CN109385439A - The one recombination TM4 phage library and its application for constructing nadh dehydrogenase gene family missing mycobacterium tuberculosis - Google Patents
The one recombination TM4 phage library and its application for constructing nadh dehydrogenase gene family missing mycobacterium tuberculosis Download PDFInfo
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Abstract
The recombination TM4 phage library for being used to construct nadh dehydrogenase gene family missing mycobacterium tuberculosis the invention discloses one and its application.Present invention firstly provides recombinant shuttle vector libraries needed for a building recombination TM4 phage library, the recombinant shuttle vector library includes 16 recombinant shuttle vectors, inserts the homology arm sequence of 16 member gene two sides of mycobacterium tuberculosis nadh dehydrogenase gene family respectively.The present invention is in order to facilitate nadh dehydrogenase gene functional research and vaccine development, construct a homologous recombination phage library, the library can be used for mycobacterium tuberculosis nadh dehydrogenase gene knockout, verify gene whether indispensable gene, nadh dehydrogenase knock-out bacterial strain is constructed, for BCG vaccine optimization improvement.When being knocked out using phage library of the present invention, it is specific good to knock out, and positive rate is high.
Description
Technical field
The present invention relates to tuberculosis technical fields, more particularly, to one for constructing nadh dehydrogenase gene family
Lack recombination TM4 phage library and its application of mycobacterium tuberculosis.
Background technique
Tuberculosis is caused by mycobacterium tuberculosis infection, is the premier infectious disease killer in the whole world, is seized daily
The life of more than 4500 people.There is following vision in World Health Organization to control lungy: making tuberculosis death toll than 2015 by 2035
Year reduces by 95%, so that incidence of tuberculosis was reduced by 90% than 2015 by 2035, was not faced by 2035 because of tuberculosis
Catastrophic expense is influenced family by tuberculosis.However, the sternness that treatment lungy faces Drug Resistance of Mycobacterium Tuberculosis is chosen
War, research mycobacterium tuberculosis pathogenesis, resistance mechanism and the new antituberculotic of exploitation are very urgent.But progress
It allows of no optimist always.
Mycobacterium tuberculosis nadh dehydrogenase family can be divided into two class of I type and II type.I type nadh dehydrogenase include NuoA,
It is 14 kinds of NuoB, NuoC, NuoD, NuoE, NuoF, NuoG, NuoH, NuoI, NuoJ, NuoK, NuoL, NuoM, NuoN, corresponding
Rv number be followed successively by Rv3145, Rv3146, Rv3147, Rv3148, Rv3149, Rv3150, Rv3151, Rv3152, Rv3153,
Rv3154,Rv3155,Rv3156,Rv3157,Rv3158；II type nadh dehydrogenase includes two kinds of NdhA, Ndh, and corresponding Rv is compiled
Number be respectively Rv0392c, Rv1854c.Nadh dehydrogenase plays weight in mycobacterium tuberculosis electron transport chain and energetic supersession
Function is wanted, is the important target spot of antituberculotic exploitation.Ndh, NdhA protein amino acid sequence have high similarity, can be by benzene
Thiazine inhibits, but the two significance level in mycobacterium tuberculosis is not identical, tuberculosis branch after knocking out researches show that NdhA
Bacillus not will receive too big influence and M. tuberculosis growth, resistance to Oxdative stress and virulence are all substantially change after Ndh knockout.
What NuoG albumen was studied in this 14 albumen of NuoA-NuoN is more, it has now been found that NuoG can influence mycobacterium tuberculosis sense
The release of the autophagy, inflammatory factor of cell and Apoptosis, influence M. tuberculosis growth, virulence etc. after dye macrophage,
And other 13 albumen researchs are comparatively less, concrete function need to be furtherd investigate.
M. tuberculosis genes knockout is the common tool of gene studies, can be used for studying gene function, building is struck
Except bacterial strain and for vaccine research and development etc..BCG (Mycobacterium bovis Bacillus Calmette-Gu é rin) is mesh
Preceding unique general prevention vaccine lungy knocks out gene further on this basis come to improve vaccine be that researcher is normal
Means are expected to improve epidemic disease than if any research, by after BCG NuoG gene knockout, discovery bacterial strain inducing cellular immunity is stronger
Whether seedling safety and preventive effect, Ndh, NdhA and other 13 kinds of Nuo albumen can be used as vaccine and knock out target spot or unknown.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide one for constructing nadh dehydrogenase gene man
Race lacks recombination TM4 phage library and its application of mycobacterium tuberculosis.
Mycobacterium tuberculosis nadh dehydrogenase gene man is knocked out for constructing the first purpose of the invention is to provide one
Recombinant shuttle vector library needed for race's phage library.
A second object of the present invention is to provide primers needed for the building recombinant shuttle vector library to combine.
Third object of the present invention is to provide one for knocking out mycobacterium tuberculosis nadh dehydrogenase gene family
Phage library.
Fourth object of the present invention is to provide the recombinant shuttle vector library and knocks out mycobacterium tuberculosis in preparation
Application in nadh dehydrogenase gene family phage library.
Fifth object of the present invention is to provide the phage libraries to knock out mycobacterium tuberculosis nadh dehydrogenase base
Because of the application in family.
6th purpose of the invention is the phage library in building nadh dehydrogenase gene family missing tuberculosis point
Application in branch bacillus.
To achieve the goals above, the present invention is achieved by the following technical programs:
One for recombination needed for constructing knockout mycobacterium tuberculosis nadh dehydrogenase gene family phage library
Shuttle vector library, the recombinant shuttle vector library include 16 recombinant shuttle vectors, insert mycobacterium tuberculosis respectively
The left and right homology arm sequence of 16 member gene two sides of nadh dehydrogenase gene family, wherein 16 member genes are
Rv0392c、Rv1854c、Rv3145、Rv3146、Rv3147、Rv3148、Rv3149、Rv3150、Rv3151、Rv3152、
Rv3153、Rv3154、Rv3155、Rv3156、Rv3157、Rv3158。
Preferably, the left and right homology arm sequence needs of the gene two sides for 16 members being inserted into 16 recombinant shuttle vectors
Meet the following conditions: the phage library using the recombinant shuttle vector library construction is for knocking out mycobacterium tuberculosis NADH
When dehydrogenase gene family, Rv0392c, Rv1854c of the mycobacterium tuberculosis knocked out, Rv3145, Rv3146, Rv3147,
Rv3148、Rv3149、Rv3150、Rv3151、Rv3152、Rv3153、Rv3154、Rv3155、Rv3156、Rv3157、Rv3158
The genetic fragment successively segment as shown in SEQ.ID.NO.65~80.
Specifically, the segment nucleotide sequence that Rv0392c is knocked out is as shown in SEQ.ID.NO:65；
The segment nucleotide sequence that Rv1854c is knocked out is as shown in SEQ.ID.NO:66；
The segment nucleotide sequence that Rv3145 is knocked out is as shown in SEQ.ID.NO:67；
The segment nucleotide sequence that Rv3146 is knocked out is as shown in SEQ.ID.NO:68；
The segment nucleotide sequence that Rv3147 is knocked out is as shown in SEQ.ID.NO:69；
The segment nucleotide sequence that Rv3148 is knocked out is as shown in SEQ.ID.NO:70；
The segment nucleotide sequence that Rv3149 is knocked out is as shown in SEQ.ID.NO:71；
The segment nucleotide sequence that Rv3150 is knocked out is as shown in SEQ.ID.NO:72；
The segment nucleotide sequence that Rv3151 is knocked out is as shown in SEQ.ID.NO:73；
The segment nucleotide sequence that Rv3152 is knocked out is as shown in SEQ.ID.NO:74；
The segment nucleotide sequence that Rv3153 is knocked out is as shown in SEQ.ID.NO:75；
The segment nucleotide sequence that Rv3154 is knocked out is as shown in SEQ.ID.NO:76；
The segment nucleotide sequence that Rv3155 is knocked out is as shown in SEQ.ID.NO:77；
The segment nucleotide sequence that Rv3156 is knocked out is as shown in SEQ.ID.NO:78；
The segment nucleotide sequence that Rv3157 is knocked out is as shown in SEQ.ID.NO:79；
The segment nucleotide sequence that Rv3158 is knocked out is as shown in SEQ.ID.NO:80.
Preferably, building process is that the left and right homology arm sequence of 16 member gene two sides is inserted into p0004S carrier
In, obtain positive p0004S recombinant plasmid；Digestion shuttle vector and p0004S recombinant plasmid, connection are distinguished with restriction enzyme
Segment obtains recombinant shuttle vector.
Preferably, the shuttle vector is phAE159.
The primer combination in one group of recombinant shuttle vector library for constructing any description above, includes 32 pairs of primers, core
Nucleotide sequence is as shown in SEQ.ID.NO.1~64.
The corresponding relationship of primer and amplified fragments is shown in Table 1.
Table 1:
Primer combination described above knocks out mycobacterium tuberculosis nadh dehydrogenase gene family phage library in preparation
Application in required recombinant shuttle vector library.
Further, one for knocking out the phage library of mycobacterium tuberculosis nadh dehydrogenase gene family, described to bite
Phage library contains 16 kinds of bacteriophages, respectively 16 containing any description above recombinant shuttle vector, also belongs to of the invention
Protection scope.
Preferably, the bacteriophage is TM4 bacteriophage.
In addition, application as described below also should be within protection scope of the present invention:
Any description above recombinant shuttle vector library knocks out mycobacterium tuberculosis nadh dehydrogenase gene family in preparation
With the application in phage library；
Phage library described above is knocking out the application in mycobacterium tuberculosis nadh dehydrogenase gene family；
Application of the phage library described above in building nadh dehydrogenase gene family missing mycobacterium tuberculosis.
In addition, the present invention also provides a kind of building recombinant shuttle vectors as a kind of selectable preferred embodiment
Library (recombinant shuttle vector library needed for knocking out mycobacterium tuberculosis nadh dehydrogenase gene family phage library)
Method, comprising the following steps:
S1. design primer, primer nucleotide sequences information is as shown in SEQ.ID.NO:1~64；
S2. using Mycobacterium tuberculosis H37Rv genome as gene template, PCR is carried out with above-mentioned primer, amplification is intended to strike respectively
Except the left and right homology arm of gene；
S3. by amplification to left and right homology arm connect in p0004S carrier, obtain positive p0004S recombinant plasmid；
S4. digestion shuttle vector and p0004S recombinant plasmid, junction fragment are distinguished with restriction enzyme, obtain recombination and wear
Shuttle carrier.
Preferably, PCR amplification system are as follows: 10 × KOD Plus buffer, 5 μ l, 2.5mM dNTP 4 μ l, MgSO4 2μ
4 μ l of l, DMSO, primers F (10 μM) 2 μ l, primer R (10 μM) 2 μ l, mycobacterium tuberculosis gene group DNA0.5 μ l, KOD
Enzyme 0.3 μ l, ddH2O 30.2μl。
Pcr amplification reaction condition are as follows: 95 DEG C of 5min, 1 circulation；95 DEG C of 30s, 60 DEG C of 30s, 68 DEG C of 1min, 5 are followed
Ring；95 DEG C of 30s, 62 DEG C of 30s, 68 DEG C of 1min, 25 circulations；68 DEG C of 10min, 1 circulation；16 DEG C of preservations.
Preferably, shuttle vector phAE159.
Meanwhile the building method for knocking out M. tuberculosis genes phage library, comprising the following steps:
S1. mycobacterium smegmatis competent cell is prepared；
S2. the recombinant shuttle vector electricity built is gone into mycobacterium smegmatis, is cultivated；
S3. plaque is selected, mycobacterium smegmatis is infected, collects phage splitting liquid.
In addition, the method for knocking out mycobacterium tuberculosis nadh dehydrogenase gene family using the phage library, including
Following steps:
S1. mycobacterium tuberculosis is cultivated；
S2. bacteriophage is added, infects mycobacterium tuberculosis；
S3. metainfective mycobacterium tuberculosis is coated on hygromycin resistance culture medium；
S4. picking individual colonies extract genome, carry out PCR and knock out verifying.
Wherein, the present invention relates to gene Knockout principle: bacteriophage mediate homologous recombination.DNA homologous recombination
Refer to the hereditary information recombination occurred in the presence of homologous recombination GAP-associated protein GAP between the DNA molecular with homologous sequence, according to
This principle can specifically knock out gene.Homology arm is chosen in gene to be knocked out two sides, two sides homology arm, which is cloned into, to be bitten
Shuttle plasmid is formed behind thallus package carrier screening-gene two sides, shuttle plasmid electricity can be formed after going in mycobacterium smegmatis and be bitten
Thallus, the phage-infect of generation, due to the presence of homology arm, can be such that plasmid sends out with to knock-out bacterial strain DNA after knock-out bacterial strain
Raw homologous recombination makes screening-gene replacement target gene to achieve the purpose that gene knockout, and knocking out successful bacterial strain can lead to
Screening-gene is crossed to be screened.
Its gene knockout experiment process (Fig. 1): building Homo~logous exchange site (AES) first is then integrated into tuberculosis
Mycobacteriophage genome obtains phasmid (phasmid)；Phasmid is imported into mycobacterium smegmatis, is integrated with
The recombinant phage in Homo~logous exchange site obtains the recombinant phage of high titre through amplification in vitro, carries out to mycobacterium tuberculosis
Transfection；Mycobacterium tuberculosis after transfection is applied to the solid medium of hygromycin resistance, 37 DEG C are cultivated 4-5 weeks, picking list
Clone, is verified by modes such as PCR.
Compared with prior art, the invention has the following beneficial effects:
The present invention constructs a homologous recombination phagocytosis in order to facilitate nadh dehydrogenase gene functional research and vaccine development
Body library, the library can be used for mycobacterium tuberculosis nadh dehydrogenase gene knockout, verifying gene whether indispensable gene, building
Nadh dehydrogenase knock-out bacterial strain, for BCG vaccine optimization improvement.When being knocked out using phage library of the present invention, strike
Except specificity is good, positive rate is high.
Detailed description of the invention
Fig. 1 is that the homologous recombination mediated using bacteriophage carries out the principle of gene knockout.
Fig. 2 is the position view for constructing TM4 bacteriophage and knocking out mycobacterium tuberculosis nadh dehydrogenase gene family.
Fig. 3 is Mycobacterium tuberculosis H37Rv Rv3154 gene knockout gene schematic diagram.
Fig. 4 is the bacterium colony after Mycobacterium tuberculosis H37Rv Rv3154 gene knockout.
Fig. 5 is PCR verification result after Mycobacterium tuberculosis H37Rv Rv3154 gene knockout.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
The design of the knockout primer of embodiment 1
According to mycobacterium tuberculosis nadh dehydrogenase family gene (Rv0392c, Rv1854c, Rv3145, Rv3146,
Rv3147、Rv3148、Rv3149、Rv3150、Rv3151、Rv3152、Rv3153、Rv3154、Rv3155、Rv3156、Rv3157、
Rv3158) the position in mycobacterium tuberculosis gene group selects suitable fragment length on selecting suitable aim sequence
It as left and right homology arm Position Design primer (600-1000bp or so) and is synthesized, further according to amplification and knockout effect
Filter out following knockout primer.
Rv0392c:
Left arm upstream primer Rv0392cLF:GTCGGTGGCATACAACTGGG (SEQ ID NO:1),
Left arm downstream primer Rv0392cLR:GGTGGGTCGTTGTCTTGGAG (SEQ ID NO:2),
Right arm upstream primer Rv0392cRL:CCTGGTCTACCTGGTCGGCTAT (SEQ ID NO:3),
Right arm downstream primer Rv0392cRR:AAGTGGTGCTGGACAACG (SEQ ID NO:4)；
Rv1854c:
Left arm upstream primer Rv1854cLF:ATACCTACCGCAGCCCGACCCT (SEQ ID NO:5),
Left arm downstream primer Rv1854cLR:TAGCCCGCCGAACCCAGAT (SEQ ID NO:6),
Right arm upstream primer Rv1854cRL:TCACCGACCAGCAGGCATTT (SEQ ID NO:7),
Right arm downstream primer Rv1854cRR:CGCCGATGGAGTCTAGTGTTCT (SEQ ID NO:8)；
Rv3145:
Left arm upstream primer Rv3145LF:CAAATTCGTCATCACCAGCT (SEQ ID NO:9),
Left arm downstream primer Rv3145LR:GATGGGTATGTAGACGTTCA (SEQ ID NO:10),
Right arm upstream primer Rv3145RL:GTGTTCCTCTACCCGTGGG (SEQ ID NO:11),
Right arm downstream primer Rv3145RR:AATGGCATCTGCTGAATCTTTT (SEQ ID NO:12)；
Rv3146:
Left arm upstream primer Rv3146LF:GTCCTTCCGCTTCGCTGCTC (SEQ ID NO:13),
Left arm downstream primer Rv3146LR:ATAGCCCGCCACCTTCTCG (SEQ ID NO:14),
Right arm upstream primer Rv3146RL:ATGCTGCTGCACGCAATCC (SEQ ID NO:15),
Right arm downstream primer Rv3146RR:GCCAGTCGTTGGTCGGATAG (SEQ ID NO:16)；
Rv3147:
Left arm upstream primer Rv3147LF:GCGTGGGACTGGAAGAACA (SEQ ID NO:17),
Left arm downstream primer Rv3147LR:CCTTCCTGGGCGTCTTGG (SEQ ID NO:18),
Right arm upstream primer Rv3147RL:TCTGCGCCGGACAGTGAT (SEQ ID NO:19),
Right arm downstream primer Rv3147RR:GCGACCAAATGCGACGAG (SEQ ID NO:20)；
Rv3148:
Left arm upstream primer Rv3148LF:CGAACCAAGACGCCCAGGAA (SEQ ID NO:21),
Left arm downstream primer Rv3148LR:GTCCACGACCTGCTGCCA (SEQ ID NO:22),
Right arm upstream primer Rv3148RL:TCAAACTGGTCACCGAGGGC (SEQ ID NO:23),
Right arm downstream primer Rv3148RR:CGGAGGTGGTTTCGTCGTG (SEQ ID NO:24)；
Rv3149:
Left arm upstream primer Rv3149LF:GCAGGACTTACCGCCCAACG (SEQ ID NO:25),
Left arm downstream primer Rv3149LR:GCGCCCTCGACGACAAACT (SEQ ID NO:26),
Right arm upstream primer Rv3149RL:GGTGGTGATGGTCAACTGGG (SEQ ID NO:27),
Right arm downstream primer Rv3149RR:GGTCGGGCAGCCATACAGA (SEQ ID NO:28)；
Rv3150:
Left arm upstream primer Rv3150LF:TGGTGCAGGGCGAGGATT (SEQ ID NO:29),
Left arm downstream primer Rv3150LR:TCGTGGCGTTGATAAGTGGC (SEQ ID NO:30),
Right arm upstream primer Rv3150RL:GGCACCTTCTGGCTGGATAA (SEQ ID NO:31),
Right arm downstream primer Rv3150RR:TGTCGCACATCGGGCAAT (SEQ ID NO:32)；
Rv3151:
Left arm upstream primer Rv3151LF:TTCACCCTGTATTCGCTGTCC (SEQ ID NO:33),
Left arm downstream primer Rv3151LR:TTGGGGACGCTGATTTCG (SEQ ID NO:34),
Right arm upstream primer Rv3151RL:GCAGGACGGCGAACCATA (SEQ ID NO:35),
Right arm downstream primer Rv3151RR:CAGTCCCAGGATGAACAGCAC (SEQ ID NO:36)；
Rv3152:
Left arm upstream primer Rv3152LF:CGTTCGTCAACTGGGAGGG (SEQ ID NO:37),
Left arm downstream primer Rv3152LR:CCACCAGCACCGTCAGCAT (SEQ ID NO:38),
Right arm upstream primer Rv3152RL:CGCTCACTACGCAACCAGG (SEQ ID NO:39),
Right arm downstream primer Rv3152RR:TGACATCTGAAGCCAGCACC (SEQ ID NO:40)；
Rv3153:
Left arm upstream primer Rv3153LF:TCCTGTTGTTGCCGTCATTC (SEQ ID NO:41),
Left arm downstream primer Rv3153LR:CGGCGTCCAGTAACTTAGTCC (SEQ ID NO:42),
Right arm upstream primer Rv3153RL:GCGCCGACCTGATCTACGAG (SEQ ID NO:43),
Right arm downstream primer Rv3153RR:GACGGCGGCGGTAATCAAC (SEQ ID NO:44)；
Rv3154:
Left arm upstream primer Rv3154LF:AAGGCACCAGCCCTGAAC (SEQ ID NO:45),
Left arm downstream primer Rv3154LR GCACTGAGCACCCAGAACAT (SEQ ID NO:46),
Right arm upstream primer Rv3154RL:CGAACTCTCCCAGGAACG (SEQ ID NO:47),
Right arm downstream primer Rv3154RR:GCACGAAGCACATGGACA (SEQ ID NO:48)；
Rv3155:
Left arm upstream primer Rv3155LF:AGCGATGTTTCTGGCGATGA (SEQ ID NO:49),
Left arm downstream primer Rv3155LR:GATAAAGGTAGTTGGCCGGATT (SEQ ID NO:50),
Right arm upstream primer Rv3155RL:GGCCGCCTGCGAAGTGGT (SEQ ID NO:51),
Right arm downstream primer Rv3155RR:GGTGCCGAAAGTGCTAAACG (SEQ ID NO:52)；
Rv3156:
Left arm upstream primer Rv3156LF:TGTCGGGTTCGGCGTTCT (SEQ ID NO:53),
Left arm downstream primer Rv3156LR:CCAGCAGCCAGGTGTAGTGAG (SEQ ID NO:54),
Right arm upstream primer Rv3156RL:GTGGTCGCCGTCGGTATC (SEQ ID NO:55),
Right arm downstream primer Rv3156RR:GGACAGGTCGTCAGCATCG (SEQ ID NO:56)；
Rv3157:
Left arm upstream primer Rv3157LF:CGCGGGCTTCTTCTCCAA (SEQ ID NO:57),
Left arm downstream primer Rv3157LR:CGGCACCAGCCAGAGCAC (SEQ ID NO:58),
Right arm upstream primer Rv3157RL:TGGTTCTGCTGGGCACTTTC (SEQ ID NO:59),
Right arm downstream primer Rv3157RR:TCTGGCGTTGCGGACTCAC (SEQ ID NO:60)；
Rv3158:
Left arm upstream primer Rv3158LF:GATGGCGGTGATGGACAAG (SEQ ID NO:61),
Left arm downstream primer Rv3158LR:ATCGGAGCGAGCAGGAAG (SEQ ID NO:62),
Right arm upstream primer Rv3158RL:ATTGCGGTATGCACGGTAGT (SEQ ID NO:63),
Right arm downstream primer Rv3158RR:ACATCGGTTTCTTCAACTCTGG (SEQ ID NO:64).
The knockout of nadh dehydrogenase gene family is carried out according to the above primer,
The segment nucleotide sequence that Rv0392c is knocked out is as shown in SEQ.ID.NO:65；
The segment nucleotide sequence that Rv1854c is knocked out is as shown in SEQ.ID.NO:66；
The segment nucleotide sequence that Rv3145 is knocked out is as shown in SEQ.ID.NO:67；
The segment nucleotide sequence that Rv3146 is knocked out is as shown in SEQ.ID.NO:68；
The segment nucleotide sequence that Rv3147 is knocked out is as shown in SEQ.ID.NO:69；
The segment nucleotide sequence that Rv3148 is knocked out is as shown in SEQ.ID.NO:70；
The segment nucleotide sequence that Rv3149 is knocked out is as shown in SEQ.ID.NO:71；
The segment nucleotide sequence that Rv3150 is knocked out is as shown in SEQ.ID.NO:72；
The segment nucleotide sequence that Rv3151 is knocked out is as shown in SEQ.ID.NO:73；
The segment nucleotide sequence that Rv3152 is knocked out is as shown in SEQ.ID.NO:74；
The segment nucleotide sequence that Rv3153 is knocked out is as shown in SEQ.ID.NO:75；
The segment nucleotide sequence that Rv3154 is knocked out is as shown in SEQ.ID.NO:76；
The segment nucleotide sequence that Rv3155 is knocked out is as shown in SEQ.ID.NO:77；
The segment nucleotide sequence that Rv3156 is knocked out is as shown in SEQ.ID.NO:78；
The segment nucleotide sequence that Rv3157 is knocked out is as shown in SEQ.ID.NO:79；
The segment nucleotide sequence that Rv3158 is knocked out is as shown in SEQ.ID.NO:80.
Recombination needed for the building of embodiment 2 knocks out mycobacterium tuberculosis nadh dehydrogenase gene family phage library is worn
Shuttle vector library
It is combined using the primer in embodiment 1, building recombinant shuttle vector library (knocks out mycobacterium tuberculosis NADH dehydrogenation
Recombinant shuttle vector library needed for enzyme gene family phage library).
1, using Mycobacterium tuberculosis H37Rv as template, to use the primer sets in embodiment 1 to be combined into primer and increase at 5 ' ends
Add corresponding restriction enzyme site, be PCR with KOD high fidelity enzyme, crosses amplification respective segments respectively.
Wherein, the amplification of left and right arms is carried out respectively according to following system:
KOD PCR reaction condition are as follows:
After the completion of PCR, 1% agarose gel electrophoresis does glue recycling with Tiangeng plastic recovery kit.Measure PCR product glue
Recycle concentration.
2, the recovery product both ends of second of PCR add restriction enzyme site, use respectively with p0004S carrier corresponding restricted
Restriction endonuclease carries out digestion, carries out agarose gel electrophoresis, and recycles required segment.Left and right arms PCR product after the recovery with
The connection of p0004S carrier segments is configured to p0004S recombinant plasmid.It extracts plasmid and sends out sequencing, just whether verifying gene left and right arms
It is really inserted into p0004S carrier, obtains positive p0004S recombinant plasmid.
3, p0004S recombinant plasmid is connected to shuttle vector phAEB159 with the product after PacI digestion, is built into recombination
PhAEB159 shuttle plasmid.
As a result: after homology arm connect conversion E. coli competent with shuttle vector phAEB159, the single colonie selected is passed through
Sequencing is crossed, homology arm sequence accuracy is demonstrated, successfully constructs shuttle vector library.
The building of embodiment 3 knocks out mycobacterium tuberculosis nadh dehydrogenase gene family phage library
The shuttle vector electricity obtained in embodiment 2 is gone in mycobacterium smegmatis, the recombination for being integrated with homology arm is obtained
Bacteriophage obtains the recombinant phage of high titre through amplification in vitro.
Concrete operations are as follows:
1, mycobacterium smegmatis competent cell is prepared；
2, the shuttle vector electricity that embodiment 2 obtains is gone in mycobacterium smegmatis competent cell；
3, the mycobacterium smegmatis after electricity turns is applied on the resistance screening culture medium containing hygromycin；
4, picking plaque infects the mycobacterium smegmatis of fresh cultured, collects phage splitting liquid.
As a result: plaque has successfully been grown, after picking plaque infects mycobacterium smegmatis, from the phage splitting of collection
The bacteriophage of high titre is obtained in liquid.
Embodiment 4 knocks out mycobacterium tuberculosis nadh dehydrogenase gene family using phage library
1, concrete operations are as follows:
By taking mycobacterium tuberculosis Rv3154 gene knockout as an example: collect 10ml mycobacterium tuberculosis to be knocked out, after resuspension and
Bacteriophage mixes, and is centrifuged afterwards for 24 hours, thallus is applied on hygromycin resistance culture medium, 37 DEG C are incubated overnight.Picking single colonie,
Genome is collected after shaking bacterium, PCR verifying knocks out effect.
It knocks out primer and PCR system, PCR condition is as follows:
Rv3154 knocks out verifying primer:
LYZFP-F:5'-CGCGATCATCCGCTCACT-3'；
LYZRP-R:5'-GTGGACCTCGACGACCCTAG-3'；
RYZFP-F:5'-TGGATCTCTCCGGCTTCACC-3'；
RYZRP-R:5'-GCCGACCGAATAGATGTGAA-3'.
KOD PCR system (50 μ l system) are as follows:
KOD PCR condition:
Step 1:95 DEG C 5min；
Step 2:95 DEG C 30s；
Step 3:60 DEG C 30s；
Step 4:68 DEG C 1min (goto step 2,5cycles)；
Step 5:95 DEG C 30s；
Step 6:62 DEG C 30s；
Step 7:68 DEG C 1min (goto step 5,25cycles)；
Step 8:68 DEG C 10min；
Step 9:16 DEG C ∞.
2, result: single colonie has successfully been grown, has proved to knock out successfully after PCR verifying.Wherein mycobacterium tuberculosis Rv3154
Gene knockout schematic diagram is shown in that Fig. 3, Rv3154c gene knockout bacterium colony are shown in that Fig. 4, Rv3154 gene knockout PCR verification result are shown in Fig. 5.
It is knocked out using other genes of the method for the invention to nadh dehydrogenase gene family and is successfully grown
Single colonie, and prove to knock out successfully.
As a result illustrate, successfully construct nadh dehydrogenase gene family knockout TM4 phage library, tuberculosis point can be used for
Branch bacillus nadh dehydrogenase gene family knocks out.
Sequence table
<110>Shanghai Jing Nuo Biotechnology Co., Ltd
<120>one for construct nadh dehydrogenase gene family missing mycobacterium tuberculosis recombination TM4 phage library and
It is applied
<160> 80
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gtcggtggca tacaactggg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtgggtcgt tgtcttggag 20
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cctggtctac ctggtcggct at 22
<210> 4
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aagtggtgct ggacaacg 18
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atacctaccg cagcccgacc ct 22
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tagcccgccg aacccagat 19
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tcaccgacca gcaggcattt 20
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cgccgatgga gtctagtgtt ct 22
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
caaattcgtc atcaccagct 20
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gatgggtatg tagacgttca 20
<210> 11
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gtgttcctct acccgtggg 19
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aatggcatct gctgaatctt tt 22
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gtccttccgc ttcgctgctc 20
<210> 14
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atagcccgcc accttctcg 19
<210> 15
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
atgctgctgc acgcaatcc 19
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gccagtcgtt ggtcggatag 20
<210> 17
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gcgtgggact ggaagaaca 19
<210> 18
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ccttcctggg cgtcttgg 18
<210> 19
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
tctgcgccgg acagtgat 18
<210> 20
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gcgaccaaat gcgacgag 18
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
cgaaccaaga cgcccaggaa 20
<210> 22
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gtccacgacc tgctgcca 18
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tcaaactggt caccgagggc 20
<210> 24
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
cggaggtggt ttcgtcgtg 19
<210> 25
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gcaggactta ccgcccaacg 20
<210> 26
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
gcgccctcga cgacaaact 19
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ggtggtgatg gtcaactggg 20
<210> 28
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ggtcgggcag ccatacaga 19
<210> 29
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
tggtgcaggg cgaggatt 18
<210> 30
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
tcgtggcgtt gataagtggc 20
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ggcaccttct ggctggataa 20
<210> 32
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tgtcgcacat cgggcaat 18
<210> 33
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
ttcaccctgt attcgctgtc c 21
<210> 34
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
ttggggacgc tgatttcg 18
<210> 35
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gcaggacggc gaaccata 18
<210> 36
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
cagtcccagg atgaacagca c 21
<210> 37
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
cgttcgtcaa ctgggaggg 19
<210> 38
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ccaccagcac cgtcagcat 19
<210> 39
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
cgctcactac gcaaccagg 19
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
tgacatctga agccagcacc 20
<210> 41
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
tcctgttgtt gccgtcattc 20
<210> 42
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
cggcgtccag taacttagtc c 21
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
gcgccgacct gatctacgag 20
<210> 44
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gacggcggcg gtaatcaac 19
<210> 45
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
aaggcaccag ccctgaac 18
<210> 46
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
gcactgagca cccagaacat 20
<210> 47
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
cgaactctcc caggaacg 18
<210> 48
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
gcacgaagca catggaca 18
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
agcgatgttt ctggcgatga 20
<210> 50
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
gataaaggta gttggccgga tt 22
<210> 51
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
ggccgcctgc gaagtggt 18
<210> 52
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
ggtgccgaaa gtgctaaacg 20
<210> 53
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
tgtcgggttc ggcgttct 18
<210> 54
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
ccagcagcca ggtgtagtga g 21
<210> 55
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
gtggtcgccg tcggtatc 18
<210> 56
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
ggacaggtcg tcagcatcg 19
<210> 57
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
cgcgggcttc ttctccaa 18
<210> 58
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
cggcaccagc cagagcac 18
<210> 59
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
tggttctgct gggcactttc 20
<210> 60
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
tctggcgttg cggactcac 19
<210> 61
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
gatggcggtg atggacaag 19
<210> 62
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
atcggagcga gcaggaag 18
<210> 63
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
attgcggtat gcacggtagt 20
<210> 64
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
acatcggttt cttcaactct gg 22
<210> 65
<211> 1057
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
tgttccagcc gctgctgtat caagtggcca ccgggatctt gtccgagggc gacattgccc 60
cgaccacccg gctgatcctg cgccggcaaa agaacgtccg ggtgttgctg ggcgaggtca 120
acgcgatcga cctgaaagcg cagacggtca cgtcgaaatt gatggacatg accacggtga 180
cgccgtacga cagcctcatc gtggccgccg gcgcacagca gtcctacttc ggcaacgacg 240
aattcgccac cttcgcgccc ggaatgaaga ccatcgacga cgcgctggag ctgcgcggcc 300
gcatcctggg cgcgttcgag gccgccgagg tcagcaccga ccatgccgaa cgggagcggc 360
gcctgacgtt cgtcgtcgtc ggcgctgggc cgaccggcgt cgaggtggct gggcagatcg 420
tcgagctcgc cgagcgcacc ctggcaggcg cgtttaggac catcacgccc agtgagtgcc 480
gggtgatcct gctcgacgcc gcacccgcgg tgttgccgcc gatgggtcca aagctgggtc 540
tcaaggcaca acggcggctg gaaaagatgg acgtcgaggt tcaactcaac gcgatggtga 600
ccgcggtcga ctacaaaggc atcaccatca aggaaaagga cggcggcgaa cgccgcatcg 660
aatgcgcgtg caaggtttgg gcggccggcg tggcggccag cccgctgggc aagatgatcg 720
ccgagggatc cgacggaacc gaaatcgacc gggccggaag ggtgatcgtg gaacccgatc 780
tcaccgtcaa gggacatccg aacgtcttcg tagtcggcga tctgatgttc gtgcccggcg 840
tacccggggt ggctcagggc gcgatccagg gggcccgata cgccaccacg gtgatcaaac 900
acatggtcaa gggcaatgac gacccagcca atcgcaagcc gttccattac ttcaacaagg 960
gcagcatggc gacgatctcc cgccacagcg ccgtcgcgca ggtcggcaag ctggagtttg 1020
ccgggtactt cgcctggctg gcgtggctgg tgctgca 1057
<210> 66
<211> 1210
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
aacgcggcaa agaagctcaa gcgggccgac gttgacatca agctgatcgc gcgcaccacc 60
catcacctgt tccagccgct gctgtaccaa gtggccaccg ggattatctc cgagggagaa 120
atcgctccgc cgacccgggt cgtgctgcgt aagcagcgca atgtccaggt actgttgggc 180
aacgtcaccc acatcgacct ggccgggcag tgcgtcgtct cggaattgct cggtcacacc 240
taccaaaccc cctacgacag cctgatcgtc gccgcgggtg ctggccagtc ttatttcggc 300
aacgaccatt tcgccgaatt cgcacccggc atgaagtcca tcgacgacgc gttggagttg 360
cgtggccgca tattgagcgc tttcgagcaa gccgaacggt ccagcgatcc ggaacggcgg 420
gccaagctac tgacattcac cgttgtcggg gctggcccca ccggtgttga aatggccgga 480
cagatcgccg agctggccga gcacacgttg aagggcgcat tccggcacat cgactcgacc 540
aaggcgcggg tgattctgct tgacgccgcc ccggcggtgc tgccaccgat gggcgcaaag 600
ctcggtcagc gggcggctgc ccggttgcag aagctgggcg tggaaatcca gctgggtgcg 660
atggtcaccg acgtcgaccg caacggcatc accgtcaagg actccgacgg caccgtccgg 720
cgcatcgagt cggcctgcaa ggtctggtcg gccggggttt cggccagtcg gttgggcagg 780
gaccttgccg agcaatcacg ggttgagctc gaccgggccg gccgggtcca agtgctgccc 840
gacctgtcca ttcccgggta cccgaacgtg ttcgtggtgg gcgatatggc cgctgtggag 900
ggtgtgccgg gtgtggcgca gggcgccatc cagggggcga aatacgtcgc cagcacgatc 960
aaggccgaac tggccggcgc caacccggcg gagcgtgagc cattccagta cttcgacaag 1020
ggatcgatgg ccacggtttc gaggttttcg gcggtggcca agatcggtcc cgttgagttc 1080
agcggcttta tcgcctggct gatttggctg gtgctgcacc tggcgtacct gatcgggttc 1140
aagaccaaga tcaccactct gctgtcgtgg acggtgactt tcctcagtac tcgccgcggc 1200
cagctgacca 1210
<210> 67
<211> 231
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
ctggtactgg cggcgctggc cgccgccttc gccgtggtgt cggtggtgat cgcgagcctg 60
gtcggcccgt cgcggttcaa ccggtcaaag caggccgcct acgaatgcgg gatcgagccc 120
gctagcactg gagccagaac ctccattggc cccggcgcgg cgagcgggca gcggttcccc 180
atcaagtact acctgaccgc gatgttgttc atcgtcttcg acatcgaaat t 231
<210> 68
<211> 345
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
gtccgcaaaa actccctgtg gccggcaaca ttcggattgg cgtgctgtgc gatcgagatg 60
atggcgaccg cgggaccaag gtttgacatt gcgcggttcg ggatggaacg gttctcggcc 120
acgccgcggc aggcagatct gatgatcgtg gcgggccggg tcagccagaa gatggcgccg 180
gtactgcgcc agatctatga ccagatggcg gagccgaaat gggttctggc catgggtgtg 240
tgcgcctcgt caggtgggat gttcaacaac tatgcgatcg tgcagggcgt ggatcatgtt 300
gttccggtcg acatctacct acccggctgc ccgccgcgcc cggag 345
<210> 69
<211> 430
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ccgcccggac tcccccaccg cggaggtggt cgacgttcgc cgcggcatgt tcggcgtctc 60
gggcaccggt gacacctccg gttacggacg gttggtgcgc caagtcgtcc tccctggcag 120
cagcccccgg ccctacggcg gctacttcga cgatatcgtc gaccggctgg ccgaggcact 180
gcggcacgag cgcgtcgaat tcgaggacgc cgtcgagaaa gtcgtggtct accgcgatga 240
actgaccctg cacgtccgcc gggatctact gccgcgggtc gcccagcggc tgcgcgacga 300
acccgaattg cgattcgagc tgtgtcttgg ggtgagcggg gtgcactacc cgcacgagac 360
gggtcgggag ctgcatgccg tctacccgct gcagtcgatc acccacaacc gtcgcctccg 420
gttggaagtg 430
<210> 70
<211> 991
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
gccgcgcgca gcgcggatcc cggtgaacgc atcgtcgtca acatggggcc ccagcacccg 60
tctacccacg gggtgttgcg gttaatcctg gagatcgagg gcgaaacagt cgtcgaagcc 120
cggtgcggaa tcggctacct gcacaccgga atcgagaaga acctcgaata ccggtactgg 180
acccagggcg tcaccttcgt gacccgaatg gattacctgt caccgttttt caacgaaacc 240
gcctactgcc tcggcgtgga gaagctgctc ggcatcaccg atgagatacc cgagcgggtc 300
aacgtcatcc gcgtgctgat gatggagctc aaccggatct cgtcgcattt ggtcgcattg 360
gcgaccgggg gcatggaatt gggcgccatg actccgatgt tcgtcggctt ccgggcacgc 420
gagatcgtgc tcacgctgtt cgaaaagatc accggtttgc ggatgaacag cgcctacatc 480
cgacccggcg gcgtggcgca ggacttaccg cccaacgcgg ccaccgaaat cgcggaagca 540
ctcaagcagt tgcgccaacc actgcgcgaa atgggcgagc tgctcaacga aaacgccatc 600
tggaaggccc gcacccaggg cgtcggatac ctggatctga ccggatgcat ggcactgggc 660
atcaccggcc cgatactgcg ttccactggg ttgccccacg acctgcggaa aagcgagccc 720
tactgcggat accagcacta tgaattcgat gtgatcaccg acgacagctg tgatgcctac 780
gggcgctaca tgattcgcgt caaagagatg tgggagtcga tgaagatcgt ggagcagtgt 840
ctggacaagt tacgacccgg cccgaccatg atctccgatc gcaagctcgc ctggccggcc 900
gacctgcagg tggggcccga cggcctgggc aactcaccca agcacatcgc caaaatcatg 960
ggctcctcga tggaagcgct gatccaccac t 991
<210> 71
<211> 405
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
tccgcggtcg tatccgccgg acgtactggc gcggctggag gtcgacgcca aggagatcat 60
cggccgctat cccgacaggc gctcggcgct gttgccgttg ctgcacctgg tgcagggcga 120
ggattcctac ctgacgccgg cgggtttgcg gttctgcgcc gatcaactcg ggctgaccgg 180
ggccgaggtg tcggcggtgg ccagcttcta caccatgtac cgccggcgcc ccaccggcga 240
gtacctggtg ggtgtgtgca cgaacacgct gtgcgccgtc atgggcggcg acgccatctt 300
cgaccgcctc aaagagcatc tcggcgtcgg ccacgacgaa accacctccg acggtgtggt 360
caccttgcaa cacatcgaat gcaacgccgc ctgcgattac gcacc 405
<210> 72
<211> 991
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
tcgctatcgg ggctatcagg cgttgcagaa agccctgacg atgccgcccg acgacgtgat 60
cagcatcgtc aaggattccg ggttacgcgg acgcggcggc gcgggctttg ccaccgggac 120
caagtggtcg ttcatcccgc agggcgacac cggcgccgcg gccaagccgc actacctggt 180
ggtcaacgcc gacgagtccg aacccggtac gtgcaaagac attccgttga tgctggcgac 240
gccacatgtg ctcatcgaag gcgtcatcat cgccgcctac gcgatccgcg cccatcacgc 300
gttcgtctac gtacgcggtg aggtggtgcc ggtattgcgc cggctgcaca acgcggtggc 360
cgaggcctat gccgccggct tcctaggccg caacatcgga ggttccggat tcgatctgga 420
gctggtggta cacgccggcg cgggcgccta catctgcggc gaggagaccg ccctgctcga 480
ctcgctggaa ggccggcgcg gccagccgcg gctgcggccc cccttccccg cggtggccgg 540
tctgtatggc tgcccgaccg tgatcaacaa cgtcgaaacg atcgccagtg tcccatcgat 600
catcctgggc ggcatcgact ggttccggtc gatgggcagc gagaaatcgc ctggcttcac 660
cctgtattcg ctgtccggcc acgtcacccg ccccggccag tacgaggcgc cgctgggcat 720
tacgctgcgc gagttgctcg actacgcagg cggggtgcgc gccgggcacc ggctgaagtt 780
ctggacaccg ggcggctcgt cgaccccgct gctcaccgac gagcatctgg atgtgccgct 840
ggactacgag ggtgtgggtg cggccggctc gatgctgggg accaaggcgc tggagatctt 900
cgacgagacc acctgcgtgg tgcgcgcggt gcgccgctgg accgagttct acaagcacga 960
atcgtgtggg aaatgcacgc cgtgccggga g 991
<210> 73
<211> 2070
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
gggcacgttg gtgattcgcg ccgccgaact gatgggaatc cagatcccgc gattctgcga 60
ccacccgctg ctggagcccg tcggcgcctg ccggcaatgc ctggtcgagg tcgaagggca 120
acgcaagccg ctggcgtcgt gcaccaccgt ggccaccgac gacatggtgg tgcgcaccca 180
actcacctcc gagattgccg acaaggccca gcacggtgtg atggaactgc tgctgatcaa 240
ccatccgctg gattgcccga tgtgcgacaa gggcggtgaa tgcccgctgc aaaaccaggc 300
aatgtctaac ggccgcacgg attctcgctt caccgaggcc aaacgtacct tcgccaaacc 360
gatcaacatc tccgcgcagg tgctgctgga ccgcgaacgt tgcatcctgt gcgcccgctg 420
cacccggttc tccgaccaga tcgccggcga tccgttcatc gatatgcagg agcgcggcgc 480
cctgcagcag gtcggtatct acgccgatga accgttcgag tcgtacttct ccggcaacac 540
ggtgcagatc tgcccggtgg gggcgctaac ggggaccgcc taccggttcc gcgcgcgtcc 600
gttcgatttg gtctccagcc ccagcgtctg cgagcactgc gcgtcgggct gcgcgcaacg 660
caccgaccat cgccgcggca aggtgctgcg gcggctggcc ggtgacgacc cggaagtcaa 720
cgaggagtgg aactgcgaca agggccggtg ggccttcacg tacgcgaccc agccggacgt 780
gatcaccact cccctgatcc gcgacggtgg ggaccccaag ggcgcgctgg tgcccacctc 840
gtggtcgcac gcaatggcgg tggccgccca gggactggcg gcagcgcggg gccgcaccgg 900
ggtgctggtc ggcggccgag tgacctggga ggacgcctac gcgtacgcca agttcgcgcg 960
gatcacgttg ggcaccaacg acatcgactt ccgcgcccgg ccgcactcgg ccgaggaggc 1020
cgacttcctg gcggcccgca tcgccgggcg gcatatggcg gtcagctatg ccgatttgga 1080
atcggctccg gtggtgctgc tggtgggatt cgagcccgaa gacgagtcgc cgatcgtgtt 1140
tctgcggtta cgcaaggccg ctcgcagaca ccgcgtcccg gtgtacacga tcgccccctt 1200
tgccactggt ggcctgcaca aaatgtcggg ccggctgatc aaaaccgttc ctggtggcga 1260
acccgcggcg ctggacgatc tggccaccgg tgcagtgggc gacctgctgg ccaccccggg 1320
cgcggtcatc atagtcgggg agcgcttggc cacggtaccg ggcggattgt cggcggccgc 1380
tcggctggcc gatacgaccg gcgcccgttt ggcgtgggtg ccgcggcggg cgggggaacg 1440
cggagcgctg gaagccggag cgttgcccac gctgttaccc ggtggccgcc cgctggccga 1500
cgaggtcgcc cgcgcgcagg tgtgtgcggc gtggcatatc gccgaattgc ctgccgcggc 1560
tggacgggac gccgacggca tcctggccgc cgctgccgac gagacgttgg ctgcgctgct 1620
ggtcgggggt atcgaacccg cggacttcgc cgacccggac gccgtgctgg ccgcgttgga 1680
cgccaccggt ttcgtggtca gcctggagct gcgacacagt acggtcaccg aacgcgccga 1740
cgtggtgttc ccggtcgcgc cgacgaccca gaaagccggc gcgttcgtca actgggaggg 1800
tcgctaccgt acattcgaac ccgcgctgcg cggcagcaca ctgcaagctg gccagtcgga 1860
tcaccgggtg ctggacgcgt tggccgacga catgggtgtc catctgggcg tgcccaccgt 1920
ggaggcggcc cgcgaggagc tggccgcgct cggtatctgg gacggcaaac acgctgccgg 1980
tccccacatc gcggccaccg ggccgaccca acccgaagct ggtgaggcga tcttgaccgg 2040
gtggcggatg ctcctcgacg agggccgcct 2070
<210> 74
<211> 923
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
cgatcctggc cgaacgcaag ctgctgggcc ggatgcagtt gcggcccggc cccaaccggg 60
ttggcccaaa aggagccctg cagagcctgg ctgacggcat caagctggcg ctcaaagaga 120
gcatcacacc cggtggcatc gatcgattcg tatattttgt ggcgccgatc atttcggtga 180
ttccggcatt caccgctttc gcgttcatcc cgtttggtcc cgaggtgtcg gtgtttggcc 240
accggacacc gttgcagata accgaccttc ccgtcgccgt gctgttcatc ctgggactgt 300
cggcgatcgg ggtatacggc atcgtgctgg gcggttgggc gtccgggtcc acctacccgc 360
tgctgggcgg ggtgcgctcc accgcgcagg tcatctccta cgaggtcgcg atgggcctgt 420
cgttcgcgac ggtgttcctt atggccggca ccatgtcgac gtcgcagatc gtggccgcac 480
aagacggtgt ctggtatgcc ttcctgttgt tgccgtcatt cgtcatctat ctcatttcta 540
tggtgggtga aaccaaccgg gcgccgttcg atttgcccga agccgagggc gagctggtcg 600
cgggattcca caccgagtac tcgtcgttga agttcgcgat gttcatgctc gccgagtacg 660
tcaatatgac tacggtttcg gcactggccg cgaccctatt cttcggtggc tggcatgctc 720
cctggccgct gaacatgtgg gcgagcgcca acaccggctg gtggccactg atctggttca 780
ccgctaaagt gtggggcttt ctgttcatct atttctggct gcgggctacg ctgccgcggc 840
tgcgctacga ccagttcatg gcgctgggct ggaagttatt gatccccgtc tcgctggtgt 900
gggtgatggt cgccgcgatc atc 923
<210> 75
<211> 357
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
tagccggatt cggggtaacg cttggttcga tgttcaaaaa gacggtcacc gaggagtatc 60
cggaaaggcc cggtccggta gcagcgcgct accacggccg tcatcagctc aaccggtatc 120
cggacggcct ggagaaatgc atcggctgcg agttgtgcgc ctgggcctgc ccggccgacg 180
caatctatgt cgagggcgcg gacaataccg aagaggagcg gttttcgccg ggcgaacgct 240
acggccgggt gtaccagatt aactatttgc gttgcatcgg ttgcggtttg tgcatcgagg 300
cgtgcccgac gcgggcgctg acgatgacct atgattacga actggccgac gacaacc 357
<210> 76
<211> 489
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
gttggcgctg ctgggcgcgg tcggggttgt gctggccgtc aacgccgtgt actcagcgat 60
gtttctggcg atgaccatga tcatcctggc ggtgttctac atggcccagg acgcgctgtt 120
tttgggtgtc gtccaggtgg ttgtctacac cggcgcggtg atgatgctgt tcctgttcgt 180
gctgatgctg atcggtgtgg actccgcgga atcactgaag gagacgctgc gcgggcagcg 240
ggtcgccgcg gtgctgaccg gtgtcgggtt cggcgttctc ctgatcagca ccatcggcca 300
ggtggcgacc cgaggttttg ccggactaac cgtcgccaac gccaacggca acgtcgaagg 360
cttggccgcg ctgatttttt cccgttacct gtgggcgttc gagttgacca gtgcgctgtt 420
gattaccgcc gccgtcgggg cgatggtgct agcgcaccgg gagcgtttcg agcgccgcaa 480
gacccagcg 489
<210> 77
<211> 175
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
tttcggtgct gctattcacc atcggagcct ccggtgtgct gctgcgacgc aacgcgatcg 60
tgatgttcat gtgcgtcgag ctcatgctca atgccgttaa cctggcgttc gtcaccttcg 120
cgcgcatgca tggccatctc gacgcccaga tgatcgcgtt cttcaccatg gtggt 175
<210> 78
<211> 1511
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
tggcactgcc actggcgggt gccgcaatct tgctgttcgg cggcagacgc accgatgcgt 60
ggggccacct gctgggctgt gccgcagcgc tggcggcatt cggggtgggc gcgatgctgc 120
tggccgacat gctcggtcgc gatgggctcg agcgcgcgat ccatcagcag gtgttcacct 180
ggatacccgc cggcggactc caagtcgact tcgggctgca gatcgatcag ttgtccatgt 240
gcttcgtgct gctgatctcc ggggtcggat cgctgattca catctattcg gtcggctaca 300
tggccgagga cccggaccgg cgcaggtttt tcggctatct caacctgttt ctggcctcga 360
tgctgctgct ggtggtcgcc gacaactatg tgttgctgta cgtcggctgg gagggtgtgg 420
gcctggcgtc gtatctgttg atcggtttct ggtaccacaa gccgtcggcg gccaccgcgg 480
ccaaaaaggc attcgtgatg aaccgggttg gggacgccgg cctagcggtg ggtatgttct 540
tgacgtttag cactttcggc accctgtcgt atgccggcgt gttcgccggc gtacccgccg 600
caagtcgcgc agtgctgacc gcgatcgggt tgttgatgct gttgggggcg tgcgccaagt 660
ccgcgcaggt tccgctgcaa gcctggcttg gcgacgcgat ggagggcccc accccggtgt 720
ccgcgctgat ccacgccgcc accatggtga ccgccggagt gtatttgatt gtgcggtcgg 780
gcccgctgta caacctggcg cccaccgccc aactggcggt cgtcatcgtc ggcgcggtga 840
cgctgctgtt tggggcgatc atcggctgcg ccaaggacga catcaaacgt gcgctggcag 900
cctcgaccat tagccagatc ggctacatgg tgctggccgc gggcctgggt ccggccggct 960
acgcgtttgc gatcatgcat ctgctcactc acggtttctt caaggccggc ctattccttg 1020
ggtccggcgc ggtgattcac gcgatgcacg aagagcagga catgcgccgt tacggtggtc 1080
tgcgcgccgc cctgccggtc acgttcgcaa ccttcggcct ggcgtatctg gcgattatcg 1140
gggtaccgcc gttcgcgggc ttcttctcca aggatgcgat catcgaggcc gcattgggcg 1200
ccggcggcat ccggggctcg ctgctgggcg gtgccgcgct gctgggtgcg ggcgtcaccg 1260
cgttctacat gacgcgagtg atgctgatga ccttcttcgg cgaaaagcgt tggacgccag 1320
gcgcccatcc gcacgaggca ccggccgtga tgacctggcc gatgatcttg ctcgccgtcg 1380
gctcggtgtt ctccggtggc ctgctcgcgg tgggtggcac gttgcggcat tggctgcagc 1440
cagttgtcgg atctcatgaa gaggccaccc atgcgctgcc gacctgggtc gccaccaccc 1500
tggcgctcgg t 1511
<210> 79
<211> 1267
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 79
ctggcaggtg cggtgctgat catcctgcta ccacccggtc ggcgccgact cgccaagtgg 60
gccggtatgg ttgtcagcgt cctgacgttg gcggtgtcga tcgtcgtcgc ggccgaattc 120
aagcccagcg ccgagccgta tcagttcgtc gaaaagcatt cctggatacc ggcgttcggc 180
gccggctata cccttggtgt ggacggcatc gcagtggtgc tggtgttgtt gaccacagtg 240
ctgattccgt tgctgctggt ggccggctgg aacgacgcaa ccgatgctga cgacctgtcc 300
cccgcaagcg ggaggtaccc ccagcgcccg gctccgccgc gcttgcgatc gtcaggtggc 360
gaacgcaccc gaggcgtgca cgcctacgtg gcattgacgc tggccatcga gtcgatggtg 420
ctgatgtcgg tgatcgcgct ggacgtgctg ctgttctacg tgttcttcga ggccatgctg 480
atcccgatgt acttcctcat cggcggcttc ggccaggggg ccggacgctc gcgtgccgcg 540
gtgaagttct tgctgtacaa cctgtttggc gggttgatca tgctggcggc ggtgatcggg 600
ctgtatgtgg tgaccgcaca gtacgattcg ggcaccttcg acttccgtga gatcgtggcc 660
ggcgtggcgg cgggccgcta cggagcggac ccggcggtgt tcaaggcgct gttcttgggc 720
ttcatgttcg cgttcgcgat caaggctccg ctgtggccgt tccatcgctg gctgccggac 780
gccgccgtcg agtccacccc agcgaccgcg gtgctgatga tggcggtgat ggacaaggtc 840
ggcaccttcg gcatgctgcg ctactgcctg cagctgtttc ctgacccgtc aacgtatttc 900
cgtccgctga tcgtgacgct ggccatcatc ggggtgatct acggcgcgat cgtggcgatc 960
ggccaaaccg acatgatgcg gctgatcgcc tacacctcga tctcgcactt cgggttcatc 1020
atcgcaggca tcttcgtcat gaccacccag ggccagagcg ggtcgacgct gtacatgctc 1080
aaccacggcc tgtccacggc ggcggtgttc ctgatcgccg gtttcttgat agcgcggcgc 1140
ggcagccgat cgatcgccga ctacggcggt gtccagaagg tggcgcccat cctggccggc 1200
acgttcatgg tctcggccat ggccaccgta tcgctgcccg gcctagcccc gtttatcagc 1260
gaattcc 1267
<210> 80
<211> 1456
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 80
gctcatcgtc ttttcggttg cggtcgccgg tgtgctggcc gaggctttcc tgccgcgccg 60
gtggcgctat ggcgcccaag tgacgctcgc ccttggcggg tcggcagtgg cactcatcgc 120
ggtcatcgtg gtggccaggt cgattcacgg gtcgggtcac gccgcggtgc tgggggccat 180
agccgtggat cgagcgaccc tgtttctgca aggcaccgta ctactggtca cgatcatggc 240
agtcgtcttc atggccgaac gcagcgcccg ggtgagtccg caacgccaga acaccctcgc 300
tgtggcgcgg ctccctggac tcgattcgtt taccccgcag gcttccgccg tgcccggcag 360
cgatgctgag cgccaagcgg aacgggcggg agccacccag acggaacttt tcccgctggc 420
gatgctgtcc gtcggcggca tgatggtgtt tcccgcgtcc aacgacctgt tgacgatgtt 480
cgttgcgctg gaggtgctat cgctgccgct gtacctgatg tgtgggctgg cccggaatcg 540
ccgcctgctg tcgcaggaag ccgcgatgaa gtacttcctg ctgggcgcct tctcgtcggc 600
gttcttcctc tacggcgtcg cgttgctata cggcgcgacc ggcacgctga ccttgccggg 660
tattcgggat gcgttggcag cgcgcaccga cgactcaatg gcgttggccg gcgtcgcgct 720
gctcgcggtc ggcctactat tcaaggtcgg cgcggtgcca ttccactcct ggattcccga 780
tgtgtaccag ggcgcaccca ccccgatcac cgggttcatg gcggccgcca ccaaggtcgc 840
ggcgttcggt gcgctgctcc gggtggtcta tgtcgcgctg ccgccgctgc acgatcagtg 900
gcgcccggtg ctgtgggcga ttgccatcct caccatgacg gtgggcaccg tcaccgcggt 960
aaaccagacc aacgtcaagc gtatgctggc ctattcatcg gtcgcgcacg tcggtttcat 1020
acttaccggc gtgatcgccg ataatccggc gggtctttcc gcgacgttgt tctatctggt 1080
cgcctacagc ttcagcacga tgggtgcgtt tgccatcgtg ggtctggtcc gaggcgccga 1140
cggctcagca ggttcagagg atgccgacct gtcccactgg gccgggctgg gacagcgttc 1200
acctatcgtg ggcgtgatgc tgtcgatgtt tctgctggcc ttcgccggca tcccgttgac 1260
cagtggattc gtcagcaagt tcgcggtgtt tagggccgcc gcttccgccg gcgcggtgcc 1320
gctggtaatc gtcggcgtga tctccagcgg cgtcgccgcc tacttctacg tgcgggtgat 1380
cgtgagcatg ttcttcaccg aagaatccgg tgacacacca cacgtggcgg cacccggcgt 1440
gctgagcaag gccgcc 1456
Claims (10)
1. one is worn for recombination needed for constructing knockout mycobacterium tuberculosis nadh dehydrogenase gene family phage library
Shuttle vector library, which is characterized in that the recombinant shuttle vector library includes 16 recombinant shuttle vectors, inserts tuberculosis respectively
The left and right homology arm sequence of 16 member gene two sides of mycobacteria nadh dehydrogenase gene family, wherein 16 member genes
For Rv0392c, Rv1854c, Rv3145, Rv3146, Rv3147, Rv3148, Rv3149, Rv3150, Rv3151, Rv3152,
Rv3153、Rv3154、Rv3155、Rv3156、Rv3157、Rv3158。
2. recombinant shuttle vector library according to claim 1, which is characterized in that be inserted into 16 recombinant shuttle vectors
It is necessary to meet following condition for the left and right homology arm sequence of the gene two sides of 16 members: utilizing recombinant shuttle vector library structure
Mycobacterium tuberculosis of the phage library built for being knocked out when knocking out mycobacterium tuberculosis nadh dehydrogenase gene family
Rv0392c、Rv1854c、Rv3145、Rv3146、Rv3147、Rv3148、Rv3149、Rv3150、Rv3151、Rv3152、
The genetic fragment of Rv3153, Rv3154, Rv3155, Rv3156, Rv3157, Rv3158 are successively as shown in SEQ.ID.NO.65~80
Segment.
3. recombinant shuttle vector library according to claim 1, which is characterized in that building process is by 16 member genes
The left and right homology arm sequence of two sides is inserted into p0004S carrier, obtains positive p0004S recombinant plasmid；Use restriction enzyme
Digestion shuttle vector and p0004S recombinant plasmid, junction fragment obtain recombinant shuttle vector respectively.
4. recombinant shuttle vector library according to claim 3, which is characterized in that the shuttle vector is phAE159.
5. one group is combined for constructing the primer in any recombinant shuttle vector library of Claims 1 to 4, feature exists
In comprising 32 pairs of primers, nucleotide sequence is as shown in SEQ.ID.NO.1~64.
6. the combination of primer described in claim 5 knocks out mycobacterium tuberculosis nadh dehydrogenase gene family bacteriophage text in preparation
Application in recombinant shuttle vector library needed for library.
7. one for knocking out the phage library of mycobacterium tuberculosis nadh dehydrogenase gene family, which is characterized in that described
Phage library contains 16 kinds of bacteriophages, respectively containing any 16 recombinant shuttle vectors of Claims 1 to 4.
8. any recombinant shuttle vector library of Claims 1 to 4 knocks out mycobacterium tuberculosis nadh dehydrogenase base in preparation
Because of the application in family's phage library.
9. phage library described in claim 7 is knocking out the application in mycobacterium tuberculosis nadh dehydrogenase gene family.
10. the answering in building nadh dehydrogenase gene family missing mycobacterium tuberculosis of phage library described in claim 7
With.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111269894A (en) * | 2020-03-05 | 2020-06-12 | 苏州十一方生物科技有限公司 | Construction method of genetic engineering recombinant phage |
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