CN102784389A - Preparation method of Mycobacterium tuberculosis Ag85A oral DNA vaccine - Google Patents

Abstract

Relating to a vaccine preparation method, the invention provides a preparation method of a Mycobacterium tuberculosis Ag85A oral DNA vaccine. The method comprises: designing a pair of PCR primers according to a gene sequence of Mycobacterium tuberculosis Ag85A, taking the DNA of a human Mycobacterium tuberculosis H37Rv standard virulent strain as a template, conducting PCR amplification to obtain an Ag85A gene, subjecting a recycled PCR product to enzyme digestion by restriction endonuclease Xhol and BamHI, then connecting the product with a eukaryotic vector pCDNA3.1+ through a T4DNA ligase action, proving an obtained positive clone as the Ag85A gene by a DNA sequencing identification, cloning the Ag85A gene to the downstream of a CMV promoter in the vector pCDNA3.1+, constructing a recombinant plasmid pCDNA3.1+/Ag85A, which is then transformed into Escherichia coli and amplified, thus obtaining the Mycobacterium tuberculosis Ag85A oral DNA vaccine. The method lays a foundation for clinical application research of oral DNA vaccines.

Description

A kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine

Technical field

The present invention relates to a kind of method for preparing of vaccine, particularly relate to a kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine.

Background technology

In recent years along with movement of population increase, spread of aids and novel mycobacterium tuberculosis strain, the appearance of multiple antibiotic resistant strain; Tuberculosis is changed over the trend of successively decreasing year by year, gets damp again, and case load obviously increases; Sickness rate becomes the highest infectious disease of mortality rate with the speed increase in every year 20%.Therefore prophylactic immunization plays critical effect to control lungy and elimination.

Prophylactic immunization at present prevents and treats tuberculosis with the vaccine of bacillus calmette-guerin vaccine (BCG) as unique clinical use, and preventive effect is also unstable.BCG suffers from the Mycobacterium tuberculosis var.bovis that the milch cow of tuberculosis property mastitis is separated on one's body by a strain, lost the antigen gene that some codings have immanoprotection action through 230 continuous passage attenuations.The inoculation of BCG simultaneously can make tuberculin test positive, diagnosis lungy is produced disturb, and therefore delay treating time develops the top priority that novel vaccine becomes current tuberculosis prevention and treatment.

Oral vaccine not only can be induced the specific immunity of film system effectively; And the approach of oral administration is simple, safety; Avoided the needed strict standard of vaccinate; Do not need the health officer of height professional training can carry out a large amount of crowds' immunity simultaneously, so oral DNA vaccine is a kind of generally accepted vaccination form that is easy to.It is Ag85A that the present invention selects the purpose antigen gene for use, and relative molecular mass is 32 KD, be present in the cell wall and culturing filtrate of tubercule bacillus and BCG, but the significant stimulation cellular immune function strengthens.The present invention relates to the structure of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A; Its encoding proteins can be located to express at intestinal mucosa, spleen and lymph node etc.; But the cellular immunization of obvious stimulation general and HI produce like cytotoxic activity and specific IgG antibodies, can also make the local specific secretion type IgA that produces of intestinal mucosa; Greatly enhance immunity is replied, and can not induce the secretion of this kind secretory IgA through injecting pathway.Used naked DNA vaccine in the past merely and have very big defective, it is poor at the gastrointestinal tract internal stability that oral administration is taken in, and gastrointestinal mucosa immunne response that antigen presentation is produced a little less than, so the required antigen amount of oral immunity is big, this has just limited the application of oral vaccine.It is carrier parcel oral vaccine that the present invention selects liposome for use, prevents degraded, effectively induces body to produce immunne response, and liposome can be used as the protective agent of vaccine simultaneously, can prevent that nucleic acid from being degraded by substance in vivo, can its specificity be delivered in the target cell; Nontoxic, disimmune have biologically inert, and be biodegradable; Be easy to preparation, easy to use, can be with in the big dna segment transporte to cells; The gene transfection rate is high, 100% isolated cells can moment expression alien gene.

Summary of the invention

The object of the present invention is to provide a kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine; It with the liposome immunoprophylaxis effect that the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier possesses the stably express destination protein; Because the delivery of liposome reaches the oral requirement of nontoxic glitch-free safety; But the significant stimulation cellular immune function strengthens, for the clinical application research of oral DNA vaccine lays the foundation.

The objective of the invention is to realize through following technical scheme:

A kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine; Its said method comprises that structure, the film dispersion method of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A prepare liposome, liposome is the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier; Detailed process is: the structure of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A comprise design of primers, Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium, PCR amplification Ag85A gene, purification polymerase chain reaction product TA be cloned into carrier pUCm-T carrier, the screening of blue white macula, sub-clone go into carrier for expression of eukaryon pCDNA3.1+ identify correct after with recombinant plasmid transformed competence colibacillus coli strain DH5 α; Make up eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A; From recipient bacterium DH 5 α, extract purification pUCm-Ag85A, the alkaline denaturation method is extracted eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A; Film dispersion method prepares liposome and comprises the preparation of phosphate buffer and obtain liposome; Liposome is that the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier comprises in 5 milliliters of centrifuge tubes of 1.5 milliliters of addings of solution; The eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A that adds 1.5 milliliters simultaneously; Putting into 4 ℃ of refrigerators after 5 milliliters of centrifuge tubes are placed eddy mixer fully to mix to jolt 5 minutes left standstill 2 hours; Make into uniform DNA liposome turbid liquor; Take out 5 milliliters of centrifuge tubes, place 4 ℃ of refrigerators preservations subsequent use with plug is airtight behind the inflated with nitrogen, the suspension in this centrifuge tube is Mycobacterium tuberculosis Ag85A oral DNA vaccine.

The method for preparing of described a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine, its said Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium is 37 ℃ and cultivated for 8 weeks that extracting DNA is a template in a small amount; PCR amplification Ag85A gene; Be 94 ℃ of preparatory degeneration 15 minutes; Change 94 ℃ of degeneration 1 minute after the thermal starting over to, annealed 1.5 minutes for 60 ℃, 72 ℃ were extended 2 minutes; Carry out 30 circulations; 72 ℃ are extended cessation reaction after 10 minutes, and reaction is got 5 microlitre products in 1% agarose gel electrophoresis after finishing, and identify that correctly the back adopts glue to reclaim test kit purification polymerase chain reaction product.

The method for preparing of described a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine; Its said purification polymerase chain reaction product TA is cloned into carrier pUCm-T carrier: set up reaction system by pUCm-T carrier pcr amplification test kit: 10x buffer 1 microlitre; PUCm-T carrier 1 microlitre, polymerase chain reaction product 3 microlitres, T4 dna ligase 1 microlitre; Add distilled water to 10 microlitre, 25 ℃ connect 30 minutes; Connect product and be transformed into recipient bacterium DH 5a competent cell; The bacterium liquid of getting after 100 microlitres transform is laid on 10 milliliters of meat soup peptone agar plates that contain 600 microgram ampicillin, 800 microgram IPTG, 80 microgram X-gal; After keeping flat 30 minutes, be inverted for 37 ℃ and cultivated 12-16 hour; Blue white macula screening: treat that blueness fully manifests; The picking white colony is in 10 milliliters of meat soup protein culture mediums that contain 600 microgram ampicillin; In 37 ℃ of water-baths with 200 rev/mins of speed jolting overnight incubation; And amplification amicillin resistance clone, plasmid extraction kit is the extracting plasmid in a small amount.

The method for preparing of described a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine; Extract purification pUCm-Ag85A among its said recipient bacterium DH 5 α; Through restricted enzyme Xho I and BamH I double digestion; Reclaim small fragment as genes of interest, its sub-clone is gone into the big fragment that carrier for expression of eukaryon pCDNA3.1+ is reclaimed equally behind Xho I and BamH I double digestion; The coupled reaction system: carrier 1 microlitre, genes of interest 4 microlitres, 10x buffer 1 microlitre, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 4 ℃ are spent the night.Get the connection product in second day and be transformed into recipient bacterium DH 5a competent cell, the picking positive colony, capable double digestion of extracting plasmid and order-checking are identified; The alkaline denaturation method is extracted eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A, and the gained plasmid is stored in pH8.0 Tris hydrochloride buffer, and making its final concentration is 250 mcg/ml.

The method for preparing of described a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine, its said film dispersion method prepares liposome and comprises:

(1) preparation of phosphate buffer: take by weighing sodium hydrogen phosphate 0.37 gram and sodium dihydrogen phosphate 2.0 grams; The adding distil water dissolving also is diluted to 1000 milliliters, and regulating this solution pH value is 5.7;

(2) take by weighing injection fabaceous lecithin 0.9 gram, cholesterol 0.3 gram; Add in the small beaker of 50ml capacity, add 2 milliliters of dehydrated alcohol, place 65～70 ℃ of water-baths; With the Glass rod stirring fabaceous lecithin and cholesterol are fully dissolved; Rotate this small beaker and make ethanol liquid film forming on wall of cup of phospholipid,, the ethanol volatilization is removed with rubber pipette bulb featheriness wind;

(3) with placing 65～70 ℃ of water-bath preheatings in 30 milliliters of addings of the 1st described phosphate buffer small beaker in the step 2 and with small beaker; After 20 minutes; 30 milliliters of the phosphate buffers of preheating are added in the step 2 in the 2nd the prepared small beaker that contains phospholipid and cholesterol ester plasma membrane 65～70 ℃ of stirred in water bath aquations 10 minutes; Subsequently small beaker is placed on the magnetic stirring apparatus, stirred 30 minutes under the room temperature, if liquor capacity reduces, can mend and add water to 30 milliliters, fully mixing can obtain liposome.

Technical scheme of the present invention is further specified:

One, the structure of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A:

1. design of primers

According to Ag85A gene order in the gene library, the polymerase chain reaction the primer: forward primer 5 '-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3 ' contains restricted enzyme BamHI restriction enzyme site; Downstream primer 5 '-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3 ' contains restricted enzyme Xho I restriction enzyme site, and after open reading frame termination codon, Ag85A contains signal peptide sequence, guarantees that the clone gene opening code-reading frame is correct;

2. Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium cultivated for 8 weeks for 37 ℃, and extracting DNA is a template in a small amount;

3. PCR amplification Ag85A gene;

94 ℃ of preparatory degeneration 15 minutes; Change 94 ℃ of degeneration 1 minute after the thermal starting over to, annealed 1.5 minutes for 60 ℃, 72 ℃ were extended 2 minutes; Carry out 30 circulations; 72 ℃ are extended cessation reaction after 10 minutes, and reaction is got 5 microlitre products in 1% agarose gel electrophoresis after finishing, and identify that correctly the back adopts glue to reclaim test kit purification polymerase chain reaction product;

4. purification polymerase chain reaction product TA is cloned into carrier pUCm-T carrier:

Set up reaction system by pUCm-T carrier pcr amplification test kit: 10x buffer 1 microlitre, pUCm-T carrier 1 microlitre, polymerase chain reaction product 3 microlitres, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 25 ℃ connect 30 minutes; Connect product and be transformed into recipient bacterium DH 5a competent cell; The bacterium liquid of getting after 100 microlitres transform is laid on 10 milliliters of meat soup peptone agar plates that contain 600 microgram ampicillin, 800 microgram IPTG, 80 microgram X-gal; After keeping flat 30 minutes, be inverted for 37 ℃ and cultivated 12-16 hour;

5. blue white macula screening:

Treat that blueness fully manifests; The picking white colony is in 10 milliliters of meat soup protein culture mediums that contain 600 microgram ampicillin; In 37 ℃ of water-baths with 200 rev/mins of speed jolting overnight incubation, and amplification amicillin resistance clone, plasmid extraction kit is the extracting plasmid in a small amount;

Sub-clone go into carrier for expression of eukaryon pCDNA3.1+ identify correct after with recombinant plasmid transformed competence colibacillus coli strain DH5 α, make up eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A;

7. from above-mentioned recipient bacterium DH 5 α, extract purification pUCm-Ag85A; Through restricted enzyme Xho I and BamH I double digestion; Reclaim small fragment as genes of interest, its sub-clone is gone into the big fragment that carrier for expression of eukaryon pCDNA3.1+ is reclaimed equally behind Xho I and BamH I double digestion; The coupled reaction system: carrier 1 microlitre, genes of interest 4 microlitres, 10x buffer 1 microlitre, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 4 ℃ are spent the night.Get the connection product in second day and be transformed into recipient bacterium DH 5a competent cell, the picking positive colony, capable double digestion of extracting plasmid and order-checking are identified;

8. the alkaline denaturation method is extracted eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A, and the gained plasmid is stored in pH8.0 Tris hydrochloride buffer, and making its final concentration is 250 mcg/ml;

The eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A that adopts the method to obtain confirms successfully to have made up the eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A that carries the Ag85A gene through PCR and double digestion, latter's success transformed competence colibacillus bacillus coli DH 5 alpha; With T7 promoter primer and BGH universal sequence recombiant plasmid is carried out positive and negative two-way order-checking respectively, show that Ag85A DNA has been cloned into the downstream of CMV promoter among the carrier for expression of eukaryon pCDNA3.1+, and inserting the back, to read frame correct; The amino acid identity of mycobacterium tuberculosis Ag85A nucleotide coding is 100% in Ag85A that sequencing analysis is cloned and gene library;

Two. film dispersion method prepares liposome:

1. the preparation of phosphate buffer: take by weighing sodium hydrogen phosphate 0.37 gram and sodium dihydrogen phosphate 2.0 grams; The adding distil water dissolving also is diluted to 1000 milliliters, and regulating this solution pH value is 5.7;

2. take by weighing injection fabaceous lecithin 0.9 gram, cholesterol 0.3 gram; Add in the small beaker of 50ml capacity, add 2 milliliters of dehydrated alcohol, place 65～70 ℃ of water-baths; With the Glass rod stirring fabaceous lecithin and cholesterol are fully dissolved; Rotate this small beaker and make ethanol liquid film forming on wall of cup of phospholipid,, the ethanol volatilization is removed with rubber pipette bulb featheriness wind;

3. the 1st described phosphate buffer in the step 2 added in the small beaker and with small beaker for 30 milliliters and place 65～70 ℃ of water-bath preheatings; After 20 minutes; 30 milliliters of the phosphate buffers of preheating are added in the step 2 in the 2nd the prepared small beaker that contains phospholipid and cholesterol ester plasma membrane 65～70 ℃ of stirred in water bath aquations 10 minutes; Subsequently small beaker is placed on the magnetic stirring apparatus, stirred 30 minutes under the room temperature, if liquor capacity reduces, can mend and add water to 30 milliliters, fully mixing can obtain liposome;

Three. liposome is the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier:

The 3rd described solution in the step 2 is added in 5 milliliters of centrifuge tubes for 1.5 milliliters; Add the 8th described eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A in 1.5 milliliters the step 1 simultaneously; Putting into 4 ℃ of refrigerators after 5 milliliters of centrifuge tubes are placed eddy mixer fully to mix to jolt 5 minutes left standstill 2 hours; Make into uniform DNA liposome turbid liquor; Take out 5 milliliters of centrifuge tubes, place 4 ℃ of refrigerators preservations subsequent use with plug is airtight behind the inflated with nitrogen, the suspension in this centrifuge tube is Mycobacterium tuberculosis Ag85A oral DNA vaccine.

The present invention has designed one couple of PCR primers voluntarily according to the gene order of mycobacterium tuberculosis Ag85A, is template with the DNA of Bacillus tuberculosis H37Rv standard virulent strain, goes out its Ag85A genes of interest through pcr amplification; With the PCR product that reclaims with the restriction endonuclease Xhol and the BamHI pair of enzyme enzyme action after; Through the effect of T4 dna ligase, pCDNA3.1+ is connected with carrier for expression of eukaryon, and the positive colony that screening obtains is identified through dna sequencing and turned out to be the Ag85A gene; And be cloned into the downstream of the CMV promoter among the carrier pCDNA3.1+; Construction recombination plasmid pCDNA3.1+/Ag85A. is with its transformed into escherichia coli and make it a large amount of amplifications, and adopts the alkaline denaturation method to extract eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A, and the applied film dispersion method prepares liposome and wraps up; Make tubercule bacillus Ag85A oral DNA vaccine; The immunoprophylaxis effect that possesses the stably express destination protein, and because the delivery of liposome reaches the oral requirement of nontoxic glitch-free safety, but the significant stimulation cellular immune function strengthens; Reach prevention final purpose lungy, for the clinical application research of oral DNA vaccine lays the foundation.

Advantage of the present invention and effect are:

The present invention provides a kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine; The preparation of this method be the immunoprophylaxis effect that the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier possesses the stably express destination protein with the liposome; And because the delivery of liposome reaches the oral requirement of nontoxic glitch-free safety; But the significant stimulation cellular immune function strengthens, and reaches prevention final purpose lungy, for the clinical application research of oral DNA vaccine lays the foundation.

The specific embodiment

Below in conjunction with embodiment the present invention is elaborated.

Embodiment:

A kind of method for preparing of Mycobacterium tuberculosis Ag85A oral DNA vaccine comprises three successive steps, and concrete grammar is following:

One, the structure of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A

1. design of primers

According to Ag85A gene order in the gene library, the polymerase chain reaction the primer: forward primer 5 '-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3 ' contains the BamHI restriction enzyme site; Downstream primer 5 '-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3 ' contains Xho I restriction enzyme site, and after open reading frame termination codon.Ag85A contains signal peptide sequence.Guarantee that the clone gene opening code-reading frame is correct.

2. Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium cultivated for 8 weeks for 37 ℃, and extracting DNA is a template in a small amount.

3. PCR amplification Ag85A gene

94 ℃ of preparatory degeneration 15 minutes change 94 ℃ of degeneration 1 minute over to after the thermal starting, 60 ℃ of annealing 1.5 minutes, and 72 ℃ were extended 2 minutes, carried out 30 circulations, and 72 ℃ are extended cessation reaction after 10 minutes.Reaction is got 5 microlitre products in 1% agarose gel electrophoresis after finishing, and identifies that correctly the back adopts glue to reclaim test kit purification polymerase chain reaction product.

4. purification polymerase chain reaction product TA is cloned into carrier pUCm-T carrier

Set up reaction system by pUCm-T carrier pcr amplification test kit: 10x buffer 1 microlitre, pUCm-T carrier 1 microlitre, PCR product 3 microlitres, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 25 ℃ connect 30 minutes.Connect product and be transformed into recipient bacterium DH 5a competent cell; The bacterium liquid of getting after 100 microlitres transform is laid on 10 milliliters of meat soup peptone agar plates that contain 600 microgram ampicillin, 800 microgram IPTG, 80 microgram X-gal; After keeping flat 30 minutes, be inverted for 37 ℃ and cultivated 14 hours.

5. blue white macula screening

Treat that blueness fully manifests; The picking white colony is in 10 milliliters of meat soup protein culture mediums that contain 600 microgram ampicillin; In 37 ℃ of water-baths with 200 rev/mins of speed jolting overnight incubation, and amplification amicillin resistance clone, plasmid extraction kit is the extracting plasmid in a small amount.

Sub-clone go into carrier for expression of eukaryon pCDNA3.1+ identify correct after with the recombinant plasmid transformed competence colibacillus bacillus coli DH 5 alpha, make up eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A.

7. from above-mentioned recipient bacterium DH 5 α, extract purification pUCm-Ag85A; Through Xho I and BamH I double digestion; Reclaim small fragment as genes of interest, its sub-clone is gone into the big fragment that carrier for expression of eukaryon pCDNA3.1+ is reclaimed equally behind Xho I and BamH I double digestion.The coupled reaction system: carrier 1 microlitre, genes of interest 4 microlitres, 10x buffer 1 microlitre, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 4 ℃ are spent the night.Get the connection product in second day and be transformed into recipient bacterium DH 5a competent cell, the picking positive colony, capable double digestion of extracting plasmid and order-checking are identified.

Confirm successfully to have made up the eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A that carries the Ag85A gene through PCR and double digestion, latter's success transformed competence colibacillus bacillus coli DH 5 alpha.With T7 promoter primer and BGH universal sequence recombiant plasmid is carried out positive and negative two-way order-checking respectively, show that Ag85A DNA has been cloned into the downstream of CMV promoter among the carrier for expression of eukaryon pCDNA3.1+, and inserting the back, to read frame correct.The amino acid identity of cloning mycobacterium tuberculosis Ag85A nucleotide coding in 1141bp Ag85A and the gene library through sequencing analysis is 100%.

Two. film dispersion method prepares liposome

1. the preparation of phosphate buffer: take by weighing sodium hydrogen phosphate 0.37 gram and sodium dihydrogen phosphate 2.0 grams, the adding distil water dissolving also is diluted to 1000 milliliters, and regulating this solution pH value is 5.7.

2. take by weighing injection fabaceous lecithin 0.9 gram, cholesterol 0.3 gram; Add in the small beaker of 50ml capacity, add 2 milliliters of dehydrated alcohol, place 70 ℃ of water-baths; With the Glass rod stirring fabaceous lecithin and cholesterol are fully dissolved; Rotate this small beaker and make ethanol liquid film forming on wall of cup of phospholipid,, the ethanol volatilization is removed with rubber pipette bulb featheriness wind.

3. the 1st described phosphate buffer in the step 2 added in the small beaker and with small beaker for 30 milliliters and place 70 ℃ of water-bath preheatings; After 20 minutes; 30 milliliters of the phosphate buffers of preheating are added in the step 2 in the 2nd the prepared small beaker that contains phospholipid and cholesterol ester plasma membrane 70 ℃ of stirred in water bath aquations 10 minutes.Subsequently small beaker is placed on the magnetic stirring apparatus, stirred 30 minutes under the room temperature, if liquor capacity reduces, can mend and add water to 30 milliliters, fully mixing can obtain liposome.

Three. liposome is the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier

The 3rd described solution in the step 2 is added in 5 milliliters of centrifuge tubes for 1.5 milliliters; Add the 8th described eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A in 1.5 milliliters the step 1 simultaneously; Putting into 4 ℃ of refrigerators after 5 milliliters of centrifuge tubes are placed eddy mixer fully to mix to jolt 5 minutes left standstill 2 hours; Make into uniform DNA liposome turbid liquor; Take out 5 milliliters of centrifuge tubes, place 4 ℃ of refrigerators preservations subsequent use with plug is airtight behind the inflated with nitrogen, the suspension in this centrifuge tube is Mycobacterium tuberculosis Ag85A oral DNA vaccine.

Claims (5)

1. the method for preparing of a Mycobacterium tuberculosis Ag85A oral DNA vaccine; It is characterized in that; Said method comprises that structure, the film dispersion method of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A prepare liposome, liposome is the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier; Detailed process is: the structure of eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A comprise design of primers, Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium, PCR amplification Ag85A gene, purification polymerase chain reaction product TA be cloned into carrier pUCm-T carrier, the screening of blue white macula, sub-clone go into carrier for expression of eukaryon pCDNA3.1+ identify correct after with recombinant plasmid transformed competence colibacillus coli strain DH5 α; Make up eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A; From recipient bacterium DH 5 α, extract purification pUCm-Ag85A, the alkaline denaturation method is extracted eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A; Film dispersion method prepares liposome and comprises the preparation of phosphate buffer and obtain liposome; Liposome is that the preparation of the Mycobacterium tuberculosis Ag85A oral DNA vaccine of carrier comprises in 5 milliliters of centrifuge tubes of 1.5 milliliters of addings of solution; The eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A that adds 1.5 milliliters simultaneously; Putting into 4 ℃ of refrigerators after 5 milliliters of centrifuge tubes are placed eddy mixer fully to mix to jolt 5 minutes left standstill 2 hours; Make into uniform DNA liposome turbid liquor; Take out 5 milliliters of centrifuge tubes, place 4 ℃ of refrigerators preservations subsequent use with plug is airtight behind the inflated with nitrogen, the suspension in this centrifuge tube is Mycobacterium tuberculosis Ag85A oral DNA vaccine.

2. the method for preparing of a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine according to claim 1 is characterized in that, said Mycobacterium tuberculosis H37Rv strain inoculation modified Russell medium is 37 ℃ and cultivated for 8 weeks that extracting DNA is a template in a small amount; PCR amplification Ag85A gene; Be 94 ℃ of preparatory degeneration 15 minutes; Change 94 ℃ of degeneration 1 minute after the thermal starting over to, annealed 1.5 minutes for 60 ℃, 72 ℃ were extended 2 minutes; Carry out 30 circulations; 72 ℃ are extended cessation reaction after 10 minutes, and reaction is got 5 microlitre products in 1% agarose gel electrophoresis after finishing, and identify that correctly the back adopts glue to reclaim test kit purification polymerase chain reaction product.

3. the method for preparing of a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine according to claim 1; It is characterized in that said purification polymerase chain reaction product TA is cloned into carrier pUCm-T carrier: set up reaction system: 10x buffer 1 microlitre, pUCm-T carrier 1 microlitre by pUCm-T carrier pcr amplification test kit; Polymerase chain reaction product 3 microlitres; T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 25 ℃ connect 30 minutes; Connect product and be transformed into recipient bacterium DH 5a competent cell; The bacterium liquid of getting after 100 microlitres transform is laid on 10 milliliters of meat soup peptone agar plates that contain 600 microgram ampicillin, 800 microgram IPTG, 80 microgram X-gal; After keeping flat 30 minutes, be inverted for 37 ℃ and cultivated 12-16 hour; Blue white macula screening: treat that blueness fully manifests; The picking white colony is in 10 milliliters of meat soup protein culture mediums that contain 600 microgram ampicillin; In 37 ℃ of water-baths with 200 rev/mins of speed jolting overnight incubation; And amplification amicillin resistance clone, plasmid extraction kit is the extracting plasmid in a small amount.

4. the method for preparing of a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine according to claim 1; It is characterized in that; Extract purification pUCm-Ag85A among said recipient bacterium DH 5 α; Through restricted enzyme Xho I and BamH I double digestion, reclaim small fragment as genes of interest, its sub-clone is gone into the big fragment that carrier for expression of eukaryon pCDNA3.1+ is reclaimed equally behind Xho I and BamH I double digestion; The coupled reaction system: carrier 1 microlitre, genes of interest 4 microlitres, 10x buffer 1 microlitre, T4 dna ligase 1 microlitre adds distilled water to 10 microlitre, and 4 ℃ are spent the night; Get the connection product in second day and be transformed into recipient bacterium DH 5a competent cell, the picking positive colony, capable double digestion of extracting plasmid and order-checking are identified; The alkaline denaturation method is extracted eukaryotic expression recombinant plasmid pCDNA3.1+/Ag85A, and the gained plasmid is stored in pH8.0 Tris hydrochloride buffer, and making its final concentration is 250 mcg/ml.

5. the method for preparing of a kind of Mycobacterium tuberculosis Ag85A oral DNA vaccine according to claim 1 is characterized in that, said film dispersion method prepares liposome and comprises:

(1) preparation of phosphate buffer: take by weighing sodium hydrogen phosphate 0.37 gram and sodium dihydrogen phosphate 2.0 grams; The adding distil water dissolving also is diluted to 1000 milliliters, and regulating this solution pH value is 5.7;

(2) take by weighing injection fabaceous lecithin 0.9 gram, cholesterol 0.3 gram; Add in the small beaker of 50ml capacity, add 2 milliliters of dehydrated alcohol, place 65～70 ℃ of water-baths; With the Glass rod stirring fabaceous lecithin and cholesterol are fully dissolved; Rotate this small beaker and make ethanol liquid film forming on wall of cup of phospholipid,, the ethanol volatilization is removed with rubber pipette bulb featheriness wind;

(3) with placing 65～70 ℃ of water-bath preheatings in 30 milliliters of addings of the 1st described phosphate buffer small beaker in the step 2 and with small beaker; After 20 minutes; 30 milliliters of the phosphate buffers of preheating are added in the step 2 in the 2nd the prepared small beaker that contains phospholipid and cholesterol ester plasma membrane 65～70 ℃ of stirred in water bath aquations 10 minutes; Subsequently small beaker is placed on the magnetic stirring apparatus, stirred 30 minutes under the room temperature, if liquor capacity reduces, can mend and add water to 30 milliliters, fully mixing can obtain liposome.