CN1250737C - PCR process for preparing CpG DNA containing livestock and bird vaccine adjuvant - Google Patents

PCR process for preparing CpG DNA containing livestock and bird vaccine adjuvant Download PDF

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CN1250737C
CN1250737C CN 200310106227 CN200310106227A CN1250737C CN 1250737 C CN1250737 C CN 1250737C CN 200310106227 CN200310106227 CN 200310106227 CN 200310106227 A CN200310106227 A CN 200310106227A CN 1250737 C CN1250737 C CN 1250737C
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recombinant plasmid
dna
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CN1554770A (en
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朱鸿飞
李光富
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Zhu Hongfei
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WEILAI OCEAN SCIENCE AND TECHN
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Abstract

The present invention relates to a preparing method of PCR containing a CpG DNA livestock and fowl vaccine adjuvant. The method comprises the following steps: synthesizing a positive chain and a negative chain according to the sequence of a D19D282006 DNA fragment formed by connecting the sequences of 5'-GGTGCATCGATGCAGGGGGG-3', 5'-GGTGCGTCGATGCAGGGGGG-3' and 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' in series; adding a basic group capable of matching with the end of a carrier at the end of synthesized oligonucleotide; mixing the oligonucleotide fragment, an annealing buffer solution and sterile redistilled water; putting the mixture in a water bath at 90 DEG C to 100 DEG C for more than 3 minutes; cooling the mixture to room temperature to form a target fragment containing the CpG gene sequence; inserting the DNA fragment into a corresponding enzyme-incision site; converting the DNA fragment into competent colibacilli to obtain recombinant plasmid; designing a specific primer according to the recombinant plasmid DNA sequences at both ends of the target fragment; adding the specific primer by using the recombinant plasmid as a template to carry out PCR proliferation with three-temperature circulations which comprise the following specific parameters: pre-modification for 5 to 10 minutes at 92 DEG C to 98 DEG C, and 30 to 35 circulations respectively at 92 DEG C to 98 DEG C for 45 to 60 seconds, 50 DEG C to 58 DEG C for 45 to 60 seconds and 72 DEG C for 45 to 60 seconds; finally extending at 72 DEG C for 10 minutes.

Description

The PCR preparation method who contains CpG DNA veterinary vaccine adjuvant
Technical field
The present invention relates to a kind of preparation method of the CpG of containing dna fragmentation, relate in particular to the PCR preparation method who contains CpG DNA (CpG DNA is meant the thymus nucleic acid that contains cytosine(Cyt) and bird pyrimidine dinucleotides motif, and CpG DNA is that those skilled in the art know and habitual technical term) veterinary vaccine adjuvant.
Technical background
The discovery of immunologic adjuvant CpG DNA comes from the immunostimulation of DNA of bacteria and particular sequence antisense oligonucleotide (ODN).Initial scientist thinks that some palindrome has immunostimulation in the DNA of bacteria, other scientists found that the particular sequence antisense oligonucleotide also had immunostimulation afterwards, and compared the immunostimulation of multiple sequence oligonucleotides, find at last no matter DNA of bacteria still is the particular sequence antisense oligonucleotide, their immunostimulation is all owing to wherein contain specific non-methylated cytosine(Cyt) guanine dinucleotides (CpG) motif.CpG DNA causes the B cytodifferentiation, stimulates cytokine and surface moleculars such as expression MHCII, B7-1, B7-2 such as B emiocytosis IL-6, IL-10, and stops the apoptosis of B cell.CpG is activated mononuclear cell, scavenger cell and dendritic cell directly, cause that Th1 samples such as these emiocytosis IL-12, TNF-α are main cytokine, and the expression of irritation cell surface molecular MHC II, B7-1, B7-2.CpG is at the collaborative NK cell that directly activates down of IL-12, and activated NK cell killing activity strengthens, and justacrine IFN-γ, IFN-γ can further promote the activation of monocyte, scavenger cell and dendritic cell conversely again.CpG can not only activate the natural immunity effectively, and can activate acquired immunity effectively, CpG can work in coordination with stimulator antigen specific b cells and T cytodifferentiation, under the Th1 effect of cytokines, significantly strengthen acquired cellular resistance, these characteristics make CpG DNA become good T cellular immunization adjuvant.Though CpG is a kind of extremely promising new and effective immunological adjuvant, can effectively improve tiring of vaccine.But to move towards actual production on a large scale, with regard to the essential too high problem of production cost that solves, produce the method that CpG ODN mainly adopts synthetic at present, need the dNTP and the dna synthesizer of high price, and turnout is limited, incompatibility large-scale industrial production and requirement of actual application.
Technology contents
The invention provides the PCR preparation method who contains CpG DNA veterinary vaccine adjuvant that low and its products therefrom of a kind of production cost can improve vaccine valence.
The present invention adopts following technical scheme to solve its technical problem:
A kind of PCR preparation method who contains CpG DNA veterinary vaccine adjuvant that can promote that veterinary vaccines is tired,
The first step: the preparation of genetically engineered recombinant plasmid: according to being by sequence:
5’----GGTGCATCGATGCAGGGGGG---3’、
5 '----GGTGCGTCGATGCAGGGGGG---3 ' reaches
5’---TCGTCGTTTTGTCGTTTTGTCGTT---3’
The sequence of the D19D282006 purpose sheet segment DNA that is in series, synthetic its oligonucleotide normal chain and minus strand, add the base sequence that can be complementary with the linear carrier end at synthetic oligonucleotide end, then with this oligonucleotide fragment, annealing buffer, and after the aseptic redistilled water mixing, in 90 ℃～100 ℃ water-baths, incubate and bathe more than 3 minutes, naturally cool to room temperature, formation contains the purpose fragment of CpG motif, after this this dna fragmentation is inserted the corresponding restriction enzyme site of linear carrier, at last, the genetically engineered recombinant plasmid that its transformed competence colibacillus intestinal bacteria are obtained;
Second step: according to the recombinant plasmid dna sequence at purpose fragment two ends, the design special primer;
The 3rd step: with the recombinant plasmid is template, adds special primer, and carries out three warm round-robin pcr amplifications, and concrete parameter is: 92 ℃～98 ℃ pre-sex change 5～10 minutes; 92 ℃～98 ℃, 45～60 seconds, 50～58 ℃, 45～60 seconds, 72 ℃, 45～60 seconds, carry out 30～35 circulations; Extended 10 minutes at 72 ℃ at last.
Consider the effect of further raising by obtained vaccine adjuvant of the present invention, and further reduce production costs, above-mentioned recombinant plasmid comprises first recombinant plasmid, and this first recombinant plasmid contains first dna fragmentation, adopt the normal chain of synthetic first dna fragmentation of method of synthetic, the base sequence of this normal chain is
5’----GGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCGA TGCAGGGGGGA---3’，
The minus strand of synthetic first dna fragmentation, the base sequence of this minus strand is
5’----CCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGC?ACCA----3’，
Annealing back forms first dna fragmentation, and 3 ' end of first dna fragmentation has outstanding VITAMIN B4 (A), so as with the T carrier in thymus gland purine (T) complementation that goes out of 5 ' distal process, connect the back and form the first recombinant plasmid PCPG1/T.Recombinant plasmid also comprises second recombinant plasmid, and this second recombinant plasmid contains second dna fragmentation, adopts the normal chain of synthetic second dna fragmentation of method of synthetic, and the base sequence of this normal chain is
5’---- CGGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GT CG?ATGCAGGGGGG C----3’
The minus strand of synthetic second dna fragmentation, the base sequence of this minus strand is
5’----CATGGCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGCACCGCATG----3
Annealing back forms second dna fragmentation, second dna fragmentation two ends be sticky end, be complementary with two ends through first recombinant plasmid of SphI, NcoI double digestion, connect the back and form the second recombinant plasmid PCPG12/T.Recombinant plasmid also comprises the 3rd reorganization plasmid, and the 3rd reorganization plasmid contains the 3rd dna fragmentation, adopts the normal chain of synthetic the 3rd dna fragmentation of method of synthetic, and the base sequence of this normal chain is
5’---- CTAGTGGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCGATGCAGGGGGG CTGCA----3’
The minus strand of synthetic the 3rd dna fragmentation, the base sequence of this minus strand is
5’----GCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGCACCA----3’
Annealing back forms the 3rd dna fragmentation, the 3rd dna fragmentation two ends be sticky end, be complementary with two ends through second recombinant plasmid of SpeI, pstI double digestion, connect the back and form the 3rd reorganization plasmid PCPG123/T.
Experimental result shows that the PCR method is produced the CpG dna fragmentation, and the purpose band that amplifies is consistent with theoretical size, and after the optimal conditions, the content of non-purpose band seldom.For carrying out the PCR reaction more easily, the present invention does template with not purified PCR product, adopts reaction conditions and the loop parameter identical with plasmid template to carry out pcr amplification, has obtained the PCR product of output and the purity suitable with plasmid template.So just do not need to produce recombinant plasmid, further reduced production cost.The present invention has selected different cycle numbers, improves PCR output to greatest extent.The present invention is a template with the distinctive recombinant plasmid of gene engineering research preparation, and designs special universal primer, produces with the PCR method to contain the CpG dna fragmentation, and heavy industrialization, low cost production CpG dna fragmentation are become a reality.With second recombinant plasmid is template, adds general upstream and downstream special primer, can amplify the CpG dna fragmentation of the about 240bp of size; With the 3rd reorganization plasmid PCPG123/T is template, adds general upstream and downstream special primer, can amplify the CpG dna fragmentation of the about 300bp of size, and the CpG dna fragmentation that amplifies is consistent with theoretical value.The cultivation of application peripheral blood mononuclear cell, [ 3H]-deoxythymidine mixes detection method, serve as to detect index with stimulation index (SI value), compared the effect that the CpG dna fragmentation stimulates pig peripheral blood mononuclear cell propagation.The result shows: contain the CpG dna fragmentation pig peripheral blood mononuclear cell is all had good immunostimulatory activity, SI＞4.5.Cooperate vaccine to use, improve the immune effect of vaccine, and can effectively avoid now using always the adverse side effects such as inflammatory reaction that cause in the adjuvant.This adjuvant low production cost is suitable for batch production production.Compared with prior art, have the following advantages and positively effect: PCR method 1. provided by the present invention is produced the CpG dna fragmentation, to CpG ODN D19, the D28 and 2006 that pig has a good immunostimulatory activity be cascaded, the IFN-γ of the wherein propagation of the stimulation pig peripheral blood mononuclear cell that D type CpG ODN D19, D28 can be moderate, and energy secreting high levels; The propagation of the stimulation pig PBMC of K type ODN 2006 energy highest levels, the series connection of K type and D type can be learnt from other's strong points to offset one's weaknesses, and improves the usefulness of CpG adjuvant.2. PCR method provided by the present invention is produced the CpG dna fragmentation, only contains CpG DNA core sequence, does not have other irrelevant sequence, has avoided the interference of non-specific sequence to CpG dna immunization stimulating activity.3. PCR method provided by the present invention is produced the CpG dna fragmentation, and the PCR method that adopts is the external synthesis method of sophisticated DNA, only need add template, dNTP, damping fluid and polysaccharase, can on the PCR instrument, produce, technology is simple, and is with low cost, accords with the needs of large-scale industrial production.Greatly reduced the production cost of CpG ODN.4. the research of ox, chicken, horse is clearly illustrated that CpG ODN can strengthen and can regulate antigenic Th 1Type or Th 2The immune response of type.Therefore, CpG ODN has substantial evidence as novel, the highly effective immunologic adjuvant of animal vaccine.The adjuvant that is used for animal vaccine with great majority is different, and CpG ODN does not cause the local inflammation reaction of domestic animal injection site, and by 10 milligrams/kg body weight dosage injection CpG ODN, monkey does not produce tangible local patholoic change.
Description of drawings
Fig. 1 is a CpG fragment recombinant clone strategic map.
Fig. 2 is recombinant plasmid agarose gel electrophoresis figure.
Fig. 3 is first recombinant plasmid PCPG1/T minus strand sequential analysis figure and the sequence thereof.
Fig. 4 is second recombinant plasmid PCPG12/T normal chain sequential analysis figure and the sequence thereof.
Fig. 5 is the 3rd reorganization plasmid PCPG123/T normal chain sequential analysis figure and sequence thereof.
The PCR product of Fig. 6 plasmid PCPG12/T.
The PCR product of Fig. 7 plasmid PCPG123/T.
Embodiment
Embodiment 1:
A kind of PCR preparation method who contains CpG DNA veterinary vaccine adjuvant that can promote that veterinary vaccines is tired,
The first step: the preparation of gene recombination plasmid: according to being by sequence:
5’----GGTGCATCGATGCAGGGGGG---3’、
5 '----GGTGCGTCGATGCAGGGGGG---3 ' reaches
5’---TCGTCGTTTTGTCGTTTTGTCGTT---3’
The sequence of the D19D282006 purpose sheet segment DNA that is in series, the normal chain and the minus strand of synthetic its oligonucleotide, add the base sequence that can be complementary with linear carrier end at synthetic oligonucleotide end, then with this oligonucleotide fragment, annealing buffer, and after the aseptic redistilled water mixing, in 90 ℃～100 ℃ water-baths, incubate and bathe more than 3 minutes, naturally cool to room temperature, formation contains the dna fragmentation of D19D282006 sequence, after this this dna fragmentation is inserted the corresponding restriction enzyme site of linear carrier, at last, the genetically engineered recombinant chou that its transformed competence colibacillus intestinal bacteria are obtained;
Second step: according to the recombinant plasmid dna sequence at purpose fragment two ends, the design special primer;
The 3rd step: with the recombinant plasmid is template, adds special primer, and carries out three warm round-robin pcr amplifications, and concrete parameter is: 92 ℃～98 ℃ pre-sex change 5～10 minutes; 92 ℃～98 ℃, 45～60 seconds, 50～58 ℃, 45～60 seconds, 72 ℃, 45～60 seconds, carry out 30～35 circulations; Extended 10 minutes at 72 ℃ at last, in the present embodiment, recombinant plasmid comprises first recombinant plasmid, and this first recombinant plasmid contains first dna fragmentation, adopts the normal chain of synthetic first dna fragmentation of method of synthetic, and the base sequence of this normal chain is
5’----GGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCGA TGCAGGGGGGA----3’，
The minus strand of synthetic first dna fragmentation, the base sequence of this minus strand is
5’----CCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGC?ACCA----3’，
Annealing back forms first dna fragmentation, and 3 ' end of first dna fragmentation has outstanding VITAMIN B4 (A), so as with the T carrier in thymus gland purine (T) complementation that goes out of 5 ' distal process, connect the back and form the first recombinant plasmid PCPG1/T; The recombinant plasmid of present embodiment also comprises second recombinant plasmid, and this second recombinant plasmid contains second dna fragmentation, adopts the normal chain of synthetic second dna fragmentation of method of synthetic, and the base sequence of this normal chain is
5’---- CGGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCG ATGCAGGGGGG C----3’
The minus strand of synthetic second dna fragmentation, the base sequence of this minus strand is
5’----CATGGCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGCACCGCATG----3’
Annealing back forms second dna fragmentation, and the two ends of second dna fragmentation are sticky end, is complementary with two ends through first recombinant plasmid of SphI, NcoI double digestion, connects the back and forms the second recombinant plasmid PCPG12/T; The recombinant plasmid of present embodiment comprises the 3rd reorganization plasmid, and the 3rd reorganization plasmid contains the 3rd dna fragmentation, adopts the normal chain of synthetic the 3rd dna fragmentation of method of synthetic, and the base sequence of this normal chain is
5’---- CTAGTGGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCGATGCAGGGGGG CTGCA----3’
The minus strand of synthetic the 3rd dna fragmentation, the base sequence of this minus strand is
5’----GCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGCACCA----3’
Annealing back forms the 3rd dna fragmentation, and the two ends of the 3rd dna fragmentation are sticky end, is complementary with two ends through second recombinant plasmid of SpeI, pstI double digestion, connects the back and forms the 3rd reorganization plasmid PCPG123/T.Present embodiment is before pcr amplification, prior to adding the LB substratum that contains penbritin in the good test tube of ventilating, insert the intestinal bacteria that contain recombinant plasmid then, cultivate 12～16h down in 37 ℃ of violent joltings, then above-mentioned cultivation bacterium being joined 2000ml contains among the LB of penbritin, cultivate 12～16h down in 37 ℃ of violent joltings, at last in 4 ℃ with 6000 rev/mins of centrifugal 15min, abandon supernatant, open wide the centrifugal mouth of pipe and be inverted centrifuge tube, supernatant is all flow to end, prepare recombinant plasmid PCPG12/T and PCPG123/T with alkaline lysis.
Embodiment 2:
The first step: the preparation 1 genetically engineered construction of recombinant plasmid of genetically engineered recombinant plasmid
1.1 the synthetic of the first dna fragmentation normal chain and minus strand oligonucleotide
The positive chain-ordering of the synthetic first fragment oligonucleotide is
5’----GGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGT GCGTCGA TGCAGGGGGGA----3’
Oligonucleotide minus strand sequence is
5’----CCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGC?ACCA----3’，
1.2 first is segmental synthetic
After the positive minus strand of above-mentioned oligonucleotide of synthetic and purifying, annealing buffer, aseptic redistilled water mixed by following prescription, centrifugal 30 seconds with 1000 rev/mins, incubate in 90 ℃ of-100 ℃ of water-baths and bathe more than 3 minutes, naturally cool to room temperature, this annealing reaction system is as follows:
Normal chain 1 μ l
Minus strand 1 μ l
5 times of annealing buffer 16 μ l
Aseptic redistilled water 2 μ l
Cumulative volume 80 μ l
1.3DNA fragment and the ligation of T carrier
After 10 times of dilutions of above-mentioned dna fragmentation, with the T carrier under the effect of e. coli dna ligase, 4 ℃ of about 16h of ligation obtain recombinant plasmid PCPG1/T.Contrast fragment in the promega T support agent box of buying with market is connected with the T carrier does the sun ginseng, and T carrier self connects to be done the moon and join, and the ligation system is as follows:
Dna fragmentation T support C ontrol
T carrier 0.5 μ l 0.5 μ l 0.5 μ l
Dna fragmentation 1.25 μ l 0.0 μ l 0.0 μ l
Control?DNA(4ng/μl) 0.0μl 0.0μl 1.5μl
10×buffer 1.0μl 1.0μl 1.0μl
T4?DNA?ligase(3unit/μl) 0.5μl 0.5μl 0.5μl
ddH 2O 6.75μl 8.0μl 6.5μl
Total?volume 10.0μl 10.0μl 10.0μl
1.4 recombinant plasmid transformed bacillus coli DH 5 alpha
1.4.1LB the preparation of agar plate and amicillin resistance LB agar plate:
Press following formulated LB liquid nutrient medium, LB agar solution earlier, 15 pounds, 20min, autoclaving is laid in the culture dish that diameter is 90mm, is prepared into the LB agar plate; By above-mentioned same method; Preparation LB agar solution is cooled to 50 ℃, and adding final concentration is the penbritin of 75 μ g/ml, behind the mixing, is laid in the culture dish that diameter is 90mm, is prepared into to contain amicillin resistance LB agar plate, and the rearmounted 4 ℃ of placements of cooling are standby.
The LB liquid nutrient medium:
Peptone (Bacto-Tryptone) 8g
Yeast extract (Bacto-Yeast Extract) 5g
NaCl 5g
Add ddH 2The O dissolving is transferred pH to 7.4 with 10N NaOH, is settled to 1000ml, is the LB liquid nutrient medium.
LB agar solution: take by weighing the agar powder of 15g, add the LB liquid nutrient medium of 1000ml, be the LB agar solution.
1.4.2 the preparation of competence bacillus coli DH 5 alpha:
From-70 ℃ of frozen bacillus coli DH 5 alphas, pick a little with platinum loop, cultivate lining out, 37 ℃ of overnight incubation at this LB agar plate with precooling.Picking one single colony inoculation is in the LB of the Amp that contains 75 μ g/ml liquid nutrient medium (prescription is as follows), and 37 ℃ of overnight incubation are gone into the LB liquid nutrient medium with 2% ratio transferred species, 240rpm, and 37 ℃ of shaking culture are to OD 600About 0.5, put 10min in the frozen water; 4 ℃, the centrifugal 2min of 6000rpm abandons supernatant, and bacterial sediment is with the 100mmol/L CaCl of 0.5 times of thalline volume, frozen water precooling 2Suspend; Incubate in the frozen water and bathe 30min, 4 ℃, the centrifugal 2min of 6000rpm abandons supernatant, and bacterial sediment is with 0.1 times of thalline volume, frozen water 100mmol/L CaCl 2Suspend, put in the frozen water standby.
1.4.3 recon transformed into escherichia coli DH5 α:
With 10 μ l connection product and after 100 μ l competence bacterias mix, put to incubate in the frozen water and bathe 30min, again in 42 ℃ of water-bath 90sec, put immediately and place 2min in the frozen water, add the LB substratum of 0.8ml, 150rpm, 37 ℃ of shaking culture 1h.
1.4.4 bed board
Get standby amicillin resistance LB agar plate, place 1h in 37 ℃ of incubators, the bacterium liquid of getting 200 μ l recon transformed into escherichia coli DH5 α evenly is applied to amicillin resistance LB agar plate, puts in 37 ℃ of incubators and spends the night.
The present invention adopts following method to verify:
1.5 the preparation of recombinant plasmid
1.5.1 the results of bacterium:
1) the LB capacity of joining that 2ml is contained 75 μ g/ml penbritins is in the 15ml and the good test tube of ventilating, and inserts from above-mentioned amicillin resistance LB agar plate picking one single bacterium colony, in 37 ℃ of concuss overnight incubation.
2) the 1.5ml culture is poured in the centrifuge tube, with Eppendorf centrifuge in 4 ℃ with the centrifugal 30sec of 12000g, remaining culture is stored in 4 ℃.
3) draw nutrient solution, make bacterial sediment dry as far as possible.
1.5.2 alkaline lysis
1) bacterial precipitation is resuspended in the ice-cold solution I of 100 μ l concuss.
Solution I
50mmol/L
50mmol/L?Tris.Cl(pH8.0)
10mmol/L?EDTA(pH8.0)
At 10bf/in 2(6.895 * 10 4Pa) vapor sterilization 15min under the high pressure is stored in 4 ℃.
2) add the new solution II of preparing of 200 μ l.
Solution II
0.2mol/L NaOH (facing) with preceding now with the current with the 10mol/L stock solution
1％SDS
Cover the tight mouth of pipe, put upside down centrifuge tube fast 5 times, with the mixed content thing, contact with solution II with the total inner surface of guaranteeing centrifuge tube, concussion does not place centrifuge tube on ice.
3) add the ice-cold solution III of 150 μ l.
Solution III
5mol/L potassium acetate 60ml
Glacial acetic acid 11.5ml
Water 28.5ml
The solution of being prepared is 3mol/L to potassium, is 5mol/L to acetate moiety.Cover the tight mouth of pipe, will manage the 10sec that leniently vibrates after being inverted, solution III is uniformly dispersed in the heavy-gravity bacterial lysate, afterwards pipe is placed 3～5min on ice.
4) with Eppendorf centrifuge in 4 ℃ with the centrifugal 5min of 12000g, supernatant is transferred in another centrifuge tube.
5) add the phenol of equivalent: chloroform, the vibration mixing, with Eppendorf centrifuge in 4 ℃ with the centrifugal 2min of 12000g, supernatant is transferred in another centrifuge tube.
6) dehydrated alcohol of 2 times of volumes of adding, the vibration mixing is placed 2min in room temperature.
7) with Eppendorf centrifuge in 4 ℃ with the centrifugal 5min of 12000g.
8) the careful suction removed supernatant liquor, centrifuge tube is inverted on a piece of paper towel, so that all liquid flows out.The drop that will invest tube wall again eliminates.
9) with 4 ℃ of 70% washing with alcohol double-stranded DNA of 1ml precipitation, set by step 8) described method is removed supernatant, makes nucleic acid precipitate dry 10min in air.
10) TE that contains the Pancreatic RNase (20 μ g/ml) of no DNA enzyme with 40 μ l dissolves nucleic acid again, vibration, be stored in-20 ℃ standby.
1.6 the agarose gel electrophoresis of plasmid DNA
1) with adhesive plaster will clean, the horizontal glue plate of exsiccant opening seals, and forms a rubber moulding, be placed on the horizontal table.
2) 1 * TAE electrophoretic buffer of preparation q.s.Get 1 * TAE electrophoretic buffer of 100ml and put into Erlenmeyer flask, add the agarose of the 1g of accurate weighing.
1×TAE
0.04mol/LTris-acetate
0.001mol/LEDTA
3) in microwave oven suspension being heated to agarose dissolves.
4) make solution be cooled to 60 ℃.Adding final concentration is the ethidium bromide of 0.5 μ g/ml, fully mixing.
5) warm agarose is poured in the rubber moulding, the thickness of gel is at 3～5min, and the insertion comb.
6) after gel solidifies fully, carefully remove comb and adhesive plaster, gel is put into electrophoresis chamber.
7) add competent DH5 α of electrophoretic buffer sheet glass and the BL21 (DE3) that did not have the dark capacity of the about 1mm of glue face just, be used separately as the clone bacterium and express bacterium, recombinant plasmid is changed over to respectively, be used to identify and express.
8) get 5 μ l plasmid DNA samples, after adding an amount of sample loading buffer and mixing, slowly mixture is added in the sample cell with micro sample adding appliance.
9) cover electrophoresis chamber and energising, electrophoresis 40～60min under an amount of voltage.
10) outage stream is checked gel under ultraviolet lamp, and with the gel imaging instrument (see figure 2) of photographing.
1.7 the nucleotide sequencing of recombinant plasmid and analysis
The present invention is with the recombinant clone bacterial strain that filters out, plasmid extraction kit with the energy lottery industry of Shen, Shanghai extracts plasmid, as template, use general T7 promoter primer, SP6 primer respectively after the nucleic acid quantification analysis, carry out the sequencing (see figure 3) at the full-automatic sequenator of ABI377.
Second step: design of primers
The PCR primer design: in this recombinant plasmid, cytosine(Cyt) (C), guanine (G) content are very high, so according to the recombinant plasmid dna sequence at purpose fragment two ends, carry out Primer with GeneTool software and analyze, therefrom filter out and meet best primer.And can betting office synthesize by the Shen, Shanghai, the PCR test kit is that worker bio-engineering corporation product is given birth in Shanghai.
Upstream primer: 5 '---CCCAAGCTTGGGCCCGACGTCGCATGC---3 '
Downstream primer: 5 '---GCGGATCCCCCATATGGTCGACCTGCAGCC---3 '
The 3rd step: pcr amplification
A large amount of preparations of the first recombinant plasmid PCPG1/T
(1) the LB capacity of joining that 8ml is contained Amp is in the 15ml and the good test tube of ventilating, and inserts a single bacterium colony then, cultivates 12～16h down in 37 ℃ of violent joltings.
(2) above-mentioned cultivation bacterium is joined 2000ml and contain among the LB of Amp, cultivate 12～16h down in 37 ℃ of violent joltings.
(3) in 4 ℃ with 6000 rev/mins of centrifugal 15min, abandon supernatant, open wide the centrifugal mouth of pipe and also be inverted centrifuge tube, supernatant is all flow to end.
(4) with vacuum residual supernatant is exhausted.
(5) with 50ml damping fluid P1 suspension bacterial aggregate.
(6) add 50ml damping fluid P2, cover the tight mouth of pipe, gently put upside down 4～6 times, the mixing content is placed 5min in room temperature.
(7) add the ice-cold damping fluid P3 of 50ml, seal bottleneck, shake centrifuge tube, put upside down 4～6 times, the mixing content forms a white flocks, and lysate is thickness no longer.
(8) pour split product into filter, room temperature is placed 10min.
(9) open vacuum pump switch, with the supernatant suction filtration to collection container.
(10) add 50ml damping fluid FWB2, the precipitation that suspends is gently opened vacuum pump switch, with the supernatant suction filtration to collection container.
(11) add 12.5ml damping fluid ER to filterable cracking supernatant, put upside down mixing 10 times, put and place 30min on ice.
(12) with 35ml damping fluid QBT balance QIAGEN-tip 25000 posts.
(13) the cracking supernatant with step (11) adds balance columns.
(14) add 200ml damping fluid QC and wash post.
(15) DNA of adding 35ml damping fluid QN elution of bound.
(16) in elutriant, add the 24.5ml Virahol, mix, in 4 ℃ with 16,000 rev/mins of centrifugal 30min, supernatant carefully inclines.
(17) adding 7ml does not have endotoxic 70% ethanol, in 4 ℃ with 16,000 rev/mins of centrifugal 10min, supernatant carefully inclines.
(18) open wide the mouth of pipe, room temperature is placed 10～20min, dissolve again with no endotoxic damping fluid TE,
In this step, (4)-(18) operation can adopt the no intracellular toxin plasmid of QIAgen company to prepare test kit;
PCR produce contain the second recombinant plasmid PCPG1/T CpG dna fragmentation product
Present embodiment is a template with the 3rd reorganization plasmid PCPG1/T, and PCR reaction system: PCR reaction cumulative volume is 100 μ l, and moiety is as follows:
10×PCRbuffer 10μl
Upstream primer P1 2.5 μ l
Downstream primer P2 2.5 μ l
Template 8 μ l,
10mM?dNTP 2μl，
10mMMgCl 2 6μl
5unit/ μ lTaq enzyme 0.5 μ l,
Supply volume to 100 μ l with aseptic redistilled water;
Loop parameter is as follows: 94 ℃ of sex change 7min; 94 ℃, 45s; 52 ℃, 45s; 72 ℃, 1min; Circulate altogether 30 times; 72 ℃ are extended 7min
1.4PCR the purifying of product
(1) after PCR finishes, from the PCR reaction tubes with reaction solution to clean 1.5ml Eppendorf centrifuge tube, add the Solution BS mixing of 4 times of volumes.No matter sample size what, minimumly need to use 400ul Solution BS.
(2) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifuge tube lid (lid can not cover yet, if still you have a lot of samples simultaneously, worries to obscure or turn solution over, advises that you cover the centrifuge tube lid), use desk centrifuge high speed centrifugation (10000rpm) 1 minute.
(3) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600ul WashSolution, high speed centrifugation (10000rpm) 1 minute.
(4) repeating step 3 once.
(5) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes.
(6) the 3S post is put into a new 1.5ml centrifuge tube, central authorities add 30ul TE or water at 3S post film, do not cover centrifuge tube lid, and room temperature or 37 ℃ were placed 2 minutes.Cover centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
1.5DNA quantitative analysis
With the TE damping fluid dilution DNA solution of an amount of volume, measure its OD with ultraviolet spectrophotometer 260And OD 280Value is calculated dna content.
1OD 260The double-stranded DNA of=50 μ g/ml
Embodiment 3:
The first step: the preparation of genetically engineered recombinant plasmid
2.1 genetically engineered construction of recombinant plasmid
2.1.1 the synthetic of the second fragment normal chain and minus strand oligonucleotide
The positive chain-ordering of the synthetic second fragment oligonucleotide is
5’----- CGGTGC ATCGATGCAGGGGGGTC GTCGTTTT GTCGTTTT GTCGTTGGTGC GTCG ATGCAGGGGGG C----3’
Oligonucleotide minus strand sequence is
5’---CATGGCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGATGCACCGCATG----3
2.2 second is segmental synthetic
After the positive minus strand of above-mentioned oligonucleotide of synthetic and purifying, annealing buffer, aseptic redistilled water mixed by following prescription, descended centrifugal 30 seconds at 1000 rev/mins, incubate in 90 ℃ of-100 ℃ of water-baths and bathe more than 3 minutes, naturally cool to room temperature, this annealing reaction system is as follows:
Normal chain 1 μ l
Anti-chain 1 μ l
5 * annealing buffer, 16 μ l
Aseptic redistilled water 72 μ l
Cumulative volume 80 μ l
2.3 ligation
The positive recombinant plasmid PCPG1/T that obtains with embodiment 2 cuts and the second fragment ligation through enzyme as carrier
In the K damping fluid, with the positive recombinant plasmid PCPG1/T restriction enzyme SphI, the NcoI difference enzymolysis that obtain, agarose gel electrophoresis shows that enzyme cuts entirely; Use restriction enzyme SphI, NcoI double digestion again, recovery purification kit that can lottery industry with the Shen, Shanghai reclaims, with second fragment under the effect of e. coli dna ligase, 4 ℃ of about 16h of ligation.Transformed into escherichia coli DH5 α extracts recombinant plasmid, measures through sequential analysis and obtains positive recombinant plasmid PCPG12/T.The restriction enzyme reaction system and the ligation system of recombinant plasmid are as follows:
The double digestion reaction:
Plasmid PCPG1/T 8 μ l
10×bufferK 5μl
0.1％BSA 5μl
SphI 2μl
NcoI 2μl
ddH 2O 28μl
Total 50μl
After mixing, of short duration centrifugal, in 37 ℃ of water-bath 2h.1% agarose gel electrophoresis is cut the recovery purification kit recovery of glue with the energy lottery industry of Shen, Shanghai.
The ligation system is as follows:
PCPG2 1.5μl
PCPG1/T 10.0μl
10×buffer 1.5μl
T4?DNAligase(3unit/μl) 1.0μl
ddH 2O 1.0μl
Total?volume 15.0μl
2.4 recombinant plasmid transformed bacillus coli DH 5 alpha
Method is with 1.4 among the embodiment 2.
The present invention adopts following method to verify::
2.5 the preparation of recombinant plasmid
Method is with 1.5 among the embodiment 2;
2.6 the agarose gel electrophoresis of plasmid DNA
Method is with 1.6 among the embodiment 2, its detected result (see figure 2)
2.7 the nucleotide sequencing of recombinant plasmid and analysis
Method is with 1.7 among the embodiment 2, its detected result (see figure 4)
Second step: design of primers
The PCR primer design: in this recombinant plasmid, cytosine(Cyt) (C), guanine (G) content are very high, so according to the recombinant plasmid dna sequence at purpose fragment two ends, carry out Primer with GeneTool software and analyze, therefrom filter out and meet best primer.And can betting office synthesize by the Shen, Shanghai, the PCR test kit is that worker bio-engineering corporation product is given birth in Shanghai.
Upstream primer: 5 '---CCCAAGCTTGGGCCCGACGTCGCATGC---3 '
Downstream primer: 5 '---GCGGATCCCCCATATGGTCGACCTGCAGCC---3 '
The 3rd step: pcr amplification
A large amount of preparations of the second recombinant plasmid PCPG12/T
(1) the LB capacity of joining that 8ml is contained Amp is in the 15ml and the good test tube of ventilating, and inserts a single bacterium colony then, cultivates 12～16h down in 37 ℃ of violent joltings.
(3) above-mentioned cultivation bacterium is joined 2000ml and contain among the LB of Amp, cultivate 12～16h down in 37 ℃ of violent joltings.
(3) in 4 ℃ with 6000 rev/mins of centrifugal 15min, abandon supernatant, open wide the centrifugal mouth of pipe and also be inverted centrifuge tube, supernatant is all flow to end.
(4) with vacuum residual supernatant is exhausted.
(5) with 50ml damping fluid P1 suspension bacterial aggregate.
(6) add 50ml damping fluid P2, cover the tight mouth of pipe, gently put upside down 4～6 times, the mixing content is placed 5min in room temperature.
(7) add the ice-cold damping fluid P3 of 50ml, seal bottleneck, shake centrifuge tube, put upside down 4～6 times, the mixing content forms a white flocks, and lysate is thickness no longer.
(8) pour split product into filter, room temperature is placed 10min.
(9) open vacuum pump switch, with the supernatant suction filtration to collection container.
(10) add 50ml damping fluid FWB2, the precipitation that suspends is gently opened vacuum pump switch, with the supernatant suction filtration to collection container.
(11) add 12.5ml damping fluid ER to filterable cracking supernatant, put upside down mixing 10 times, put and place 30min on ice.
(12) with 35ml damping fluid QBT balance QIAGEN-tip 25000 posts.
(13) the cracking supernatant with step (11) adds balance columns.
(14) add 200ml damping fluid QC and wash post.
(15) DNA of adding 35ml damping fluid QN elution of bound.
(16) in elutriant, add the 24.5ml Virahol, mix, in 4 ℃ with 16,000 rev/mins of centrifugal 30min, supernatant carefully inclines.
(17) adding 7ml does not have endotoxic 70% ethanol, in 4 ℃ with 16,000 rev/mins of centrifugal 10min, supernatant carefully inclines.
(18) open wide the mouth of pipe, room temperature is placed 10～20min, dissolve again with no endotoxic damping fluid TE,
In this step, (4)-(18) operation can adopt the no intracellular toxin plasmid of QIAgen company to prepare test kit;
1.3PCR produce contain the second recombinant plasmid PCPG12/T CpG dna fragmentation product
Present embodiment is a template with the 3rd reorganization plasmid PCPG123/T, and PCR reaction system: PCR reaction cumulative volume is 100 μ l, and moiety is as follows:
10×PCR?buffer 10μl
Upstream primer P1 2.5 μ l
Downstream primer P2 2.5 μ l
Template 8 μ l,
10mMdNTP 2μl，
10mMMgCl 2 6μl
5unit/ μ l Taq enzyme 0.5 μ l,
Supply volume to 100 μ l with aseptic redistilled water;
Loop parameter is as follows: 94 ℃ of sex change 7min; 94 ℃, 45s; 52 ℃, 45s; 72 ℃, 1min; Circulate altogether 30 times: 72 ℃ are extended 7min
1.4PCR the purifying of product
(7) after PCR finishes, from the PCR reaction tubes with reaction solution to clean 1.5ml Eppendorf centrifuge tube, add the Solution BS mixing of 4 times of volumes.No matter sample size what, minimumly need to use 400ul Solution BS.
(8) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifuge tube lid (lid can not cover yet, if still you have a lot of samples simultaneously, worries to obscure or turn solution over, advises that you cover the centrifuge tube lid), use desk centrifuge high speed centrifugation (10000rpm) 1 minute.
(9) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600ul WashSolution, high speed centrifugation (10000rpm) 1 minute.
(10) repeating step 3 once.
(11) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes.
(12) the 3S post is put into a new 1.5ml centrifuge tube, central authorities add 30ul TE or water at 3S post film, do not cover centrifuge tube lid, and room temperature or 37 ℃ were placed 2 minutes.
Cover centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
1.5DNA quantitative analysis
With the TE damping fluid dilution DNA solution of an amount of volume, measure its OD with ultraviolet spectrophotometer 260And OD 280Value is calculated dna content.
10D 260The double-stranded DNA of=50 μ g/ml
Embodiment 4
The first step: the preparation of genetically engineered recombinant plasmid
3 genetically engineered construction of recombinant plasmid
3.1 the synthetic of the 3rd fragment normal chain and minus strand oligonucleotide
The positive chain-ordering of synthetic the 3rd fragment oligonucleotide is
5’---- CTAGTGGTGC ATCGATGCAGGGGGGTC GCGTTTT GTCGTTTT GTCGTTGGTGC GTCGATGCAGGGGGG CTGCA----3’
Oligonucleotide minus strand sequence is
5’----GCCCCCCTGCATCGACGCACCAACGACAAAACGACAAAACGACGACCCCCCTGCATCGGATGCACCA----3’
3.2 the 3rd is segmental synthetic
After the positive minus strand of above-mentioned oligonucleotide of synthetic and purifying, annealing buffer, aseptic redistilled water mixed by following prescription, descended centrifugal 30 seconds at 1000 rev/mins, incubate in 90 ℃ of-100 ℃ of water-baths and bathe more than 3 minutes, naturally cool to room temperature, this annealing reaction system is as follows:
Normal chain 1 μ l
Anti-chain 1 μ l
5 * annealing buffer, 16 μ l
Aseptic redistilled water 72 μ l
Cumulative volume 80 μ l
3.3 ligation
The positive recombinant plasmid PCPG12/T that obtains with embodiment 3 cuts and the 3rd fragment ligation through enzyme as carrier
In the K damping fluid, with the positive recombinant plasmid PCPG12/T restriction enzyme SphI, the NcoI difference enzymolysis that obtain, agarose gel electrophoresis shows that enzyme cuts entirely; Use restriction enzyme SphI, NcoI double digestion again, recovery purification kit that can lottery industry with the Shen, Shanghai reclaims, with the 3rd fragment under the effect of e. coli dna ligase, 4 ℃ of about 16h of ligation.Transformed into escherichia coli DH5 α extracts recombinant plasmid, measures through sequential analysis and obtains positive recombinant plasmid PCPG123/T.The restriction enzyme reaction system and the ligation system of recombinant plasmid are as follows:
The double digestion reaction:
Plasmid PCPG1/T 8 μ l
10×buffer?K 5μl
pstI 2μl
SpeI 2μl
ddH 2O 33μl
Total 50μl
After mixing, of short duration centrifugal, in 37 ℃ of water-bath 2h.1% agarose gel electrophoresis is with the recovery purification kit recovery of Shen, Shanghai energy lottery industry.
The ligation system is as follows:
PCPG2 1.5μl
PCPG1/T 10.0μl
10×buffer 1.5μl
T4?DNAligase(3unit/μl) 1.0μl
ddH 2O 1.0μl
Total?volume 15.0μl
3.4 recombinant plasmid transformed bacillus coli DH 5 alpha
Method is with 1.4 among the embodiment 2
3.5 the preparation of recombinant plasmid
Method is with 1.5 among the embodiment 2.
The present invention adopts following method to verify:
3.6 the agarose gel electrophoresis of plasmid DNA
Method is with 1.6 among the embodiment 2, its detected result (see figure 2)
3.6 the nucleotide sequencing of recombinant plasmid and analysis
Method is with 1.6 among the embodiment 2, its detected result (see figure 5).
Second step: design of primers
The PCR primer design: in this recombinant plasmid, cytosine(Cyt) (C), guanine (G) content are very high, so according to the recombinant plasmid dna sequence at purpose fragment two ends, carry out Primer with GeneTool software and analyze, therefrom filter out and meet best primer.And can betting office synthesize by the Shen, Shanghai, the PCR test kit is that worker bio-engineering corporation product is given birth in Shanghai.
Upstream primer: 5 '---CCCAAGCTTGGGCCCGACGTCGCATGC---3,
Downstream primer: 5 '---GCGGATCCCCCATATGGTCGACCTGCAGCC---3,
The 3rd step: pcr amplification
A large amount of preparations of the 3rd reorganization plasmid PCP6123/T
(1) the LB capacity of joining that 8ml is contained Amp is in the 15ml and the good test tube of ventilating, and inserts a single bacterium colony then, cultivates 12～16h down in 37 ℃ of violent joltings.
(4) above-mentioned cultivation bacterium is joined 2000ml and contain among the LB of Amp, cultivate 12～16h down in 37 ℃ of violent joltings.
(3) in 4 ℃ with 6000 rev/mins of centrifugal 15min, abandon supernatant, open wide the centrifugal mouth of pipe and also be inverted centrifuge tube, supernatant is all flow to end.
(4) with vacuum residual supernatant is exhausted.
(5) with 50ml damping fluid P1 suspension bacterial aggregate.
(6) add 50ml damping fluid P2, cover the tight mouth of pipe, gently put upside down 4～6 times, the mixing content is placed 5min in room temperature.
(7) add the ice-cold damping fluid P3 of 50ml, seal bottleneck, shake centrifuge tube, put upside down 4～6 times, the mixing content forms a white flocks, and lysate is thickness no longer.
(8) pour split product into filter, room temperature is placed 10min.
(9) open vacuum pump switch, with the supernatant suction filtration to collection container.
(10) add 50ml damping fluid FWB2, the precipitation that suspends is gently opened vacuum pump switch, with the supernatant suction filtration to collection container.
(11) add 12.5ml damping fluid ER to filterable cracking supernatant, put upside down mixing 10 times, put and place 30min on ice.
(12) with 35ml damping fluid QBT balance QIAGEN-tip 25000 posts.
(13) the cracking supernatant with step (11) adds balance columns.
(14) add 200ml damping fluid QC and wash post.
(15) DNA of adding 35ml damping fluid QN elution of bound.
(16) in elutriant, add the 24.5ml Virahol, mix, in 4 ℃ with 16,000 rev/mins of centrifugal 30min, supernatant carefully inclines.
(17) adding 7ml does not have endotoxic 70% ethanol, in 4 ℃ with 16,000 rev/mins of centrifugal 10min, supernatant carefully inclines.
(18) open wide the mouth of pipe, room temperature is placed 10～20min, dissolve again with no endotoxic damping fluid TE,
In this step, (4)-(18) operation can adopt the no intracellular toxin plasmid of QIAgen company to prepare test kit;
1.3PCR produce contain the 3rd reorganization plasmid PCPG123/T CpG dna fragmentation product
Present embodiment is a template with the 3rd reorganization plasmid PCPG123/T, and PCR reaction system: PCR reaction cumulative volume is 100 μ l, and moiety is as follows:
10×PCRbuffer 10μl
Upstream primer P1 2.5 μ l
Downstream primer P2 2.5 μ l
Template 8 μ l,
10mM?dNTP 2μl，
10mM?MgCl 2 6μl
5unit/ μ l Taq enzyme 0.5 μ l,
Supply volume to 100 μ l with aseptic redistilled water;
Loop parameter is as follows: 94 ℃ of sex change 7min; 94 ℃, 45s; 52 ℃, 45s; 72 ℃, 1min; Circulate altogether 30 times; 72 ℃ are extended 7min
1.4PCR the purifying of product
(13) after PCR finishes, from the PCR reaction tubes with reaction solution to clean 1.5ml Eppendorf centrifuge tube, add the Solution BS mixing of 4 times of volumes.No matter sample size what, minimumly need to use 400ul Solution BS.
(14) the 3S post is put into collection tube, mixed solution is transferred in the post, do not cover the centrifuge tube lid, room temperature was placed 2 minutes; Cover centrifuge tube lid (lid can not cover yet, if still you have a lot of samples simultaneously, worries to obscure or turn solution over, advises that you cover the centrifuge tube lid), use desk centrifuge high speed centrifugation (10000rpm) 1 minute.
(15) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, add 600ul WashSolution, high speed centrifugation (10000rpm) 1 minute.
(16) repeating step 3 once.
(17) outwell waste liquid in the collection tube, the 3S post is put into same collection tube, high speed centrifugation (10000rpm) 2 minutes.
(18) the 3S post is put into a new 1.5ml centrifuge tube, central authorities add 30ul TE or water at 3S post film, do not cover centrifuge tube lid, and room temperature or 37 ℃ were placed 2 minutes.
Cover centrifuge tube lid, 10000rpm high speed centrifugation 1 minute, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.
1.5DNA quantitative analysis
With the TE damping fluid dilution DNA solution of an amount of volume, measure its OD with ultraviolet spectrophotometer 260And OD 280Value is calculated dna content.
10D 260The double-stranded DNA of=50 μ g/ml
1.6CpG DNA strengthens the immune effect of aftosa vaccine
1. the immunity of foot and mouth disease vaccine: Schweineseuche O-shaped deactivation is carried out immunity available from Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. by product description.With 20, body weight is the weanling pig about 15 kilograms, is divided into control group and test group at random, uses aftosa vaccine and aftosa vaccine+PCPG fragment (every of 200 μ g/) muscle immunity behind the basal part of the ear respectively.
2. antibody titer detects in the foot and mouth disease serum: the 3rd week blood sampling of immunity back, separation of serum adopts the forward indirect hemagglutination test to detect antibody titer.Foot and mouth disease forward indirect hemagglutination antigen, positive serum, negative serum are all available from the Lanzhou veterinary institute.By the operation of working method by specification.On blood-coagulation-board with serum to be checked with diluent dilute successively be 1: 0,1: 2,1: 4 ..., 1: 512, if the positive serum of the negative serum of dilution in 1: 16, dilution in 1: 512, diluent contrast, every hole adds 25 μ l foot and mouth disease forward indirect hemagglutination antigens, and mixing fully vibrates.Room temperature leaves standstill result of determination after 2 hours.Agglutinative maximum dilution multiple tiring for this serum appears with 50% red corpuscle.
3. result: 10 parts of blood of control group please in only have 2 parts of antibody titers to reach 1: 128, the antibody of all the other 8 parts of serum is all less than 1: 128,10 parts of blood of test group please in have 8 parts of antibody titers to reach 1: 128, the antibody of all the other 2 parts of serum reaches 1: 64.

Claims (1)

1, a kind ofly can improve the PCR preparation method who contains CpG DNA veterinary vaccine adjuvant that veterinary vaccines is tired, it is characterized in that:
The first step: this genetically engineered recombinant plasmid prepares as follows, according to
5’-GGTGC ATCGATGCAGGGGGG-3’、
5 '-GGTGC GTCGATGCAGGGGGG-3 ' reaches
5 '-TC GTCGTTTT GTCGTTTT GTCGTT-3 ' the target DNA sequence that is in series, synthetic its normal chain and minus strand, add the base sequence that can be complementary with linear carrier end at synthetic oligonucleotide end, after the oligonucleotide fragment that will so obtain, annealing buffer and aseptic redistilled water mix then, in 90 ℃～100 ℃ water-baths, incubate and bathe more than 3 minutes, naturally cool to room temperature, the one or more such double chain DNA fragment that forms is thus inserted the corresponding restriction enzyme site of linear carrier, at last, the genetically engineered recombinant plasmid that its transformed competence colibacillus intestinal bacteria are obtained.
Second step: according to the recombinant plasmid dna sequence at purpose fragment two ends, the design special primer.
The 3rd step: with the recombinant plasmid is template, adds special primer, and carries out three warm round-robin pcr amplifications, and concrete parameter is: 92 ℃～98 ℃ pre-sex change 5～10 minutes; 92 ℃～98 ℃, 45～60 seconds, 50～58 ℃, 45～60 seconds, 72 ℃, 45～60 seconds, carry out 30～35 circulations; Extended 10 minutes at 72 ℃ at last.
CN 200310106227 2003-11-07 2003-11-07 PCR process for preparing CpG DNA containing livestock and bird vaccine adjuvant Expired - Lifetime CN1250737C (en)

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