CN100355895C - Plasmid with bar streptomycete Lat gene loss, derivative and constructing method thereof - Google Patents

Abstract

The present invention relates to a plasmid with clavate streptomycete lat genes deleted, a derivant and a construction method thereof, which belongs to biological engineering. The present invention particularly relates to a PCR amplification in vitro primer of lysine epsilon-transaminase lat genes encoded by clavate streptomycete, a plasmid with lat genes deleted, a derivant and a construction method thereof. The present invention designs an upstream primer and a downstream primer, realizes mass amplification in vitro of lat genes and constructs a plasmid and a derivant having the capabilities of single exchange and integration. In addition, the present invention implements the deletion mutation of lat genes of clavate streptomycete having influence on the yield of clavulanic acid and completes the construction work in colibacillus. The magnitude of the plasmid is about 7.8 kb or 7.0 kb, and the plasmid contains a part of encoding lysine epsilon-transaminase genes in clavate streptomycete, a colibacillus source plasmid, a duplication starting point of a streptomycete source plasmid and a temperature sensitive replicon gene with an ambylan resistance selective marker capable of being expressed in colibacillus and streptomycete and pSW344E.

Description

The plasmid of bar streptomycete Lat gene loss, derivative and construction process thereof

Technical field

The invention belongs to biotechnology, relate to a kind of with the genetic engineering technique realization clavuligerus coding Methionin ε-PCR amplification in vitro primer of transaminase lat gene and plasmid, derivative and construction process thereof of lat genetically deficient.

Background technology

Since penicillin came out, β-Nei Xiananleikangshengsu was widely used so far, and many beta-lactam verieties with new feature constantly come out.But along with the widespread use of β-Nei Xiananleikangshengsu, various bacteriums are also increased day by day to the antibiotic resistance of this class.Bacterial resistance mechanism has multiple, and wherein producing β-Nei Xiananmei is topmost resistance mechanism, and it is significant for solving the drug-resistance of bacteria problem therefore to develop beta-lactamase inhibitor.This class enzyme inhibitors and β-Nei Xiananleikangshengsu combined utilization by beta-lactamase inhibitor deactivation β-Nei Xiananmei, and make β-Nei Xiananleikangshengsu bring into play original anti-microbial effect.Clavulanic acid is that first is found and is applied to clinical beta-lactamase inhibitor, and is synthetic by the clavuligerus metabolism because of it, so have another name called clavulanic acid.Clavuligerus (Streptomyces clavuligerus) is that a kind of clavulanic acid that is widely known by the people produces bacterium.Its anti-microbial effect is faint, but has the enzyme effect that presses down preferably.The β-Nei Xiananmei that clavulanic acid produces streptococcus aureus, plasmid-mediated enzyme (TEM and SHV etc.) and Klebsiella that gram-negative bacteria produces, the karyomit(e) enzyme that proteus vulgaris, bacteroides fragilis produce all has restraining effect.The in-vitro antibacterial that amoxycilline Trihydrate bp and clavulanic acid share is discovered, clavulanic acid improve the amoxycilline Trihydrate bp minority enteron aisles such as Klebsiella, proteus vulgaris are produced inha enzyme etc. anti-microbial effect 4-32 doubly, the anti-microbial activity of bloodthirsty hemophilus influenza is improved 32 times.This very effect of clavulanic acid has determined it to be in great demand, and the therefore relevant method that improves clavuligerus clavulanic acid output is the hot fields of studying both at home and abroad.

The approach of domestic raising clavulanic acid output mainly is an optimization of fermentation conditions, but its output that can improve is limited.Though clavuligerus is carried out the main means that protoplast regeneration and traditional ultraviolet mutagenesis method screening superior strain are still strain improvement, exist the seed selection cycle long, efficient is low and problems such as blindness, randomness still have to be solved.Because the clavulanic acid fermentation yield is low, complex manufacturing, therefore the main all the time dependence on import of the chemical synthesis industrialization of still being unrealized, the clavulanic acid preparation of China increases substantially it and produces bacterial strain to produce the acid amount be a vital task in the current clavulanic acid exploitation.The research that utilizes engineered method to improve clavuligerus clavulanic acid output at present still belongs to blank at home.

More to the research of clavuligerus clavulanic acid synthetic gene bunch abroad, clavuligerus clavulanic acid synthetic approach is clear and definite substantially, for the output of utilizing engineered method to improve clavulanic acid provides may.Because the biosynthesizing of clavulanic acid is subjected to the regulation and control of gene usually, so the sudden change of this gene of clavuligerus just seems very important to the influence of clavulanic acid productive rate.Start with from influencing the metabolic lat gene of clavuligerus clavulanic acid, the transgenation of certain enzyme of coding in another metabolism branch road that will be parallel with clavulanic acid metabolism branch road, thus block this metabolism branch road and then can improve clavulanic acid output.The pathways metabolism of the synthetic cephamycin C of clavuligerus is with L-Methionin, L-halfcystine and Xie Ansuan are the (see figure 5) of precursor substance, the gene cluster of the enzyme of coding cephamycin C route of synthesis is now clear and definite, the lat genes encoding Methionin ε-transaminase of clavuligerus, it is first gene of cephamycin C synthetic, although the route of synthesis of cephamycin C and clavulanic acid is not subjected to identical enzyme catalysis, but coding and the adjacent coding of gene cluster of the synthetic relevant enzyme of clavulanic acid and the gene cluster of the synthetic relevant enzyme of cephamycin C, and two pathways metabolism is subjected to identical adjusting PROTEIN C caR and the regulation and control of ClaR, therefore, the cephamycin C synthetic stops and will effectively improve the output of clavulanic acid, it also is the preferred genes of regulating and control, the inactivation of this enzyme will cause the cephamycin C synthetic to stop, therefore, purpose the lat gene is suddenlyd change will make the cephamycin C route of synthesis be obstructed, and then can effectively improve the output of clavulanic acid.

The method that people such as Paradkar once adopted gene substitution has carried out inserting sudden change to the lat gene of clavuligerus, and clavulanic acid output is improved.The main thought of its experiment is, at first the lat gene of clavuligerus is inserted the multiple clone site of escherichia coli plasmid pUC119, then the lat gene that inserts is carried out single endonuclease digestion, be connected through the apramycin resistant gene of modifying with end, make and insert one section apramycin resistant gene in the lat gene, to insert lat gene (lat::apr) fragment of resistant gene through double digestion, end modified back is connected with intestinal bacteria-streptomycete shuttle vector pIJ486, obtains the recombinant plasmid that the lat gene inserts sudden change.After being used in this plasmid that builds in the intestinal bacteria and transforming original clavulanic acid and produce bacterium, the karyomit(e) of plasmid and streptomycete is by double exchange generation homologous recombination, thereby realization suddenlys change to the lat gene of clavuligerus.But, because its insertion mutation method step that adopts is more loaded down with trivial details relatively, and used shuttle vector pIJ486, do not have single cross and change and be incorporated into chromosomal ability, and double exchange is integrated and certainly will be had certain difficulty.

But, the lat gene of clavuligerus is carried out deletion mutantion, and selects for use and have single cross and change the plasmid that the shuttle vector of integration ability makes up the lat transgenation and do not appear in the newspapers at present.

Summary of the invention

The problem that solves:

At above-mentioned situation, the objective of the invention is to adopt engineered technology, two upstream and downstream primers that are used for pcr amplification clavuligerus coding Methionin ε-transaminase lat gene have been designed, realized that the lat gene in vitro increases in a large number, and the lat gene of the clavuligerus that influences clavulanic acid output carried out deletion mutantion, made up and be used to transform clavuligerus and then realize two kinds of disappearance plasmid pKCLES, pKCLHS and the derivative that integration ability is changed in single cross that have, and finished the structure work in intestinal bacteria clavuligerus lat gene disruption.

Technical scheme:

The present invention has at first carried out the PCR amplification in vitro to the lat gene of clavuligerus, and the terminally modified rear clone of lat gene after will increasing is to the multiple clone site (MCS) of intestinal bacteria-streptomycete shuttle vector pKC1139, be built into recombinant plasmid pKCLES and pKCLHS from connecting respectively after double digestion is removed in the lat gene one section EcoRI-ScaI fragment or HindIII-ScaI fragment, two kinds of recombinant plasmids are relaxed plasmid.

The plasmid and the derivative thereof of clavuligerus (Streptomyces clavuligerus) the lat genetically deficient that the present invention makes up, a part that comprises coding Methionin ε-aminotransferase gene in the clavuligerus, also comprised on the plasmid Escherichia coli source plasmid and streptomycete source plasmid replication origin, have all effable apramycin resistance selective marker in intestinal bacteria and streptomycete, and have pSW344E temperature sensitive type replicon gene.Above-mentioned clavuligerus (Streptomyces clavuligerus) comes from Chinese antibiotic microorganism DSMZ preserving number: CACC NO.SIIA 2.111.

Need to prove:

The plasmid of described clavuligerus (Streptomyces clavuligerus) lat genetically deficient, the pKCLHS size is about 7.8kb, and is represented by the estriction map of Fig. 1; Plasmid pKCLES size is about 7.0kb, and is represented by the estriction map of Fig. 2.

The plasmid pKCLHS of described clavuligerus (Streptomyces clavuligerus) lat genetically deficient and the derivative of pKCLES size are the 6.5kb-8.3kb except that 7.0kb, 7.8kb.

Plasmid pKCLHS, the pKCLES of clavuligerus (Streptomyces clavuligerus) lat genetically deficient and the construction process of derivative thereof

The construction process of plasmid pKCLHS and pKCLES:

At first make up the recombinant plasmid pUCm-T-lat contain goal gene lat,, obtain the lat gene fragment that size is about 1.8kb, select for use pUCm-T carrier with multiple clone site and the lat gene fragment behind the purifying with T by the design primer performing PCR amplification in vitro of going forward side by side ₄Dna ligase connects and obtains recombinant plasmid pUCm-T-lat; With EcoRI-HindIII double digestion pUCm-T-lat, purified back of the lat gene fragment that obtains and the carrier pKC1139 behind EcoRI-HindIII double digestion and purifying are with T ₄Dna ligase connects, and obtains recombinant plasmid pKC1139-lat; PKC1139-lat reclaims the dna fragmentation of about 7.8kb or 7.0kb respectively through HindIII-ScaI or EcoRI-ScaI double digestion, the recovery product behind two kinds of purifying is at first all with T ₄Archaeal dna polymerase is mended flat terminal, again with T ₄Dna ligase carries out from connecting, obtain one section HindIII-ScaI of lat genetically deficient and segmental recombinant plasmid pKCLHS of EcoRI-ScaI and pKCLES respectively, building process is represented by Fig. 4, whole plasmid construction process is finished in intestinal bacteria Escherichia coli (DH5 α), this bacterial classification is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preserving number is: CGMCC NO.0428.1.

The construction process of this derivative:

The restriction enzyme that adopts is to remove used HindIII-ScaI of structure plasmid pKCLHS and pKCLES and any two kinds of enzymes in all the other the restriction enzyme site (see figure 3)s the EcoRI-ScaI on the lat gene, lat gene among the plasmid pKC1139-lat is cut and reclaim remaining dna fragmentation after the lat genetically deficient, and all the other methods are with the structure of plasmid pKCLHS and pKCLES.

The lat gene the primer of above-mentioned PCR amplification in vitro coding Methionin ε-transaminase has the dna sequence dna shown in SEQ NO ID:1 or 2, or has and the dna sequence dna 90% shown in SEQ NO ID:1 or 2 or the dna sequence dna of higher homology.

Primer 1:5 '-CCATAATTCAGCCTGATCCCCCAGGAGTTCTCACCCATG-3 ' (SEQ NO ID:1);

Primer 2: 5 '-ATCGCGGGCCGGCCGGCCCACGGGGTCGCCCCGGGCCGG-3 ' (SEQ NO ID:2).

Beneficial effect:

The present invention has utilized engineered method, compares with traditional mutagenesis to have the advantage that purpose is strong, working strength is little, the cycle is short, efficient is high.This method is carried out deletion mutantion to the lat gene that influences clavulanic acid output, has designed the plasmid of bar streptomycete Lat gene loss sudden change, and finishes the structure work in intestinal bacteria.The building process of bar streptomycete Lat gene loss plasmid of the present invention, compare with the construction process that inserts mutant plasmid, construction process of the present invention is more simple and easy to do, and because the shuttle vector pKC1139 that the present invention adopts has pSW344E temperature sensitive type replicon gene, therefore the plasmid of the bar streptomycete Lat gene loss that builds is the temperature sensitive type shuttle plasmid.This plasmid transforms clavuligerus and the homology single cross can take place changes, on the easier karyomit(e) that is incorporated into clavuligerus of homology double exchange.The present invention further transforms clavuligerus, improves its clavulanic acid output, lays a good foundation for obtaining the clavulanic acid superior strain, and is significant to the update of China's clavulanic acid industry.

Description of drawings

Fig. 1 is the restriction figure of plasmid pKCLIIS of the present invention;

Fig. 2 is the restriction figure of plasmid pKCLES of the present invention;

Fig. 3 is the restriction figure of lat gene fragment;

Fig. 4 is the building process figure of plasmid pKCLHS of the present invention and pKCLES;

Fig. 5 is the route of synthesis of cephamycin C;

Fig. 6 is a clavuligerus chromosomal DNA electrophorogram;

Fig. 7 is a pcr amplification lat gene fragment electrophorogram of the present invention;

Fig. 8 is the restriction enzyme digestion and electrophoresis figure of recombinant plasmid pUCm-T-lat of the present invention;

Fig. 9 is the restriction enzyme digestion and electrophoresis figure of recombinant plasmid pKC1139-lat of the present invention;

Figure 10 is the restriction enzyme digestion and electrophoresis figure of recombinant plasmid pKCLHS of the present invention and pKCLES.

Embodiment

Embodiment 1

The structure of recombinant plasmid pKCLHS

1. the extraction of the total DNA of clavuligerus

Picking solid ISP (ISP (Difco): 0.8%, agar powder: 2%, pH=7.3) dull and stereotyped last 28 ℃ of clavuligerus of cultivating 3 days are inoculated in the sub-liquid ISP substratum, and 28 ℃ of shaking tables are cultivated 2d, centrifugal results thalline, with the EDTA of 10mM pH=8.0 washing once or wash thalline with water twice.Add SET (10mmol/LTris-HCl (pH8.0), 10mmol/L NaCl, 1mmol/LEDTA (pH8.0)) solution to 1mL, and add 20 μ L 50mg/mL (or N,O-Diacetylmuramidase of other concentration of a great deal of), 37 ℃ of water-bath 30-60min.Add the Proteinase K 28 μ L mixings of 20mg/mL, add 120 μ L 10%SDS, the upset mixing, 55 ℃ of temperature are bathed 2h.The mixture branch is installed to (0.8mL/ pipe) in two clean centrifuge tubes, add 500 μ L phenol/chloroforms respectively, the abundant mixing of fierce concussion, the careful supernatant liquor of drawing in centrifugal back is in another centrifuge tube.If albumen precipitation is incomplete, can repeat above two steps.Add the dehydrated alcohol of 600 μ L ice, the abundant mixing that overturns leaves standstill 3min, high speed centrifugation deposit D NA (if the more available application of sample rifle of karyomit(e) is chosen in another centrifuge tube).Dehydrated alcohol with ice is washed once, and gained DNA places 10min in room temperature, makes it dry.Dried DNA is dissolved in the 20 μ L TE damping fluids, is stored in-20 ℃.The electrophorogram of the total DNA of clavuligerus is seen Fig. 6, wherein: a. clavuligerus chromosomal DNA; B.10kb Marker (be followed successively by the normal size dna fragmentation of 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb and 1.5kb in the drawings from top to bottom, below herewith).

2. according to the dna sequence dna of clavuligerus lat gene design primer, and with the total DNA of clavuligerus is template PCR amplification in vitro lat gene, and the PCR product is carried out purifying

According to two primers of the analysis design pcr amplification lat gene of the lat gene order (SEQ NO ID:3) of clavuligerus coding Methionin ε-transaminase, as follows respectively:

Primer 1:5 '-CCATAATTCAGCCTGATCCCCCAGGAGTTCTCACCCATG-3 ';

Primer 2: 5 '-ATCGCGGGCCGGCCGGCCCACGGGGTCGCCCCGGGCCGG-3 ';

Utilize the lat gene of PCR reaction system composite coding Methionin ε-transaminase to carry out the PCR reaction, reaction system is as follows: clavuligerus template DNA 3 μ L (100ng/ μ L), 10 * PCR buffer, 5 μ L, 2mM dNTP 5 μ L, primer 1:2 μ L (100ng/ μ L), primer 2: 2 μ L (100ng/ μ L), Taq enzyme (5u/ μ L) 1 μ L, DMSO 2.5 μ L, MgCl ₂3 μ L (25mM), ddH ₂O 26.5 μ L, reaction system is totally 50 μ L.Cycling condition: with above-mentioned system in 94 ℃ of constant temperature 5 minutes, 94 ℃ of constant temperature 2 minutes, 61 ℃ of constant temperature 1 minute, 72 ℃ of constant temperature 2 minutes, second goes on foot the 4th step totally 40 circulations, 72 ℃ of constant temperature 15 minutes is stored in 4 ℃.Above PCR reacts each reagent and is reagent in the Sangon SK2491 test kit, PCR reacts the synthetic of two primers to be finished by Sangon company, the purifying of PCR product adopts the Sangon UNIQ-10 of company pillar PCR product purification test kit, and purification process is referring to UNIQ-10 pillar PCR product purification test kit specification sheets.Pcr amplification obtains the lat gene fragment that size is about 1.8kb, and the lat gene fragment electrophorogram behind the purifying is seen Fig. 7, wherein: Marker a.10kb; The b.lat gene fragment.

3. the lat gene behind the purifying is connected with the pUCm-T carrier and obtains recombinant plasmid pUCm-T-lat

At first the PCR product lat gene fragment behind the purifying is connected with the pUCm-T carrier, the ligation system is as follows: 10 * connection damping fluid (Ligation buffer): 1 μ L, PEG4000:1 μ L, PUCm-T carrier: 1 μ L, purified pcr product lat gene fragment: 4 μ L, sterilization distilled water (Sterile ddH ₂O): 2 μ L, T ₄Dna ligase: 1 μ L; Whole ligation system is totally 10 μ L, and above-mentioned linked system is spent the night in 16 ℃ of constant temperature, connects product and is used for Transformed E .coliDH5 α.Above linked system agents useful for same all adopts Sangon SK2211PCR to produce clone's test kit.Electric shock Transformed E .coliDH5 α:

Prepare E.coliDH5 α electric shocking method competent cell and above-mentioned connection product is adopted electric shock conversion method Transformed E .coliDH5 α.With the cell after transforming in 1mL SOC substratum (SOB substratum: Tryptones; 20%, yeast extract: 0.5%, NaCl 0.05%, KCl:0.25mM, MgCl ₂: 2mM, 121 ℃ of sterilization 20min.Add 20mL 1mol/L glucose solution (final concentration 20mmol/L) in every liter of SOB substratum before using, be the SOC substratum) in 37 ℃, shaking table was cultivated after 1 hour, get cultured thalline, be applied on the LB solid plate that contains X-gal and IPTG and 100 μ g/mL penbritins, with above-mentioned flat board in 37 ℃ of constant temperature culture, selecting white clone (carrier pUCm-T contains the multiple clone site district of the lacZ gene that can be used for the white screening of intestinal bacteria medium blue) transfers in the liquid LB substratum that contains 50 μ g/mL penbritins, 37 ℃ of shaking tables are cultivated 12h, centrifugal collection thalline also extracts plasmid, lat gene fragment size is 1.8kb, pUCm-T carrier size is 2.8kb, connects the correct recombinant plasmid pUCm-T-lat of back gained size and should be 4.6kb.Institute's upgrading grain is carried out single endonuclease digestion with EcoRI respectively and EcoRI-HindIII carries out double digestion, restriction enzyme digestion and electrophoresis figure sees Fig. 8, wherein: a.EcoRI-HindIII double digestion pUCm-T-lat; B.KpnI single endonuclease digestion pUCm-T-lat; C. linear pUCm-T carrier; D.lat gene; E.10kb Marker.Size is the dna fragmentation of a 4.6kb behind the single endonuclease digestion, is that the plasmid that two sizes are respectively the dna fragmentation of 2.8kb and 1.8kb can be defined as correct pUCm-T-lat recombinant plasmid behind the double digestion.Above-mentioned used T ₄Dna ligase and restriction enzyme are Sangon company product, the endonuclease reaction system is with reference to the restriction enzyme specification sheets, the preparation and the electric shock transformation method of intestinal bacteria electric shocking method transformed competence colibacillus cell, the preparation method who contains the LB flat board of X-gal and IPTG, and alkaline process extracts the method for e. coli plasmid dna all referring to document [Sambrook J., Fritsch E.F., Maniatis T. (Jin Dongyan etc. translate), molecular cloning molecular cloning experiment guide, second edition, Beijing: Science Press, 1999]

4. recombinant plasmid pUCm-T-lat is carried out double digestion, be connected with the linear plasmid carrier pKC1139 that obtains through same double digestion and obtain recombinant plasmid pKC1139-lat

With gained recombinant plasmid pUCm-T-lat EcoRI-HindIII double digestion, reclaim the lat gene fragment (the DV 805A of Takara company glue reclaims test kit) of 1.8kb, make lat gene two ends have EcoRI and HindIII restriction enzyme site respectively, be connected with the T4 dna ligase with intestinal bacteria-streptomycete shuttle vector pKC1139 (Institute of Medicinal Biological Technique provides by Beijing) through same double digestion, linked system is as follows: the lat gene fragment: 3 μ L, plasmid pKC1139:3 μ L, connect damping fluid (10 *): 1 μ L, T4 dna ligase: 1 μ L, sterilization distilled water: 2 μ L, the total system of ligation: 10 μ L; Above-mentioned linked system is connected 12h in 16 ℃ of constant temperature, and the connection product of gained is used for Transformed E .coliDH5 α competent cell.

The preparation of competent escherichia coli cell and method for transformation are referring to the step in the present embodiment 3., just the yeast culture of electric shock conversion is after 1 hour, be applied on the LB solid plate that contains X-gal and IPTG and 25 μ g/mL apramycins, selecting white clone (carrier pKC1139 contains the multiple clone site district of the lacZ gene that can be used for the white screening of intestinal bacteria medium blue) after 37 ℃ of constant temperature culture transfers in the liquid LB substratum that contains 12.5 μ g/mL apramycins, 37 ℃ of shaking tables are cultivated 12h, centrifugal collection thalline also extracts plasmid, lat gene fragment size is 1.8kb, intestinal bacteria-streptomycete shuttle vector pKC1139 size is 6.5kb, connects the correct recombinant plasmid pKC1139-lat of back gained size and should be 8.3kb.Institute's upgrading grain is carried out single endonuclease digestion with EcoRI respectively, and restriction enzyme digestion and electrophoresis figure sees Fig. 9, wherein: a.10kbMarker; B, c.EcoRI single endonuclease digestion pKC1139-lat; The d.lat gene fragment; E.EcoRI single endonuclease digestion pKC1139 carrier.Size is that a segmental plasmid of 8.3kbDNA can be defined as correct pKC1139-lat recombinant plasmid behind the single endonuclease digestion pKC1139-lat.Above-mentioned used T4 dna ligase and restriction enzyme are Sangon company product, the endonuclease reaction system is with reference to the restriction enzyme specification sheets, the preparation and the electric shock transformation method of intestinal bacteria electric shocking method transformed competence colibacillus cell, the preparation method who contains the LB flat board of X-gal and IPTG, alkaline process extracts the method for e. coli plasmid dna all referring to document [Sambrook J., Fritsch E.F., Maniatis T. (Jin Dongyan etc. translate), molecular cloning molecular cloning experiment guide, second edition, Beijing: Science Press, 1999]

5. the disappearance of the clavuligerus lat Gene Partial in pKC1139-lat back is got continuously recombinant plasmid pKCLHS certainly

PKC1139-lat carries out the HindIII-ScaI double digestion with the gained recombinant plasmid, the one section size that cuts the lat gene is about the HindIII-ScaI gene fragment of 0.4kb, reclaim the linear pKCLHS fragment of about 7.8kb (the DV 805A of Takara company glue reclaims test kit), mend flat with T4 archaeal dna polymerase (Takara company product) to its two sticky end, it is as follows to mend flat system: sterilization distilled water: 3.5 μ L, T4 dna polymerase buffer liquid (10 *): 1 μ L, 0.1%BSA:1 μ L, reclaim pKCLHS:2.5 μ L, dNTP (2mM): 1 μ L, the total system of filling-in: 9 μ L; This system at first is transferred to 37 ℃ and add the T4 archaeal dna polymerase of 0.5 μ L 4U/ μ L then in 70 ℃ of insulation 5min, gently mixing with mixture in 37 ℃ the insulation 5min after, be placed in the ice through the vibrator concuss, the benefit of the gained thing of showing no increases in output is used for ligation immediately.

With the pKCLHS T that mends after putting down ₄Dna ligase (Takara company product) carries out from connecting, linked system is as follows: T4 dna ligase damping fluid (10 *): 2 μ L, mend flat pKCLHS:9.5 μ L, T4 dna ligase (10U/ μ L): 1.5 μ L, sterilization distilled water: 7 μ L, the total system of ligation: 20 μ L; This system is connected 12h in 16 ℃ of constant temperature, connect product and be used for Transformed E .coliDH5 α.

The method of the preparation of E.coliDH5 α electric shocking method competent cell and electric shock Transformed E .coliDH5 α referring to the step in the present embodiment 3..The gained transformant is extracted plasmid and carry out enzyme and cut checking, the size of the linear plasmid pKCLHS before connecting is about 7.8kb, connects the correct recombinant plasmid pKCLHS of back gained size and also should be 7.8kb.Institute's upgrading grain is carried out single endonuclease digestion with EcoRI, and restriction enzyme digestion and electrophoresis figure sees Figure 10, wherein: a.HindIII single endonuclease digestion pKCLES; B.HindIII/KpnI double digestion pKCLES; C.EcoRI single endonuclease digestion pKC1139-lat; D.10kb Marker; E.EcoRI single endonuclease digestion pKCLHS; E.KpnI single endonuclease digestion pKCLHS.Size is that the plasmid of the dna fragmentation of a 7.8kb can be defined as correct pKCLHS from connecting the gained recombinant plasmid behind the single endonuclease digestion.Above-mentioned used restriction enzyme is Sangon company product, the endonuclease reaction system is with reference to the restriction enzyme specification sheets, the preparation and the electric shock transformation method of intestinal bacteria electric shocking method transformed competence colibacillus cell, and alkaline process extracts the method for e. coli plasmid dna all referring to document [Sambrook J., Fritsch E.F., Maniatis T. (Jin Dongyan etc. translate), molecular cloning molecular cloning experiment guide, second edition, Beijing: Science Press, 1999]

Embodiment 2

The structure of recombinant plasmid pKCLES

Concrete grammar is referring to 1. 2. 3. 4. four steps among the embodiment 1, the 5. the among step and the embodiment 1 5. to go on foot used restriction enzyme different, specific as follows:

PKC1139-lat carries out the EcoRI-ScaI double digestion with the gained recombinant plasmid, the one section size that cuts the lat gene is about the EcoRI-ScaI gene fragment of 1.4kb, reclaim the linear pKCLES fragment of about 7.0kb (DV805A of Takara company glue reclaims test kit), to its two viscosity art end T ₄Archaeal dna polymerase (Takara company product) is mended flat, and it is as follows to mend flat system: sterilization distilled water: 3.5 μ L, T4 dna polymerase buffer liquid (10 *): 1 μ L, 0.1%BSA:1 μ L, reclaim pKCLES:2.5 μ L, dNTP (2mM): 1 μ L, the total system of filling-in: 9 μ L; This system at first is transferred to 37 ℃ and add the T of 0.5 μ L 4U/ μ L then in 70 ℃ of insulation 5min ₄Archaeal dna polymerase, mixing behind 37 ℃ of insulation 5min, is placed on mixture in the ice through the vibrator concuss gently, the benefit thing of showing no increases in output be used for ligation immediately.

With the pKCLES T that mends after putting down ₄Dna ligase (Takara company product) carries out from connecting, and linked system is as follows: T ₄Dna ligase damping fluid (10 *): 2 μ L, mend flat pKCLES:9.5 μ L, T4 dna ligase (10U/ μ L): 1.5 μ L, sterilization distilled water: 7 μ L, the total system of ligation: 20 μ L; This system is connected 12h in 16 ℃ of constant temperature, connect product and be used for Transformed E .coliDH5 α.

The method of the preparation of E.coliDH5 α electric shocking method competent cell and electric shock Transformed E .coliDH5 α referring to step among the embodiment 1 3..The gained transformant is extracted plasmid and carry out enzyme and cut checking, the size of the linear plasmid pKCLES before connecting is about 7.0kb, connects the correct recombinant plasmid pKCLES of back gained size and also should be 7.0kb.Institute's upgrading grain is carried out single endonuclease digestion with HindIII, and restriction enzyme digestion and electrophoresis figure sees Figure 10.Size is that the plasmid of the dna fragmentation of a 7.0kb can be defined as correct pKCLES from connecting the gained recombinant plasmid behind the single endonuclease digestion.Above-mentioned used restriction enzyme is Sangon company product, the endonuclease reaction system is with reference to the restriction enzyme specification sheets, the preparation and the electric shock transformation method of intestinal bacteria electric shocking method transformed competence colibacillus cell, and alkaline process extracts the method for e. coli plasmid dna all referring to document [Sambrook J., Fritsch E.F., Maniatis T. (Jin Dongyan etc. translate), molecular cloning molecular cloning experiment guide, second edition, Beijing: Science Press, 1999]

Embodiment 3

The derivative 1 and the construction process thereof that contain the intestinal bacteria-streptomycete shuttle plasmid of clavuligerus lat Gene Partial disappearance

Concrete grammar is referring to 1. 2. 3. 4. four steps among the embodiment 1, the 5. the among step and the embodiment 1 5. to go on foot used restriction enzyme different, concrete steps are as follows:

Adopt and to remove used HinoIII-ScaI of structure plasmid pKCLHS and pKCLES and any two kinds of enzymes in all the other the restriction enzyme site (see figure 3)s the EcoRI-ScaI on the lat gene, as adopt EcoNI-HindIII that the lat gene among the plasmid pKC1139-lat is cut, the one section size that cuts the lat gene is about the EcoNI-HindIII gene fragment of 0.7kb, reclaim about 7.6kb linear fragment, mend flat with T4 archaeal dna polymerase (Takara company product) to its two sticky end, it is as follows to mend flat system: sterilization distilled water: 3.5 μ L, T4 dna polymerase buffer liquid (10 *): 1 μ L, 0.1%BSA:1 μ L, reclaim dna fragmentation: 2.5 μ L, dNTP (2mM): 1 μ L, the total system of filling-in: 9 μ L; This system at first is transferred to 37 ℃ and add the T4 archaeal dna polymerase of 0.5 μ L 4U/ μ L then in 70 ℃ of insulation 5min, gently mixing with mixture in 37 ℃ the insulation 5min after, be placed in the ice through the vibrator concuss, the benefit thing of showing no increases in output be used for ligation immediately.

With the dna fragmentation T behind the end-filling ₄Dna ligase (Takara company product) carries out from connecting, and linked system is as follows: T4 dna ligase damping fluid (10 *): 2 μ L, mend flat dna fragmentation: 9.5 μ L, T ₄Dna ligase (10U/ μ L): 1.5 μ L, sterilization distilled water: 7 μ L, the total system of ligation: 20 μ L; This system is connected 12h in 16 ℃ of constant temperature, connect product and be used for Transformed E .coliDH5 α.

The method of the preparation of E.coliDH5 α electric shocking method competent cell and electric shock Transformed E .coliDH5 α referring to step among the embodiment 1 3..The gained transformant is extracted plasmid and carry out enzyme and cut checking.Above-mentioned used restriction enzyme is Sangon company product, the endonuclease reaction system is with reference to the restriction enzyme specification sheets, the preparation and the electric shock transformation method of intestinal bacteria electric shocking method transformed competence colibacillus cell, and alkaline process extracts the method for e. coli plasmid dna all referring to document [Sambrook J., Fritsch E.F., Maniatis T. (Jin Dongyan etc. translate), molecular cloning molecular cloning experiment guide, second edition, Beijing: Science Press, 1999]

Embodiment 4

The derivative 2 and the construction process thereof that contain the intestinal bacteria-streptomycete shuttle plasmid of clavuligerus lat Gene Partial disappearance

Adopt and to remove used HinIII-ScaI of structure plasmid pKCLHS and pKCLES and any two kinds of enzymes in all the other the restriction enzyme site (see figure 3)s the EcoRI-ScaI on the lat gene, as adopt SplI-EcoNI that the lat gene among the plasmid pKC1139-lat is cut, the one section size that cuts the lat gene is about the SplI-EcoNI gene fragment of 0.6kb, reclaim about 7.7kb linear fragment, all the other methods are with embodiment 3.

Sequence table

SEQUENCE?LISTING

＜110〉University Of Science and Technology Of Tianjin

＜120〉plasmid of bar streptomycete Lat gene loss, derivative and construction process thereof

<130>041012

<160>3

<170>PatentIn?version?3.1

<210>1

<211>39

<212>DNA

＜213〉artificial sequence

<400>1

ccataattca?gcctgatccc?ccaggagttc?tcacccatg 39

<210>2

<211>39

<212>DNA

＜213〉artificial sequence

<400>2

atcgcgggcc?ggccggccca?cggggtcgcc?ccgggccgg 39

<210>3

<211>1779

<212>DNA

＜213〉clavuligerus

<220>

<221>gene

<222>(1)..(1779)

<223>

<400>3

gaattcccct?gaacacgaag?ctgagcaaca?gctcgtcacg?cgctcccgag?ctggccattc 60

agggcagttc?acaaagagcc?atcgagaggc?gtccgagaga?gctggaagag?gggtccaaga 120

gcatggtggg?tcattattgt?gatcctaaaa?tgtccagttc?accgccatga?cagcagaggc 180

tggaaagtcc?cccataattc?agcctgatcc?cccaggagtt?ctcacccatg?ggcgaagcag 240

cacgccaccc?cgacggcgat?ttctcggacg?tgggaaacct?ccacgctcag?gacgtgcacc 300

aggcacttga?gcagcatatg?ctcgtcgacg?ggtacgacct?cgttctcgac?ctcgacgcca 360

gctccggcgt?ctggctcgtc?gacgccgtca?cccagaagcg?gtatctcgac?ctcttttcct 420

tctttgcctc?ggcgccgctc?ggaatcaacc?cgcccagcat?tgtcgaggac?ccggcattca 480

tgcgggagct?ggccgtggcc?gcggtcaaca?agccgtcgaa?ccccgatctt?tattcggtgc 540

cgtacgcccg?tttcgtcaag?accttcgccc?gggtcctcgg?cgacccccgg?ctgcggcggc 600

tgttcttcgt?ggacggcggg?gcgctggccg?tggagaacgc?gctcaaggcg?gccctcgact 660

ggaaggccca?gaagctgggc?ctcgccgagc?cggacaccga?ccggctccag?gtgctgcatc 720

tggagcgctc?gttccacggc?cgcagcggct?acaccatgtc?gctgacgaac?accgagccgt 780

ccaagaccgc?ccgcttcccc?aagttcggct?ggccacggat?ctcgtccccc?gccctccagc 840

acccgccggc?cgagcacacc?ggcgccaacc?aggaggccga?gcgacgggcg?ctggaggccg 900

cccgggaggc?gttcgcagcg?gcggacggca?tgatcgcctg?cttcatcgcg?gagcccatcc 960

agggcgaggg?cggcgacaac?cacctcagcg?cggagttcct?ccaggccatg?cagcggctct?1020

gccacgagaa?cgacgccctg?ttcgtcctgg?acgaggtgca?gagcggctgc?ggcatcaccg?1080

gtaccgcctg?ggcctaccag?cagctcggcc?tccagcccga?cctggtggcc?ttcggcaaga?1140

agacccaggt?ctgcggggtg?atgggcggcg?gccggatcga?cgaggtcccc?gagaacgtct 1200

tcgccgtctc?ctcccggatc?agctccacct?ggggcggcaa?cctcgccgac?atggtccgcg 1260

ccacccggct?gctggagacg?atcgagcgca?cccaggtctt?cgacaccgtc?gtccagcgcg 1320

gcaagtactt?ccgggacggc?ctggaggacc?tggccgcccg?ccacccctcc?gtcgtgacca 1380

acgcccgcgg?ccggggcctg?atgtgcgcgg?tcgacctgcc?ggacacccgg?acccgcaatg 1440

aggtgctgcg?gctcatgtac?acggagcacc?aggtcatcgc?cctgccctgc?ggcgggcgca 1500

gcctccggtt?ccgccccgcg?ctgacgatcg?cggagcacga?gatcgaccag?gcccttcagg 1560

cgctggcgag?cagtgtcacg?ccggtcgccg?agagcgtctg?acgcccggcc?ggccgggtgc 1620

cccagcgggg?cccggccggc?cgcaccgcgg?aacggaacac?ccctggcgcg?tcgcccggtg 1680

acacgctctc?cgggcggctc?aaccgcgcca?cccccacggc?acgttcgcct?tcacccgcac 1740

ggagagccca?cgaatgatgt?cagcacggta?caagcttat 1779

Claims (6)

1. the plasmid of clavuligerus (Streptomyces clavuligerus) lat genetically deficient, it is characterized in that upstream EcoRI that plasmid comprises the coding Methionin ε-aminotransferase gene in the clavuligerus partial sequence to the ScaI in the partial sequence of ScaI or downstream to HindIII, the replication origin that comprises Escherichia coli source plasmid and streptomycete source plasmid on the plasmid, for having the shuttle plasmid of all effable apramycin resistance selective marker in intestinal bacteria and streptomycete, and has pSW344E temperature sensitive type replicon gene; Wherein plasmid pKCLHS size is about 7.8kb, and is represented by the estriction map of Fig. 1; Plasmid pKCLES size is about 7.0kb, and is represented by the estriction map of Fig. 2.

2. the plasmid derivative thing of clavuligerus (Streptomyces clavuligerus) lat genetically deficient is characterized in that the plasmid derivative thing comprises and sees Fig. 3 except that making up used HindIII-ScaI of plasmid pKCLHS and pKCLES and the partial sequence between all the other any two restriction enzyme sites the EcoRI-ScaI; The derivative size of plasmid pKCLHS and pKCLES is the 6.5kb-8.3kb except that 7.0kb, 7.8kb.

3. the construction process of the plasmid of clavuligerus (Streptomyces clavuligerus) lat genetically deficient, the construction process that it is characterized in that plasmid pKCLHS and pKCLES: at first make up the recombinant plasmid pUCm-T-lat that contains goal gene lat, by the design primer performing PCR amplification in vitro of going forward side by side, obtain the lat gene fragment that size is about 1.8kb, select for use pUCm-T carrier with multiple clone site and the lat gene fragment behind the purifying with T ₄Dna ligase connects and obtains recombinant plasmid pUCm-T-lat; With EcoRI-HindIII double digestion pUCm-T-lat, purified back of the lat gene fragment that obtains and the carrier pKC1139 behind EcoRI-HindIII double digestion and purifying are with T ₄Dna ligase connects, and obtains recombinant plasmid pKC1139-lat; PKC1139-lat reclaims the dna fragmentation of about 7.8kb or 7.0kb respectively through HindIII-ScaI or EcoRI-ScaI double digestion, the recovery product behind two kinds of purifying is at first all with T ₄Archaeal dna polymerase is mended flat terminal, again with T ₄Dna ligase carries out from connecting, obtain one section HindIII-ScaI of lat genetically deficient and segmental recombinant plasmid pKCLHS of EcoRI-ScaI and pKCLES respectively, building process is represented that by Fig. 4 whole plasmid construction process is finished in intestinal bacteria Escherichia coli DH5 α.

4. the construction process of the plasmid derivative thing of clavuligerus (Streptomyces clavuligerus) lat genetically deficient, it is characterized in that: at first make up the recombinant plasmid pUCm-T-lat that contains goal gene lat, by the design primer performing PCR amplification in vitro of going forward side by side, obtain the lat gene fragment that size is about 1.8kb, select for use pUCm-T carrier with multiple clone site and the lat gene fragment behind the purifying with T ₄Dna ligase connects and obtains recombinant plasmid pUCm-T-lat; With EcoRI-HindIII double digestion pUCm-T-lat, purified back of the lat gene fragment that obtains and the carrier pKC1139 behind EcoRI-HindIII double digestion and purifying are with T ₄Dna ligase connects, and obtains recombinant plasmid pKC1139-lat; The restriction enzyme that adopts is that the remove used HindIII-ScaI of structure plasmid pKCLHS and pKCLES and all the other restriction enzyme sites the EcoRI-ScaI on the lat gene are seen any two kinds of restriction enzymes among Fig. 3, lat gene among the plasmid pKC1139-lat is cut and reclaim remaining dna fragmentation after the lat genetically deficient, and the recovery product behind the purifying is at first with T ₄Archaeal dna polymerase is mended flat terminal, again with T ₄Dna ligase carries out obtaining the plasmid derivative thing of lat genetically deficient from connecting, and whole plasmid derivative thing building process is finished in intestinal bacteria Escherichia coli DH5 α.

5. according to the construction process of the plasmid of the described clavuligerus of claim 3 (Streptomyces clavuligerus) lat genetically deficient, it is characterized in that the lat gene the primer of PCR amplification in vitro coding Methionin ε-transaminase has the dna sequence dna shown in SEQ NO ID:1 or 2;

Primer 1:5 '-CCATAATTCAGCCTGATCCCCCAGGAGTTCTCACCCATG-3 '

Primer 2: 5 '-ATCGCGGGCCGGCCGGCCCACGGGGTCGCCCCGGGCCGG-3 '.

6. according to the construction process of the plasmid derivative thing of the described clavuligerus of claim 4 (Streptomyces clavuligerus) lat genetically deficient, it is characterized in that the lat gene the primer of PCR amplification in vitro coding Methionin ε-transaminase has the dna sequence dna shown in SEQ NO ID:1 or 2;

Primer 1:5 '-CCATAATTCAGCCTGATCCCCCAGGAGTTCTCACCCATG-3 '

Primer 2: 5 '-ATCGCGGGCCGGCCGGCCCACGGGGTCGCCCCGGGCCGG-3 '.

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