CN106834331A - Express the rBCG and its construction method of sheep kind Brucella sp P39 and L7/L12 fusion - Google Patents
Express the rBCG and its construction method of sheep kind Brucella sp P39 and L7/L12 fusion Download PDFInfo
- Publication number
- CN106834331A CN106834331A CN201710084435.2A CN201710084435A CN106834331A CN 106834331 A CN106834331 A CN 106834331A CN 201710084435 A CN201710084435 A CN 201710084435A CN 106834331 A CN106834331 A CN 106834331A
- Authority
- CN
- China
- Prior art keywords
- brucella
- bcg
- fusion
- rbcg
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/098—Brucella
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The present invention provides a kind of rBCG for expressing sheep kind Brucella sp P39 and L7/L12 fusion, and it is that the expression vector of sheep kind Brucella sp P39 and the L7/L12 fusion that will carry codon optimization is transferred in BCG to build and obtains.The kytoplasm associated proteins PBP39 (encoding gene is P39) and Brucella sp ribosome protein L 7/L/L12 produced by Brucella sp are T cell antigen.BCG vaccine BCG is the vaccine of the prevention tuberculosis of Unique Product so far, and it not only has significant immunologic adjuvant effect, and is a kind of functional, safe exogenous gene expression host.Brucella sp P39 and the L7/L12 fusion optimized using BCG expression codons can both improve the expression quantity of genes of interest, gained rBCG vaccines can simulate the characteristic of Brucella sp intracellular infection and parasitism again, more effectively induction body produces immune response, can also play BCG as expressive host high security, prepare simple, with low cost the advantages of and the immunologic adjuvant effects that have of BCG in itself, be expected to turn into new brucella disease vaccine.
Description
Technical field
The present invention relates to genetic engineering and vaccine preparation technology field, specifically, it is related to a kind of expression sheep kind Brucella sp
The rBCG and its construction method of P39 and L7/L12 fusions.
Background technology
Brucellosis (Brucellosis) abbreviation cloth disease, is that a kind of infecting both domestic animals and human caused by Brucella sp infection is infected
Disease.Brucella sp is a kind of facultative intracellular bacterial parasite, has hyperinfection and pathogenic to people and mammal.The Pseudomonas is main
Including sheep kind, ox kind, pig kind and 6 biological species etc. kind of dog, popular mainly first 3 kinds of China, wherein with sheep kind Brucella sp sense
Dye is most common.The circulation way of cloth disease is animal-animal and animal-crowd.The domestic animals such as sheep, ox, pig most easy infection, animal sense
Reproductive organs and fetal membrane inflammation, miscarriage, sterile and various lesion tissues can be caused after dye, substantially increase what is propagated to crowd
Probability.Crowd is generally susceptible to Brucella sp, and general sheep kind Brucella sp endangers maximum to people.People and ill domestic animal and contaminated livestock products
After contact, Brucella sp can be infected by approach such as alimentary canal, respiratory tract, urogenital tract, skin and eye conjunctivas.
Over nearly 20 years, China's cloth disease epidemic situation is on the rise and the trend by spreading rapidly.The cloth disease vaccine of domestic and foreign current
Attenuated live vaccine is, vaccine generally existing toxicity is larger, protection period is shorter for these, cannot be distinguished by natural infection and vaccine connects
Many deficiencies such as kind, it is impossible to meet the current demand of cloth disease prevention and control.Therefore, the research and development of novel cloth disease vaccine are for control
Spreading for cloth disease epidemic situation is significant, is also the focus of domestic and international cloth disease research.Existing certified Brucella sp T cell antigen
Including PBP39, L7/L12 and Cu-Zn SOD etc., these albumen and its encoding gene have been used for cloth disease subunit vaccine and DNA epidemic diseases
The experimental study of seedling.
BCG vaccine (Bacillus Calmette-Gu é rin, BCG) be it is currently the only for preventing vaccine lungy,
Widely used in the global extension Immunization programme of the World Health Organization.There are BCG many decades largely to use record, thermally-stabilised safely
Property good, low production cost, significant adjuvant effect and can induce by oral administration potent mucosal it is immune the features such as, while BCG is also
A kind of live vaccine vectors of very attractive, it is tempting as the expressive host prospect of new generation vaccine by the use of BCG.In recent years, with BCG
For the recombinant BCG vaccine research and development of carrier are increasingly subject to the concern and attention of domestic and international researcher, for pathogen be related to carefully
Bacterium, virus, parasite and tumour etc., with vast potential for future development.
At present both at home and abroad there is not yet relevant structure carries M5 plants of 39kDa kytoplasm associated proteins PBP39 coding of sheep kind Brucella sp
The fusion (P39-L7/L12 genes) of gene (P39 genes) and ribosome protein L 7/L/L12 encoding genes (L7/L12 genes)
The report of recombinant BCG.
The content of the invention
It is an object of the invention to provide a kind of rBCG and its structure for expressing sheep kind Brucella sp P39 and L7/L12 fusion
Method.
It is a further object of the present invention to provide applications of the rBCG in brucella disease vaccine is prepared.
In order to realize the object of the invention, present invention firstly provides a kind of expression casette, comprising being optimized by codon
Sheep kind Brucella sp P39 genes and codon optimization L7/L12 genomic constitutions fusion.
In the present invention, P39, L7/L12 gene for constituting sheep kind Brucella sp P39 and L7/L12 fusion are all from
Optimize in brucella melitensis (Brucella melitensis) M5 bacterial strains (CMCC55009), and codon, it is described to melt
Close the nucleotide sequence such as SEQ ID NO of gene:Shown in 1.
The present invention also provides a kind of expression vector, and the expression vector carries sheep kind Brucella sp P39 and L7/L12 fusion
The expression cassette of gene.
Preferably, the carrier that sets out of the expression vector is for Escherichia coli-mycobacterium shuttle expression carrier pMV361.
It is highly preferred that the nucleotide sequence of the expression vector such as SEQ ID NO:Shown in 2.
The present invention also provides a kind of rBCG for expressing sheep kind Brucella sp P39 and L7/L12 fusion, and it is to carry
The expression vector of sheep kind Brucella sp P39 and L7/L12 track fusion box is transferred in BCG, and screening acquisition can secreting, expressing sheep kind
The rBCG of Brucella sp P39 and L7/L12 fusion.
The rBCG of present invention expression sheep kind Brucella sp P39 and L7/L12 fusion, is built by following methods and obtained：
1) according to the sequence of published sheep kind Brucella sp M5 pnca genes P39 and L7/L12, carried out using Jcat softwares close
After numeral optimization, P39 the and L7/L12 genes after artificial synthesized complete sequence optimization will build the fusion P39-L7/ for obtaining
L12 is used as genes of interest；
2) structure of the recombinant expression carrier of the sheep kind Brucella sp P39-L7/L12 fusions after optimization is carried：Will be upper
State genes of interest and insert shuttle expression carrier pMV361 by I two restriction enzyme sites of Pvu II and BstB；
3) Host Strains conversion：By step 2) build the recombinant expression carrier that obtains and convert into BCG, screening positive clone,
Obtain final product can secreting, expressing sheep kind Brucella sp P39-L7/L12 fusions rBCG.
The present invention also provides applications of the rBCG in brucella disease vaccine is prepared.
The present invention also provides the brucella disease vaccine prepared by the rBCG.
RBCG of the present invention can be used directly as brucella disease vaccine, or be aided with immunologic adjuvant, be made
Brucella disease vaccine.
The present invention has advantages below：
(1) the kytoplasm associated proteins PBP39 (encoding gene is P39) and Bu Shi of about 39kDa sizes are produced by Brucella sp
Bacterium ribosomal protein L7/L12 (relative molecular weight size is about 15kDa, and its encoding gene is L7/L12 genes) is T cell and resists
Original, can stimulate body to produce effective immune response.
(2) BCG (BCG vaccine) is the vaccine of the prevention tuberculosis of Unique Product so far, used as the expression place of foreign gene
The vaccine of main preparation has the advantages that safe, heat endurance is good, it is simple, cheap to prepare.
(3) BCG has significant immunologic adjuvant effect in itself, therefore rBCG vaccines need not add vaccine adjuvant, further
Cost is saved.
(4) BCG is also a kind of facultative intracellular bacterial parasite, and rBCG can simulate Brucella sp infection and the entozoic feature of born of the same parents,
More effectively induction body produces immune response.
(5) after the optimization of P39-L7/L12 fusions codon, it is possible to increase expression quantity of the foreign gene in BCG
(after being induced 4 hours through 60 DEG C, expression quantity can improve more than 50%), solves because of the caused restructuring not high of exogenous gene expression amount
The not good problem of BCG immune effects.
(6) results of animal shows that the rBCG of structure being capable of the inducing mouse obvious immune response of generation.
(7) there is important Research Significance using sheep kind Brucella sp P39-L7/L12 fusion Prepare restructuring BCG vaccines
With potential application value.
Brief description of the drawings
Fig. 1 is the recombinant BCG of P39-L7/L12 Fusion gene constructions optimized and not optimized in the embodiment of the present invention 2
The SDS-PAGE electrophoresis results of middle destination protein；Wherein, 1：Carry not optimized P39-L7/L12 fusion recombinant BCGs；
2：P39-L7/L12 fusion recombinant BCGs after carrying is optimized；3：P39-L7/L12 fusions after carrying is optimized
Recombinant BCG (temperature-induced)；4：The BCG of untransfected；M：Albumen Marker.
Fig. 2 is that the different recombinant BCGs of structure in the embodiment of the present invention 3 compare the immune effect of Balb/c mouse.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or the condition advised according to manufacturer's specification.
Embodiment 1 carries the structure of the recombinant expression carrier of sheep kind Brucella sp P39 and L7/L12 fusion
Comprise the following steps：
1st, according to the sequence (GenBank of published brucella melitensis M5 pnca genes P39 and L7/L12:
EF189139.1；GenBank:EF173477.1), after carrying out codon optimization using Jcat softwares, artificial synthesized complete sequence is excellent
P39-L7/L12 fusions after change are used as genes of interest.(the nucleotide sequence of fusion such as SEQ ID NO:Shown in 1).
2nd, the structure of the recombinant expression carrier of the M5 bacterial strain P39-L7/L12 fusions after optimization is carried：By above-mentioned mesh
Gene insert shuttle expression carrier pMV361 by I two restriction enzyme sites of Pvu II and BstB.
3rd, the checking of correct recombinant expression carrier is inserted.
By determining nucleic acid sequence, it was demonstrated that carry the restructuring of the sheep kind Brucella sp P39-L7/L12 fusions after optimization
Expression vector establishment success.
The structure of the rBCG of expression sheep kind Brucella sp P39 and the L7/L12 fusion of embodiment 2
1st, using BCG as Host Strains, recombinant expression carrier (the complete sequence such as SEQ ID NO that will be built in embodiment 1:2 institutes
Show) convert into BCG.
Converted using electric method for transformation, experiment condition is：2500V, 25 μ F, 1000 Ω, electricity turn time 5ms, 0.1cm
Electric shock cup.Electric conversion reaction system is：The μ l of plasmid 3 (concentration is 0.68 μ g/ μ l), (concentration is about 1 to the μ l of competence BCG bacterium solutions 100
×1010CFU/ml)。
2nd, the screening of positive colony
After electricity turns, being inoculated on the culture medium (inclined-plane) containing 50 μ g/ml kanamycins carries out positive colony screening.
3rd, the detection of destination gene expression amount
The positive colony (i.e. recombinant BCG) that obtains will be screened be inoculated into carries out amplification cultivation in fluid nutrient medium, collect training
Support supernatant, the expression quantity of SDS-PAGE electrophoresis detection genes of interest.P39-L7/L12 fusions after optimization are temperature-induced
Under (60 DEG C induce 4 hours), expression quantity is significantly raised.Result is shown in Fig. 1.
The effect experiment of the brucella disease vaccine of embodiment 3
By the immune 6-8 week old female Balb/c mouse of recombinant BCG, hypodermic injection, injection dosage is 4 × 108CFU/ only, exempts from
After epidemic disease 4 weeks, the expression of Th1/Th2 cytokines in detection each group mice serum.Test result indicate that, with carry without
The recombinant BCG (rBCG-P39-L7/L12 (wild)) for optimizing P39-L7/L12 fusions is compared with unconverted BCG, is taken
Recombinant BCG (rBCG-P39-L7/L12) with codon optimization P39-L7/L12 fusions can effectively induce IL-
2nd, the generation of the Th1 cytokines such as IL-12 and IFN-γ.(Fig. 2)
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Inner Mongolia Medical University
<120>Express the rBCG and its construction method of sheep kind Brucella sp P39 and L7/L12 fusion
<130> KHP161118993.5
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1578
<212> DNA
<213>Artificial sequence
<400> 1
atggccccgg tggccaacgc ccaggagaag cagaacgtgg aggtgctgca ctggtggacc 60
tcgggcggcg aggcctcggc cctggaggtg ctgaagaagg acctggagtc gaagggcatc 120
tcgtggaccg acatgccggt ggccggcggc ggcggcaccg aggccatgac cgtgctgcgc 180
gcccgcgtga ccgccggcaa cgccccgacc gccgtgcaga tgctgggctt cgacatccgc 240
gactgggccg agcagggcgc cctgggcaac ctggacaccg tggcctcgaa ggagggctgg 300
gagaaggtga tcccggcccc gctgcaggag ttcgccaagt acgacggcca ctggatcgcc 360
gccccggtga acatccactc gaccaactgg atgtggatca acaaggccgc cctggacaag 420
gccggcggca aggagccgac caactgggac gagctgatcg ccctgctgga caacttcaag 480
gcccagggca tcaccccgat cgcccacggc ggccagccgt ggcaggacgc caccatcttc 540
gacgccgtgg tgctgtcgtt cggcccggac ttctacaaga aggccttcat cgacctggac 600
ccggaggccc tgggctcgga caccatgaag caggccttcg accgcatgtc gaagctgcgc 660
acctacgtgg acgacaactt ctcgggccgc gactggaacc tggcctcggc catggtgatc 720
gagggcaagg ccggcgtgca gttcatgggc gactgggcca agggcgagtt cctgaaggcc 780
ggcaagaagc cgggcgagga cttcgtgtgc atgcgctacc cgggcaccca gggcgccgtg 840
accttcaact cgggcatgtt cgccatgttc aaggtgtcgg aggacaaggt gccggcccag 900
ctggagatgg cctcggccat cgagtcgccg gccttccagt cggccttcaa cgtggagaag 960
ggctcggccc cggcccgcac cgacgtgccg gacaccgcct tcgacgcctg cggcaagaag 1020
accatcgccg acgtgaagga ggccaactcg aagggcaccc tgctgggctc gatggcccac 1080
ggctacgcca acccggccgc cgtgaagaac gccatctacg acgtggtgac ccgccagttc 1140
aacggccagc tgtcgtcgga ggacgccgtg aaggagctgg tggtggccgt ggaggccgcc 1200
aagatggccg acctggccaa gatcgtggag gacctgtcgg ccctgaccgt gctggaggcc 1260
gccgagctgt cgaagctgct ggaggagaag tggggcgtgt cggccgccgc cccggtggcc 1320
gtggccgccg ccggcggcgc cgccccggcc gccgccgccg aggagaagac cgagttcgac 1380
gtggtgctgg ccgacggcgg cgccaacaag atcaacgtga tcaaggaggt gcgcgccctg 1440
accggcctgg gcctgaagga ggccaaggac ctggtggagg gcgccccgaa ggccgtgaag 1500
gagggcgcct cgaaggacga ggccgagaag atcaaggccc agctggaggc cgccggcgcc 1560
aaggtggagc tgaagtaa 1578
<210> 2
<211> 6023
<212> DNA
<213>Recombinant expression carrier
<400> 2
gctagcaaca aagcgacgtt gtgtctcaaa atctctgatg ttacattgca caagataaaa 60
atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta 120
tgagccatat tcaacgggaa acgtcttgct cgaggccgcg attaaattcc aacatggatg 180
ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct 240
atcgcttgta tgggaagccc catgcgccag agttgtttct gaaacatggc aaaggtagcg 300
ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc 360
ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 420
tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg 480
ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt 540
ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg 600
ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 660
aaatgcataa tcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 720
ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg 780
gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc 840
cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat 900
tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac 960
actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt 1020
tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa 1080
gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc 1140
cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc 1200
ttcacgaggc agacctcact agttccactg agcgtcagac cccgtagaaa agatcaaagg 1260
atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 1320
gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 1380
tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 1440
ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 1500
ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 1560
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 1620
aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc 1680
cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 1740
gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 1800
ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 1860
cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 1920
tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 1980
cgctcgccgc agccgaacga ccgagcgcaa cgcgtgcggc cgcggtaccc ggggatcctc 2040
tagagtcgac caccaagggc accatctctg cttgggccac cccgttggcc gcagccagct 2100
cgctgagagc cgtgaacgac agggcgaacg ccagcccgcc gacggcgagg gttccgaccg 2160
ctgcaactcc cggtgcaacc ttgtcccggt ctattctctt cactgcacca gctccaatct 2220
ggtgtgaatg cccctcgtct gttcgcgcag gcggggggct ctattcgttt gtcagcatcg 2280
aaagtagcca gatcagggat gcgttgcaac cgcgtatgcc caggtcagaa gagtcgcaca 2340
agagttgcag acccctggaa agaaaaatgg ccagagggcg aaaacaccct ctgaccagcg 2400
gagcgggcga cgggaatcga acccgcgtag ctagtttgga agaatgggtg tctgccgacc 2460
acatatgggc cggtcaagat aggtttttac cccctctcgg ctgcatcctc taagtggaaa 2520
gaaattgcag gtcgtagaag cgcgttgaag cctgagagtt gcacaggagt tgcaacccgg 2580
tagccttgtt cacgacgaga ggagacctag ttggcacgtc gcggatgggg atcgctgaag 2640
actcagcgca gcgggaggat ccaagcctca tacgtcaacc cgcaggacgg tgtgaggtac 2700
tacgcgctgc agacctacga caacaagatg gacgccgaag cctggctcgc gggcgagaag 2760
cggctcatcg agatggagac ctggacccct ccacaggacc gggcgaagaa ggcagccgcc 2820
agcgccatca cgctggagga gtacacccgg aagtggctcg tggagcgcga cctcgcagac 2880
ggcaccaggg atctgtacag cgggcacgcg gagcgccgca tctacccggt gctaggtgaa 2940
gtggcggtca cagagatgac gccagctctg gtgcgtgcgt ggtgggccgg gatgggtagg 3000
aagcacccga ctgcccgccg gcatgcctac aacgtcctcc gggcggtgat gaacacagcg 3060
gtcgaggaca agctgatcgc agagaacccg tgccggatcg agcagaaggc agccgatgag 3120
cgcgacgtag aggcgctgac gcctgaggag ctggacatcg tcgccgctga gatcttcgag 3180
cactaccgga tcgcggcata catcctggcg tggacgagcc tccggttcgg agagctgatc 3240
gagcttcgcc gcaaggacat cgtggacgac ggcatgacga tgaagctccg ggtgcgccgt 3300
ggcgcttccc gcgtggggaa caagatcgtc gttggcaacg ccaagaccgt ccggtcgaag 3360
cgtcctgtga cggttccgcc tcacgtcgcg gagatgatcc gagcgcacat gaaggaccgt 3420
acgaagatga acaagggccc cgaggcattc ctggtgacca cgacgcaggg caaccggctg 3480
tcgaagtccg cgttcaccaa gtcgctgaag cgtggctacg ccaagatcgg tcggccggaa 3540
ctccgcatcc acgacctccg cgctgtcggc gctacgttcg ccgctcaggc aggtgcgacg 3600
accaaggagc tgatggcccg tctcggtcac acgactccta ggatggcgat gaagtaccag 3660
atggcgtctg aggcccgcga cgaggctatc gctgaggcga tgtccaagct ggccaagacc 3720
tcctgaaacg caaaaagccc ccctcccaag gacactgagt cctaaagagg ggggtttctt 3780
gtcagtacgc gaagaaccac gcctggccgc gagcgccagc accgccgctc tgtgcggaga 3840
cctgggcacc agccccgccg ccgccaggag cattgccgtt cccgccagaa atctagacgg 3900
tgaccacaac gacgcgcccg ctttgatcgg ggacgtctgc ggccgaccat ttacgggtct 3960
tgttgtcgtt ggcggtcatg ggccgaacat actcacccgg atcggagggc cgaggacaag 4020
gtcgaacgag gggcatgacc cggtgcgggg cttcttgcac tcggcatagg cgagtgctaa 4080
gaataacgtt ggcactcgcg accggtgagt cgtaggtcgg gacggtgagg ccaggcccgt 4140
cgtcgcagcg agtggcagcg aggacaactt gagccgtccg tcgcgggcac tgcgcccggc 4200
cagcgtaagt agcggggttg ccgtcacccg gtgacccccg gtttcatccc cgatccggag 4260
gaatcacttc gcaatggcca agacaattgc ggatccagct ggccacatgg ccccggtggc 4320
caacgcccag gagaagcaga acgtggaggt gctgcactgg tggacctcgg gcggcgaggc 4380
ctcggccctg gaggtgctga agaaggacct ggagtcgaag ggcatctcgt ggaccgacat 4440
gccggtggcc ggcggcggcg gcaccgaggc catgaccgtg ctgcgcgccc gcgtgaccgc 4500
cggcaacgcc ccgaccgccg tgcagatgct gggcttcgac atccgcgact gggccgagca 4560
gggcgccctg ggcaacctgg acaccgtggc ctcgaaggag ggctgggaga aggtgatccc 4620
ggccccgctg caggagttcg ccaagtacga cggccactgg atcgccgccc cggtgaacat 4680
ccactcgacc aactggatgt ggatcaacaa ggccgccctg gacaaggccg gcggcaagga 4740
gccgaccaac tgggacgagc tgatcgccct gctggacaac ttcaaggccc agggcatcac 4800
cccgatcgcc cacggcggcc agccgtggca ggacgccacc atcttcgacg ccgtggtgct 4860
gtcgttcggc ccggacttct acaagaaggc cttcatcgac ctggacccgg aggccctggg 4920
ctcggacacc atgaagcagg ccttcgaccg catgtcgaag ctgcgcacct acgtggacga 4980
caacttctcg ggccgcgact ggaacctggc ctcggccatg gtgatcgagg gcaaggccgg 5040
cgtgcagttc atgggcgact gggccaaggg cgagttcctg aaggccggca agaagccggg 5100
cgaggacttc gtgtgcatgc gctacccggg cacccagggc gccgtgacct tcaactcggg 5160
catgttcgcc atgttcaagg tgtcggagga caaggtgccg gcccagctgg agatggcctc 5220
ggccatcgag tcgccggcct tccagtcggc cttcaacgtg gagaagggct cggccccggc 5280
ccgcaccgac gtgccggaca ccgccttcga cgcctgcggc aagaagacca tcgccgacgt 5340
gaaggaggcc aactcgaagg gcaccctgct gggctcgatg gcccacggct acgccaaccc 5400
ggccgccgtg aagaacgcca tctacgacgt ggtgacccgc cagttcaacg gccagctgtc 5460
gtcggaggac gccgtgaagg agctggtggt ggccgtggag gccgccaaga tggccgacct 5520
ggccaagatc gtggaggacc tgtcggccct gaccgtgctg gaggccgccg agctgtcgaa 5580
gctgctggag gagaagtggg gcgtgtcggc cgccgccccg gtggccgtgg ccgccgccgg 5640
cggcgccgcc ccggccgccg ccgccgagga gaagaccgag ttcgacgtgg tgctggccga 5700
cggcggcgcc aacaagatca acgtgatcaa ggaggtgcgc gccctgaccg gcctgggcct 5760
gaaggaggcc aaggacctgg tggagggcgc cccgaaggcc gtgaaggagg gcgcctcgaa 5820
ggacgaggcc gagaagatca aggcccagct ggaggccgcc ggcgccaagg tggagctgaa 5880
gtaattcgaa gcttatcgat gtcgacgtag ttaactagcg tacgatcgac tgccaggcat 5940
caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcc 6000
ggccatcatg gccgcggtga tca 6023
Claims (9)
1. a kind of expression casette, it is characterised in that comprising the sheep kind Brucella sp P39 genes optimized by codon and process
The fusion of the L7/L12 genomic constitutions of codon optimization.
2. expression cassette according to claim 1, it is characterised in that the P39 genes and the L7/L12 genes are all from
In sheep kind Brucella sp M5 bacterial strains, the nucleotide sequence such as SEQ ID NO of the fusion:Shown in 1.
3. a kind of expression vector, it is characterised in that the expression vector carries the expression casette described in claim 1 or 2.
4. expression vector according to claim 3, it is characterised in that the carrier that sets out is that Escherichia coli-mycobacterium is shuttled
Expression vector pMV361.
5. expression vector according to claim 4, it is characterised in that the nucleotide sequence of the expression vector such as SEQ ID
NO:Shown in 2.
6. the rBCG of sheep kind Brucella sp P39 and L7/L12 fusion is expressed, it is characterised in that it is to appoint claim 3-5
Expression vector described in one is transferred in BCG, and screening is obtained can secreting, expressing sheep kind Brucella sp P39 and L7/L12 fusion
rBCG。
7. the construction method of rBCG described in claim 6, it is characterised in that comprise the following steps：
1) according to the sequence of published sheep kind Brucella sp M5 pnca genes P39 and L7/L12, codon is carried out using Jcat softwares
After optimization, P39 the and L7/L12 genes after artificial synthesized complete sequence optimization will build the fusion P39-L7/L12 for obtaining and make
It is purpose gene；
2) structure of the recombinant expression carrier of the sheep kind Brucella sp P39-L7/L12 fusions after optimization is carried：By above-mentioned mesh
Gene insert shuttle expression carrier pMV361 by I two restriction enzyme sites of Pvu II and BstB；
3) Host Strains conversion：By step 2) build the recombinant expression carrier that obtains and convert into BCG, screening positive clone is obtained final product
Can secreting, expressing sheep kind Brucella sp P39-L7/L12 fusions rBCG.
8. applications of the rBCG described in claim 6 in brucella disease vaccine is prepared.
9. the brucella disease vaccine for being prepared as rBCG described in claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710084435.2A CN106834331B (en) | 2017-02-16 | 2017-02-16 | rBCG for expressing fusion gene of Brucella melitensis P39 and L7/L12 and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710084435.2A CN106834331B (en) | 2017-02-16 | 2017-02-16 | rBCG for expressing fusion gene of Brucella melitensis P39 and L7/L12 and construction method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106834331A true CN106834331A (en) | 2017-06-13 |
| CN106834331B CN106834331B (en) | 2020-04-07 |
Family
ID=59128574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710084435.2A Active CN106834331B (en) | 2017-02-16 | 2017-02-16 | rBCG for expressing fusion gene of Brucella melitensis P39 and L7/L12 and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106834331B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314709A (en) * | 2018-03-22 | 2018-07-24 | 吉林农业大学 | Recombinant glycosylated albumen P39 and its preparation method and application |
| CN109486846A (en) * | 2018-12-29 | 2019-03-19 | 山东农业大学 | A kind of three kinds of gene recombination plasmids of brucella, construction method and its expression and application in Escherichia coli |
| CN113637703A (en) * | 2021-08-06 | 2021-11-12 | 河北科技师范学院 | Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector |
-
2017
- 2017-02-16 CN CN201710084435.2A patent/CN106834331B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| AYMAN AL-MARIRI等: "Protection of BALB/c mice against Brucella melitensis 16 M infection induced by vaccination with live Escherchia coli expression Brucella P39 protein", 《VACCINE》 * |
| LUO DE-YAN等: "DNA vaccine encoding L7/L12-P39 of Brucella abortus induces protective immunity in BALB/c mice", 《CHINESE MEDICAL JOURNAL》 * |
| NCBI: "GenBank登录号：EF173477.1", 《NCBI GENBANK》 * |
| NCBI: "GenBank登录号：EF189139.1", 《NCBI GENBANK》 * |
| 张亚丽等: "布鲁氏菌omp25重组BCG的构建及其疫苗作用检测", 《中国病原生物学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314709A (en) * | 2018-03-22 | 2018-07-24 | 吉林农业大学 | Recombinant glycosylated albumen P39 and its preparation method and application |
| CN109486846A (en) * | 2018-12-29 | 2019-03-19 | 山东农业大学 | A kind of three kinds of gene recombination plasmids of brucella, construction method and its expression and application in Escherichia coli |
| CN109486846B (en) * | 2018-12-29 | 2021-08-20 | 山东农业大学 | Three-gene recombinant plasmid of Brucella, construction method and expression and application thereof in escherichia coli |
| CN113637703A (en) * | 2021-08-06 | 2021-11-12 | 河北科技师范学院 | Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector |
| CN113637703B (en) * | 2021-08-06 | 2023-08-25 | 河北科技师范学院 | Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106834331B (en) | 2020-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101106253B1 (en) | A Echerichia coli comprising a polynucleotide encoding psicose 3-epimerase and method of producing psicose using the same | |
| CN111235080B (en) | Gene recombination escherichia coli and production method of 5-hydroxytryptamine | |
| CN106834331B (en) | rBCG for expressing fusion gene of Brucella melitensis P39 and L7/L12 and construction method thereof | |
| CN111154707B (en) | Method for producing genetically engineered escherichia coli and melatonin | |
| CN109943581A (en) | The continuously-directional evolutionary system and directed evolution method of a kind of plasmid and bacteriophage auxiliary | |
| CN103740744B (en) | A kind of zeaxanthin synthetic gene recombinant plasmid and its production and use | |
| CN105754958B (en) | A kind of mycobacteriophage that delivering autonomous light-emitting component and its application | |
| CN109295123A (en) | A kind of biological production of betaxanthin | |
| CN104278031B (en) | Promoter A regulated by xanthine as well as recombinant expression vector and application of promoter A | |
| CN106676124B (en) | rBCG for expressing ovine brucella P39 gene and construction method and application thereof | |
| CN106854654B (en) | rBCG for expressing sheep brookfield strain L7/L12 gene and construction method and application thereof | |
| CN110241098A (en) | The truncated-type high specific variant of the CRISPR nuclease SpCas9 of streptococcus pyogenes and its application | |
| CN112899211B (en) | Method for increasing yield of 2-KLG in gluconobacter oxydans | |
| CN111088267B (en) | Method for improving cell density of liquid fermentation of clostridium solvolyticum | |
| CN102586161B (en) | Mycobacterium smegmatis IL-17A and preparation method thereof | |
| CN112592877B (en) | Recombinant escherichia coli for over-expressing lsrC gene and construction method and application thereof | |
| CN114774472B (en) | Construction and application of recombinant escherichia coli resistant to pentanediamine | |
| KR101732026B1 (en) | An effective method for specific gene silencing using artificial small RNA | |
| CN113583928A (en) | Salmonella typhimurium green fluorescent strain and construction method thereof | |
| CN115141856B (en) | Recombinant clostridium and construction method and application thereof | |
| CN114150002A (en) | Light-operated gene switch and application thereof | |
| CN113755412B (en) | Genetically engineered bacterium for producing MK-7, method and application | |
| KR20220103619A (en) | Antibacterial Protein EFL200 Having Lytic Activity to Enterococcus faecium | |
| CN111254104B (en) | Preparation method of genetically engineered escherichia coli and indole-3-acetic acid | |
| CN114736310B (en) | Protein and biological material for producing circovirus type 2 virus-like particles and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |