two kinds of attenuated live vaccines and inactivated vaccines, and wherein attenuated live vaccines is a class of most study in China's all birds infectious disease attenuated live vaccines.Although such vaccine can prevent the generation of this disease to a certain extent, also has the problems such as safety is poor, and side effect is bigger than normal, misapplication even may cause the outburst of fowl pasteurella multocida disease.And even the immune effect of fowl pasteurella multocida disease inactivated vaccine is not as attenuated live vaccines, so must develop more effective fowl pasteurella multocida disease vaccine to prevent this disease.
Summary of the invention
The present invention is directed to the problems referred to above a kind of preparation method that can be used for preventing the fowl pasteurella multocida ptfa gene chitose nano DNA vaccine of fowl pasteurella multocida disease is provided, this vaccine preparation method is comparatively simple, easy to operate, the vaccine of preparing with the method can effectively prevent the injury of fowl pasteurella multocida disease to chicken.
The present invention solves the problems of the technologies described above adopted technical scheme to be:
The preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine, its concrete preparation method comprises the following steps:
The acquisition of step 1, fowl pasteurella multocida ptfa gene
(1), according to the genome sequence of fowl pasteurella multocida, be designed for two primer PL and the PU of amplification ptfa gene, its gene order is as sequence table sequence 1 and sequence 2:
PU：5′-TGCggattcATGAAAAAAGCCATTTTC-3′
PL：5′-TGACCTCGAGtTATGCGCAAAATCCT-3′；
(2), in PCR reaction tube, add successively PU and PL primer fowl pasteurella multocida genome aqueous solution 1 microlitre, ddNTPs 2.5 microlitres, Taq archaeal dna polymerase 1 microlitre, PCR reaction buffer 2 microlitres and water 11.5 microlitres that respectively 1 microlitre, concentration are 100-200pmol/ml, the laggard performing PCR reaction of mix homogeneously, after PCR reaction finishes, in product, the gene order of fowl pasteurella multocida ptfa gene is as sequence table sequence 3;
Described PCR response procedures is: first 95 ℃ of denaturations are 3 minutes, then enter cyclic program, cyclic program is 94 ℃ of degeneration 1 minute, 53 ℃ of renaturation 30 seconds, 72 ℃ are extended 30 seconds, extend eventually and within 5 minutes, finish PCR reaction again after above-mentioned cyclic program is repeated to 25 times with 72 ℃.
(3), in the above-mentioned PCR reaction tube that fowl pasteurella multocida ptfa gene is housed, add successively restriction endonuclease
bamh I and
xhoeach 3 microlitres of l I, restriction endonuclease buffer 4 microlitres and water 3 microlitres, after mix homogeneously, PCR reaction tube is positioned in the water-bath of 37 ℃ and reacts 6 hours, the water-bath of putting into again 65 ℃ after taking-up reacts with end for 5 minutes, product is fowl pasteurella multocida ptfa gene after enzyme action, be stored under-20 ℃ of conditions, standby;
The processing of step 2, pcDNA3.1 (+) plasmid
In the centrifuge tube of 0.5 milliliter, add successively each 2.5 microlitres of pcDNA3.1 (+) plasmid 5 microlitres, restriction endonuclease BamH I and Xhol I, restriction endonuclease buffer 2 microlitres and water 8 microlitres, after mix homogeneously, centrifuge tube is positioned in the water-bath of 37 ℃ and reacts 5 hours, after taking-up, put into again the water-bath 5 minutes of 65 ℃, pcDNA3.1 (+) plasmid after being processed, standby;
The preparation of step 3, fowl pasteurella multocida ptfa recombiant plasmid
(1), successively to adding fowl pasteurella multocida ptfa gene 7 microlitres after pcDNA3.1 (+) plasmid 1 microlitre after processing, enzyme action, T4 DNA ligase 1 microlitre and T4 DNA ligase buffer 1 microlitre in the centrifuge tube of 0.5 milliliter, after mix homogeneously, centrifuge tube is positioned in the water-bath of 16 ℃ and reacts 8 hours, take out standby;
(2), by in the centrifuge tube of 1.5 milliliters of the liquid immigrations in above-mentioned 0.5 milliliter of standby centrifuge tube, add again 100 microlitre escherichia coli JM83, after mix homogeneously, be positioned over successively lower 30 minutes of 0 ℃ of condition, lower 2 minutes of lower 1 minute of 42 ℃ of conditions and 0 ℃ of condition, then to the escherichia coli fluid medium that adds 0.8 milliliter in centrifuge tube, after mix homogeneously, being positioned over the speed oscillation with 180 revs/min in the constant-temperature table of 37 ℃ cultivates 1 hour, after cultivation finishes by centrifuge tube with the rotating speed of 2000 revs/min centrifugal 1 minute, outwell supernatant, add wherein again 0.1 ml sterile water, by the precipitation sucking-off of getting up that suspends, and to coat containing concentration be on the solid culture flat board of 60 mcg/ml ampicillin, be inverted in interior the cultivation 12 hours of constant incubator of 37 ℃,
(3), 1 bacterium colony on the solid culture flat board after the above-mentioned cultivation of picking, it is the escherichia coli fluid medium of 60 mcg/ml ampicillin containing concentration that bacterium colony is put into 5 milliliters, being placed in rotating speed is 180 revs/min, temperature is the interior shaken cultivation of the constant-temperature table of 37 ℃ 12 hours, standby;
(4), get 0.6 milliliter of bacterium liquid after above-mentioned cultivation, add wherein 0.3 milliliter of phenol and 0.3 milliliter of chloroform, after mix homogeneously with the rotating speed of 10000 revs/min centrifugal 5 minutes, centrifugal end is drawn in the centrifuge tube that supernatant liquid joins 2 milliliters afterwards, add wherein again the dehydrated alcohol of 1.2 milliliters, after mix homogeneously with the rotating speed of 13000 revs/min centrifugal 10 minutes, outwell after supernatant the centrifuge tube room temperature that contains sedimentary 2 milliliters is placed 30 minutes, add wherein after 30 microlitre sterilized water dissolution precipitations standby;
(5), get in the centrifuge tube that liquid 2 microlitres in the centrifuge tube of above-mentioned 2 milliliters join 0.5 milliliter, then add successively restriction endonuclease
bamh I and
xhoeach 1 microlitre of l I, restriction endonuclease buffer 1 microlitre and water 5 microlitres, after mix homogeneously, be positioned in the water-bath of 37 ℃ and react 2 hours, reaction finishes rear agarose gel electrophoresis and detects the fowl pasteurella multocida ptfa recombiant plasmid of determining in product, adopt its concentration of spectrophotometric determination, and adjusted to 0.1 mg/ml with sterilized water, standby;
The preparation of step 4, fowl pasteurella multocida ptfa gene chitose nano DNA vaccine
(1), getting concentration is that 0.5 milliliter of the fowl pasteurella multocida ptfa recombiant plasmid of 0.1 mg/ml adds in the centrifuge tube of 1.5 milliliters, add wherein 0.71 milligram of sodium sulfate, after sodium sulfate dissolves, be positioned in the water-bath of 55 ℃ and be incubated 20 minutes, standby;
(2), taking 4.1 milligrams of anhydrous sodium acetates is dissolved in containing in the test tube in 10 ml sterile waters; it is 5.6 that the sodium hydroxide of take regulates pH value; in test tube, add 0.2 gram of chitosan again; the water-bath that this test tube is placed in to 55 ℃ after chitosan dissolves is incubated 20 minutes; 0.5 milliliters of liquid of drawing subsequently in test tube joins in 1.5 milliliters of centrifuge tubes after above-mentioned steps (1) insulation; centrifuge tube is vibrated 1 minute on vortex oscillation device, then at room temperature place 1 hour, standby;
(3), in the standby centrifuge tube of above-mentioned steps (2), add 3 milligrams of sodium lauryl sulfates and 5 milligrams of calcium chloride successively again, after sodium lauryl sulfate and calcium chloride dissolving, add wherein again the Fructus Maydis oil of 0.1 milliliter, after mix homogeneously, at room temperature place 20 minutes, now the liquid in centrifuge tube is fowl pasteurella multocida ptfa gene chitose nano DNA vaccine.
The constituent of described escherichia coli fluid medium: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, surplus is water; The constituent of described solid culture flat board: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar powder 1.8%, surplus is water.
Described escherichia coli JM83 and fowl pasteurella multocida also can be bought and be obtained by market.
Beneficial effect is:
1, the present invention's gene used is the ptfa gene of fowl pasteurella multocida; this gene is one of main protection antigen gene of fowl pasteurella multocida, and the antigen of this gene code can produce stronger immunne response level and protection effect by induced animal body.The selected chitosan of the present invention is the unique positively charged alkaline polysaccharide of occurring in nature it is found that up to now, this alkaline polysaccharide can wrap negative charge fowl pasteurella multocida ptfa recombiant plasmid to form the nano-particle complex of combining closely be fowl pasteurella multocida ptfa gene chitose nano DNA vaccine, after entering animal body, can be combined with the anion mucin on zooblast surface by the unnecessary cationic charge in surface, engulfed into cell, proceed in nucleus and express, simultaneously, the degraded that can resist animal body endogenous dna enzyme after fowl pasteurella multocida ptfa recombiant plasmid is wrapped up by chitosan molecule, strengthen sustained-release and controlled release effect, improve immune effect.
2, the fowl pasteurella multocida ptfa gene chitose nano DNA vaccine that prepared by the present invention, except containing chitosan, is also added with sodium lauryl sulfate, calcium chloride and Fructus Maydis oil.Sodium lauryl sulfate wherein enters specificity and the nonspecific immune response reaction that can activate animal body after animal body, and enhancement antigen presenting cells is to the processing of vaccine and offer; Calcium chloride can increase the permeability of animal cell membrane, and the DNA molecular being conducive in vaccine enters in cell; Fructus Maydis oil can merge mutually with chitose nano DNA vaccine; be conducive to the slow release of vaccine; thereby the immune system of stimulating animal body constantly; extend the immune duration of vaccine; therefore; the present invention is conducive to strengthen the immune response of animal body to vaccine by sodium lauryl sulfate, calcium chloride, Fructus Maydis oil and chitosan use in conjunction, improves the immune protective effect of vaccine.
3, utilize fowl pasteurella multocida ptfa gene chitose nano DNA vaccine prepared by the inventive method not contain pathogen alive; after giving animal applications, can thereby not cause fowl pasteurella multocida disease due to the former of vaccine itself; immune protective rate is for reaching 85%; and the essence of this vaccine is the nano-particle containing recombiant plasmid, be therefore easy to storage and transport.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of the fowl pasteurella multocida ptfa gene that obtains of the present invention;
Fig. 2 is the agarose gel electrophoresis figure after the double digestion of fowl pasteurella multocida ptfa recombiant plasmid of the present invention;
In figure, labelling is: M, DL5000Marker, 1, fowl pasteurella multocida ptfa gene, 2, pcDNA3.1 (+) plasmid after double digestion.
The specific embodiment
The preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine, its concrete preparation method comprises the following steps:
The acquisition of step 1, fowl pasteurella multocida ptfa gene
(1), according to the genome sequence of fowl pasteurella multocida, be designed for two primer PL and the PU of amplification ptfa gene, its gene order is as sequence table sequence 1 and sequence 2:
PU：5′-TGCggattcATGAAAAAAGCCATTTTC-3′
PL：5′-TGACCTCGAGtTATGCGCAAAATCCT-3′；
(2), in PCR reaction tube, add successively PU and PL primer fowl pasteurella multocida genome aqueous solution 1 microlitre, ddNTPs 2.5 microlitres, Taq archaeal dna polymerase 1 microlitre, PCR reaction buffer 2 microlitres and water 11.5 microlitres that respectively 1 microlitre, concentration are 100-200pmol/ml, the laggard performing PCR reaction of mix homogeneously, after PCR reaction finishes, in product, the gene order of fowl pasteurella multocida ptfa gene is as sequence table sequence 3;
Described PCR response procedures is: first 95 ℃ of denaturations are 3 minutes, then enter cyclic program, cyclic program is 94 ℃ of degeneration 1 minute, 53 ℃ of renaturation 30 seconds, 72 ℃ are extended 30 seconds, extend eventually and within 5 minutes, finish PCR reaction again after above-mentioned cyclic program is repeated to 25 times with 72 ℃.
(3), in the above-mentioned PCR reaction tube that fowl pasteurella multocida ptfa gene is housed, add successively restriction endonuclease
bamh I and
xhoeach 3 microlitres of l I, restriction endonuclease buffer 4 microlitres and water 3 microlitres, after mix homogeneously, PCR reaction tube is positioned in the water-bath of 37 ℃ and reacts 6 hours, the water-bath of putting into again 65 ℃ after taking-up reacts with end for 5 minutes, product is fowl pasteurella multocida ptfa gene after enzyme action, be stored under-20 ℃ of conditions, standby;
The processing of step 2, pcDNA3.1 (+) plasmid
In the centrifuge tube of 0.5 milliliter, add successively each 2.5 microlitres of pcDNA3.1 (+) plasmid 5 microlitres, restriction endonuclease BamH I and Xhol I, restriction endonuclease buffer 2 microlitres and water 8 microlitres, after mix homogeneously, centrifuge tube is positioned in the water-bath of 37 ℃ and reacts 5 hours, after taking-up, put into again the water-bath 5 minutes of 65 ℃, pcDNA3.1 (+) plasmid after being processed, standby;
The preparation of step 3, fowl pasteurella multocida ptfa recombiant plasmid
(1), successively to adding fowl pasteurella multocida ptfa gene 7 microlitres after pcDNA3.1 (+) plasmid 1 microlitre after processing, enzyme action, T4 DNA ligase 1 microlitre and T4 DNA ligase buffer 1 microlitre in the centrifuge tube of 0.5 milliliter, after mix homogeneously, centrifuge tube is positioned in the water-bath of 16 ℃ and reacts 8 hours, take out standby;
(2), by in the centrifuge tube of 1.5 milliliters of the liquid immigrations in above-mentioned 0.5 milliliter of standby centrifuge tube, add again 100 microlitre escherichia coli JM83, after mix homogeneously, be positioned over successively lower 30 minutes of 0 ℃ of condition, lower 2 minutes of lower 1 minute of 42 ℃ of conditions and 0 ℃ of condition, then to the escherichia coli fluid medium that adds 0.8 milliliter in centrifuge tube, after mix homogeneously, being positioned over the speed oscillation with 180 revs/min in the constant-temperature table of 37 ℃ cultivates 1 hour, after cultivation finishes by centrifuge tube with the rotating speed of 2000 revs/min centrifugal 1 minute, outwell supernatant, add wherein again 0.1 ml sterile water, by the precipitation sucking-off of getting up that suspends, and to coat containing concentration be on the solid culture flat board of 60 mcg/ml ampicillin, be inverted in interior the cultivation 12 hours of constant incubator of 37 ℃,
(3), 1 bacterium colony on the solid culture flat board after the above-mentioned cultivation of picking, it is the escherichia coli fluid medium of 60 mcg/ml ampicillin containing concentration that bacterium colony is put into 5 milliliters, being placed in rotating speed is 180 revs/min, temperature is the interior shaken cultivation of the constant-temperature table of 37 ℃ 12 hours, standby;
(4), get 0.6 milliliter of bacterium liquid after above-mentioned cultivation, add wherein 0.3 milliliter of phenol and 0.3 milliliter of chloroform, after mix homogeneously with the rotating speed of 10000 revs/min centrifugal 5 minutes, centrifugal end is drawn in the centrifuge tube that supernatant liquid joins 2 milliliters afterwards, add wherein again the dehydrated alcohol of 1.2 milliliters, after mix homogeneously with the rotating speed of 13000 revs/min centrifugal 10 minutes, outwell after supernatant the centrifuge tube room temperature that contains sedimentary 2 milliliters is placed 30 minutes, add wherein after 30 microlitre sterilized water dissolution precipitations standby;
(5), get in the centrifuge tube that liquid 2 microlitres in the centrifuge tube of above-mentioned 2 milliliters join 0.5 milliliter, then add successively restriction endonuclease
bamh I and
xhoeach 1 microlitre of l I, restriction endonuclease buffer 1 microlitre and water 5 microlitres, after mix homogeneously, be positioned in the water-bath of 37 ℃ and react 2 hours, reaction finishes rear agarose gel electrophoresis and detects the fowl pasteurella multocida ptfa recombiant plasmid of determining in product, adopt its concentration of spectrophotometric determination, and adjusted to 0.1 mg/ml with sterilized water, standby;
The preparation of step 4, fowl pasteurella multocida ptfa gene chitose nano DNA vaccine
(1), getting concentration is that 0.5 milliliter of the fowl pasteurella multocida ptfa recombiant plasmid of 0.1 mg/ml adds in the centrifuge tube of 1.5 milliliters, add wherein 0.71 milligram of sodium sulfate, after sodium sulfate dissolves, be positioned in the water-bath of 55 ℃ and be incubated 20 minutes, standby;
(2), taking 4.1 milligrams of anhydrous sodium acetates is dissolved in containing in the test tube in 10 ml sterile waters; it is 5.6 that the sodium hydroxide of take regulates pH value; in test tube, add 0.2 gram of chitosan again; the water-bath that this test tube is placed in to 55 ℃ after chitosan dissolves is incubated 20 minutes; 0.5 milliliters of liquid of drawing subsequently in test tube joins in 1.5 milliliters of centrifuge tubes after above-mentioned steps (1) insulation; centrifuge tube is vibrated 1 minute on vortex oscillation device, then at room temperature place 1 hour, standby;
(3), in the standby centrifuge tube of above-mentioned steps (2), add 3 milligrams of sodium lauryl sulfates and 5 milligrams of calcium chloride successively again, after sodium lauryl sulfate and calcium chloride dissolving, add wherein again the Fructus Maydis oil of 0.1 milliliter, after mix homogeneously, at room temperature place 20 minutes, now the liquid in centrifuge tube is fowl pasteurella multocida ptfa gene chitose nano DNA vaccine.
The constituent of described escherichia coli fluid medium: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, surplus is water; The constituent of described solid culture flat board: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar powder 1.8%, surplus is water.
Described escherichia coli JM83 and fowl pasteurella multocida also can be bought and be obtained by market.Escherichia coli JM83 used in the present invention buys in magnificent biological engineering company limited; Fowl is bought in China Veterinery Drug Inspection Office by pasteurella multocida.
zoopery:
1, animal immune: get 1 age in days without 40 of any processing and the identical chickling of upgrowth situation, normal raising after 28 days, be divided at random matched group and immune group, every group each 20, every chicken of immune group all carries out immunity with fowl pasteurella multocida ptfa gene chitose nano DNA vaccine, the dosage of immunity is 0.2 milliliter of/chicken, immunization ways is intramuscular injection, immunity is three times altogether, when raising to the 28th day, the 49th day and the 70th day, carry out immunity respectively, the chicken of matched group does not carry out any immunity;
2, challenge test: when chicken 91 age in days of immune group and matched group, the equal intramuscular injection fowl of every chicken pasteurella multocida of two groups are carried out to counteracting toxic substances, the counteracting toxic substances dosage of every chicken is 1.0 * 10
10individual fowl pasteurella multocida, subsequently the chicken of matched group and immune group is continued to raise 2 weeks, record two groups of chickens at counteracting toxic substances the survival volume after 2 weeks, and calculate the protective rate of matched group and immune group, the higher explanation immunoprophylaxis of survival rate effect is better, illustrates and utilizes fowl pasteurella multocida ptfa gene chitose nano DNA vaccine prepared by method of the present invention to have immunoprophylaxis effect;
The computational methods of the protective rate of matched group are as follows:
(quantity/20 that the chicken of matched group still survives after 2 weeks at counteracting toxic substances) * 100%;
The computational methods of the protective rate of immune group are as follows:
(quantity/20 that the chicken of immune group still survives after 2 weeks at counteracting toxic substances) * 100%.
Result shows; the chicken of matched group is all dead after 2 weeks at counteracting toxic substances; the protective rate of matched group is 0%; the chicken of immune group survives 17 after 2 weeks at counteracting toxic substances; the protective rate of immune group is 85%, illustrates and utilizes the fowl pasteurella multocida ptfa gene chitose nano DNA vaccine that method of the present invention is prepared can effectively prevent fowl pasteurella multocida disease.
Claims (4)
1. the preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine, is characterized in that: concrete preparation method is:
The acquisition of step 1, fowl pasteurella multocida ptfa gene
(1), according to the genome sequence of fowl pasteurella multocida, be designed for two primer PL and the PU of amplification ptfa gene, its gene order is as sequence table sequence 1 and sequence 2:
PU：5′-TGCggattcATGAAAAAAGCCATTTTC-3′
PL：5′-TGACCTCGAGtTATGCGCAAAATCCT-3′；
(2), in PCR reaction tube, add successively PU and PL primer fowl pasteurella multocida genome aqueous solution 1 microlitre, ddNTPs 2.5 microlitres, Taq archaeal dna polymerase 1 microlitre, PCR reaction buffer 2 microlitres and water 11.5 microlitres that respectively 1 microlitre, concentration are 100-200pmol/ml, the laggard performing PCR reaction of mix homogeneously, after PCR reaction finishes, in product, the gene order of fowl pasteurella multocida ptfa gene is as sequence table sequence 3;
(3), in the above-mentioned PCR reaction tube that fowl pasteurella multocida ptfa gene is housed, add successively restriction endonuclease
bamh I and
xhoeach 3 microlitres of l I, restriction endonuclease buffer 4 microlitres and water 3 microlitres, after mix homogeneously, PCR reaction tube is positioned in the water-bath of 37 ℃ and reacts 6 hours, the water-bath of putting into again 65 ℃ after taking-up reacts with end for 5 minutes, product is fowl pasteurella multocida ptfa gene after enzyme action, be stored under-20 ℃ of conditions, standby;
The processing of step 2, pcDNA3.1 (+) plasmid
In the centrifuge tube of 0.5 milliliter, add successively each 2.5 microlitres of pcDNA3.1 (+) plasmid 5 microlitres, restriction endonuclease BamH I and Xhol I, restriction endonuclease buffer 2 microlitres and water 8 microlitres, after mix homogeneously, centrifuge tube is positioned in the water-bath of 37 ℃ and reacts 5 hours, after taking-up, put into again the water-bath 5 minutes of 65 ℃, pcDNA3.1 (+) plasmid after being processed, standby;
The preparation of step 3, fowl pasteurella multocida ptfa recombiant plasmid
(1), successively to adding fowl pasteurella multocida ptfa gene 7 microlitres after pcDNA3.1 (+) plasmid 1 microlitre after processing, enzyme action, T4 DNA ligase 1 microlitre and T4 DNA ligase buffer 1 microlitre in the centrifuge tube of 0.5 milliliter, after mix homogeneously, centrifuge tube is positioned in the water-bath of 16 ℃ and reacts 8 hours, take out standby;
(2), by in the centrifuge tube of 1.5 milliliters of the liquid immigrations in above-mentioned 0.5 milliliter of standby centrifuge tube, add again 100 microlitre escherichia coli JM83, after mix homogeneously, be positioned over successively lower 30 minutes of 0 ℃ of condition, lower 2 minutes of lower 1 minute of 42 ℃ of conditions and 0 ℃ of condition, then to the escherichia coli fluid medium that adds 0.8 milliliter in centrifuge tube, after mix homogeneously, being positioned over the speed oscillation with 180 revs/min in the constant-temperature table of 37 ℃ cultivates 1 hour, after cultivation finishes by centrifuge tube with the rotating speed of 2000 revs/min centrifugal 1 minute, outwell supernatant, add wherein again 0.1 ml sterile water, by the precipitation sucking-off of getting up that suspends, and to coat containing concentration be on the solid culture flat board of 60 mcg/ml ampicillin, be inverted in interior the cultivation 12 hours of constant incubator of 37 ℃,
(3), 1 bacterium colony on the solid culture flat board after the above-mentioned cultivation of picking, it is the escherichia coli fluid medium of 60 mcg/ml ampicillin containing concentration that bacterium colony is put into 5 milliliters, being placed in rotating speed is 180 revs/min, temperature is the interior shaken cultivation of the constant-temperature table of 37 ℃ 12 hours, standby;
(4), get 0.6 milliliter of bacterium liquid after above-mentioned cultivation, add wherein 0.3 milliliter of phenol and 0.3 milliliter of chloroform, after mix homogeneously with the rotating speed of 10000 revs/min centrifugal 5 minutes, centrifugal end is drawn in the centrifuge tube that supernatant liquid joins 2 milliliters afterwards, add wherein again the dehydrated alcohol of 1.2 milliliters, after mix homogeneously with the rotating speed of 13000 revs/min centrifugal 10 minutes, outwell after supernatant the centrifuge tube room temperature that contains sedimentary 2 milliliters is placed 30 minutes, add wherein after 30 microlitre sterilized water dissolution precipitations standby;
(5), get in the centrifuge tube that liquid 2 microlitres in the centrifuge tube of above-mentioned 2 milliliters join 0.5 milliliter, then add successively restriction endonuclease
bamh I and
xhoeach 1 microlitre of l I, restriction endonuclease buffer 1 microlitre and water 5 microlitres, after mix homogeneously, be positioned in the water-bath of 37 ℃ and react 2 hours, reaction finishes rear agarose gel electrophoresis and detects the fowl pasteurella multocida ptfa recombiant plasmid of determining in product, record its concentration, and adjusted to 0.1 mg/ml with sterilized water, standby;
The preparation of step 4, fowl pasteurella multocida ptfa gene chitose nano DNA vaccine
(1), getting concentration is that 0.5 milliliter of the fowl pasteurella multocida ptfa recombiant plasmid of 0.1 mg/ml adds in the centrifuge tube of 1.5 milliliters, add wherein 0.71 milligram of sodium sulfate, after sodium sulfate dissolves, be positioned in the water-bath of 55 ℃ and be incubated 20 minutes, standby;
(2), taking 4.1 milligrams of anhydrous sodium acetates is dissolved in containing in the test tube in 10 ml sterile waters; it is 5.6 that the sodium hydroxide of take regulates pH value; in test tube, add 0.2 gram of chitosan again; the water-bath that this test tube is placed in to 55 ℃ after chitosan dissolves is incubated 20 minutes; 0.5 milliliters of liquid of drawing subsequently in test tube joins in 1.5 milliliters of centrifuge tubes after above-mentioned steps (1) insulation; centrifuge tube is vibrated 1 minute on vortex oscillation device, then at room temperature place 1 hour, standby;
(3), in the standby centrifuge tube of above-mentioned steps (2), add 3 milligrams of sodium lauryl sulfates and 5 milligrams of calcium chloride successively again, after sodium lauryl sulfate and calcium chloride dissolving, add wherein again the Fructus Maydis oil of 0.1 milliliter, after mix homogeneously, at room temperature place 20 minutes, now the liquid in centrifuge tube is fowl pasteurella multocida ptfa gene chitose nano DNA vaccine.
2. the preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine as claimed in claim 1, it is characterized in that: described PCR response procedures is: first 95 ℃ of denaturations are 3 minutes, then enter cyclic program, cyclic program is 94 ℃ of degeneration 1 minute, 53 ℃ of renaturation 30 seconds, 72 ℃ are extended 30 seconds, extend eventually and within 5 minutes, finish PCR reaction again after above-mentioned cyclic program is repeated to 25 times with 72 ℃.
3. the preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine as claimed in claim 1, is characterized in that: the concentration that adopts spectrophotometric determination fowl pasteurella multocida ptfa recombiant plasmid.
4. the preparation method of fowl pasteurella multocida ptfa gene chitose nano DNA vaccine as claimed in claim 1, it is characterized in that: the constituent of described escherichia coli fluid medium: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, surplus is water; The constituent of described solid culture flat board: configuration by weight percentage, wherein peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar powder 1.8%, surplus is water.
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| CN115216478A (en) * | 2022-06-01 | 2022-10-21 | 华南农业大学 | Construction method and application of pasteurella multocida endotoxin attenuated inactivated vaccine strain |
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| CN115216478A (en) * | 2022-06-01 | 2022-10-21 | 华南农业大学 | Construction method and application of pasteurella multocida endotoxin attenuated inactivated vaccine strain |
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