CN108913644A - A kind of the attenuation recombination Listeria monocytogenes and preparation method of genome foreign gene-carrying - Google Patents

Abstract

The present invention relates to a kind of attenuation of genome foreign gene-carrying recombination Listeria monocytogenes and preparation methods, foreign gene (being not limited only to antigen gene) is integrated into Listeria monocytogenes, any antibiotic drug resistant gene is not introduced, it is complete simultaneously to knock out bacterium actA, plcB virulence gene, prepared attenuation recombination Listeria monocytogenes can induce high-caliber cellullar immunologic response, can provide reliable technical method to the preparation of Listeria monocytogenes carrier bacterin.

Description

A kind of attenuation recombination Listeria monocytogenes of genome foreign gene-carrying and preparation Method

Technical field

The invention belongs to field of biotechnology, are related to a kind of single increasing Liszt of attenuation recombination of genome foreign gene-carrying Bacterium and preparation method.

Background technique

Listeria monocytogenes (Listeria monocytogenes, hereinafter abbreviation Lm) due to it is unique can be The biological characteristics grown in the host phagocytes slurry of infection, become good T cell immune activation adjuvant, in vaccine It is widely applied in preparation, has partially entered clinical test rank by the recombination viable bacteria therapeutic tumor vaccine of carrier of Lm Section.Such as two kinds of recombinant L m live bacterial vaccines (Lm-LLO-CD105A and Lm-LLO- of Wood LM et al. report building CD105B the primary and metastatic tumo(u)r tissue volume, reduction breast cancer that) can be substantially reduced breast cancer mouse animal model are certainly Send out mouse model Tumor incidence (Wood LM, Pan ZK, Guirnalda P, et al.Targeting tumor vasculature with novel Lmsteria-based vaccines directed against CD105.Cancer Immunol Immunother.2011,60(7): 931-942).In addition, there are also some using Listeria monocytogenes as the epidemic disease of background Seedling comes into clinical test such as PSA, (Wood, Laurence M., and the Yvonne Paterson.2014. such as Her2 “Attenuated Listeria Monocytogenes:A Powerful and Versatile Vector for the Future of Tumor Immunotherapy.”Frontiers in Cellular and Infection Microbiology 4:51–51.)。

But the foreign gene that above-mentioned recombination Lm bacterium carries is introduced in a manner of plasmid, and bacterial gene is not integrated into Group, so the unstable expression of target gene, it is possible to be lost with plasmid loss, and the introducing of plasmid has also brought it into His non-tumour antigen related gene, especially above-mentioned plasmid introduce chloramphenicol resistance gene, cause to the propagation of bacterial resistance It threatens.Since Lm can cause a disease to people, pretends the Lm for vaccine carrier and need to strictly be knocked out pathogenic related gene and be attenuated, And the attenuated approach of the existing Lm bacterial strain as vaccine carrier is indefinite, only part knocks out virulence gene to some, and there are virulence Reversion may.

Summary of the invention

In view of this, the purpose of the present invention is to provide a kind of attenuations of genome foreign gene-carrying to recombinate single increasing Li Si Special bacterium and preparation method.

In order to achieve the above objectives, the present invention provides the following technical solutions：

A kind of attenuation recombination Listeria monocytogenes of genome foreign gene-carrying, the attenuation Listeria monocytogenes are single Increase Listeria (Listeria monocytogenes, abbreviation Lm) completely knock out the two virulence genes of actA and plcB and ?.

Preferably, III restriction enzyme site of upstream belt Hind of the foreign gene, I restriction enzyme site of downstream belt Xho.

A kind of preparation method of the attenuation recombination Listeria monocytogenes of above-mentioned genome foreign gene-carrying, is to be attenuated list Increasing Listeria be carrier, foreign gene-carrying and obtain.

Preferably, specific step is as follows：

(1) by the Hind III digestion position of foreign gene insertion plasmid pCW203 (patent CN103074361B) of quasi- integration Between point and Xho I restriction enzyme site, intermediate recombinant plasmid is obtained；

(2) it cuts between the BamH I restriction enzyme site for the intermediate recombinant plasmid that step (1) obtains and Xho I restriction enzyme site Segment is inserted between the BamH I restriction enzyme site and Xho I restriction enzyme site of recombinant plasmid pCW180, and target practice plasmid is obtained, described heavy The gene order of group plasmid pCW180 is shown in SEQ ID NO.1 in sequence table；

(3) the target practice plasmid electrotransformation for obtaining step (2) recombinates Listeria monocytogenes Lm Δ actAplcB-lacZ, so The bacterium that conversion is obtained afterwards hybridizes through single, double homologous recombination to be cultivated, and object bacteria is obtained.

It is further preferred that in step (1), the preparation method of the foreign gene is：From biological genome template or base Because cloned plasmids library PCR amplification obtains or DNA synthesizer directly synthesizes.

It is further preferred that the preparation method of recombinant plasmid pCW180 is as follows in step (2)：

Upstream is carried Spe I by (2-1), downstream carries Lm mpl gene (the Genbank ID of Not I：DQ054595.1) It is inserted between I restriction enzyme site of Spe I and Not of plasmid pCW154 (patent CN103074361B), obtains the first intermediate recombinant plasmid The gene order of pCW160, the first intermediate recombinant plasmid pCW160 is shown in SEQ ID NO.2 in sequence table；

Upstream is carried Xba I by (2-2), downstream carries Lm orfBAldh gene (the Genbank ID of Not I： M82881.1) I restriction enzyme site of Xba I and Not of the first intermediate recombinant plasmid pCW160 that insertion above-mentioned steps (2-1) obtains it Between, the second intermediate recombinant plasmid pCW170 is obtained, the gene order of the second intermediate recombinant plasmid pCW170 is in sequence table Shown in SEQ ID NO.3；

(2-3) uses I digested plasmid pCW154 of Not, obtains one section of gene that both ends are I restriction enzyme site of Not, is inserted into above-mentioned step Suddenly I restriction enzyme site of Not for the second intermediate recombinant plasmid pCW170 that (2-2) is obtained, obtains recombinant plasmid pCW180.

It is further preferred that recombinating the preparation method of Listeria monocytogenes Lm Δ actAplcB-lacZ such as in step (3) Under：

(3-1) lacZ gene segment (patent CN103074361B) insertion with Not I restriction enzyme site respectively by upstream and downstream Between the Not I restriction enzyme site of second intermediate recombinant plasmid pCW170, a target practice plasmid pCW190, the plasmid are obtained The gene order of pCW190 is shown in SEQ ID NO.4 in sequence table；

(3-2) by target practice plasmid pCW190 electrotransformation Lm obtained by step (3-1), the bacterium for then obtaining conversion is through single, double Homologous recombination hybridization culture to get.

It is further preferred that recombinating the genome of Listeria monocytogenes Lm Δ actAplcB-lacZ in step (3) OrfBAldh upstream region of gene has been integrated into beta-galactosidase gene, can express beta galactosidase, in the chloro- 3- containing 5- bromo- 4 Grow up to blue colonies on the culture medium of indoles-β-D- galactoside.

It is further preferred that orfBAldh upstream region of gene incorporates external source in the genome of the object bacteria in step (3) Gene.

The beneficial effects of the present invention are：

Foreign gene (being not limited only to antigen gene) is integrated into Lm by the present invention, does not introduce any antibiotic drug resistant gene, Complete simultaneously to knock out bacterium actA, plcB virulence gene, prepared attenuation recombination Listeria monocytogenes can induce high-caliber Cellullar immunologic response can provide reliable technical method to the preparation of Listeria monocytogenes carrier bacterin.It is specific as follows：

1, the recombination Listeria monocytogenes of foreign gene-carrying can induce efficient antigentic specificity CD4 in vivo⁺And CD8⁺Carefully Born of the same parents' immune response, the IFN-γ cell factor of secreting high levels, compared to the recombination sheep Listeria of foreign gene-carrying For (Listeria ivanovii, abbreviation Li), the secretory volume of the antigen specific cytokine of induction is significantly improved.

2, the Disease-causing gene actA and plcB for having knocked out bacterium, ensure that the safety of recombinant bacterium.Animal experiments show that subtracting The LD50 of bacterium after poison is significantly improved than wild strain, significant to animal vitals liver, the pathology damage of spleen after inoculation animal It reduces, is also greatly speeded up by the speed that body is removed.

3, recombinant bacterium is free of drug resistant gene, ensure that it in the environmental safety for preparing the application of the fields such as vaccine.

4, preparation manipulation is simple, and screening is easy.Preparation method only needs simple a few step common molecular clones, Bacteria Culture etc. Relevant operation, it is low in cost to the of less demanding of instrument and equipment and operating technology, after being successfully prepared, recombinant bacterium can mass propagation, It is easy to be mass produced.Object bacteria is screened using colony colour and antibiotics resistance situation, object bacteria is intolerant to the white of erythromycin Color bacterium colony, screening technique are simply easy, and naked eyes can determine whether.

5, recombination result is stablized.Fixed point integration of foreign gene is thin after integration to Lm genome orfBAldh upstream region of gene Bacterium growth is unaffected, and the foreign gene after integration is stable in the presence of bacterial genomes, will not lose, and overcoming recombinant plasmid can The risk of missing that can have.

6, label is complete, convenient for detection.CD8+ and CD4+T cell recognition epitopes are carried after exogenous origin gene integrator convenient for detection Intend specific C D8+ and the CD4+T cell response of integrator gene for external source；It carries HA western blot and detects label, just In the fusion protein of western blot detection external source integrator gene coding.

Detailed description of the invention

In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Explanation：

Fig. 1 is a kind of structure map of the first intermediate recombinant plasmid pCW160, contains ampicillin resistance gene (Amp), erythromycin resistance gene (Ery), Lm mpl gene are located between I restriction enzyme site of Spe I and Not, Li orfBAldh base Because between I restriction enzyme site of Xba I and Not.

Fig. 2 is a kind of structure map of the second intermediate recombinant plasmid pCW170, contains ampicillin resistance gene (Amp), erythromycin resistance gene (Ery), Lm mpl gene are located between I restriction enzyme site of Spe I and Not, Lm orfBAldh base Because between I restriction enzyme site of Xba I and Not.

Fig. 3 is a kind of structure map of recombinant plasmid pCW180, contains ampicillin resistance gene (Amp), erythromycin Resistant gene (Ery), Lm mpl gene are located between I restriction enzyme site of Spe I and Not, and Lm orfBAldh gene is located at I He of Xba Between I restriction enzyme site of Not, gene order box (gene cassette) is located between two I sites Not：Include promoter Phly, secreting signal peptide (secret signal), HA epitope gene, VSV-G epitope gene, GP33epitope base Cause, GP61epitope gene, PRRSV ORF5 gene, the BamH I for foreign gene insertion, I site Xho.

Fig. 4 is a kind of structure map of target practice plasmid pCW190, contains ampicillin resistance gene (Amp), erythromycin Resistant gene (Ery), Lm mpl gene are located between I restriction enzyme site of Spe I and Not, and Lm orfBAldh gene is located at I He of Xba Between I restriction enzyme site of Not, lacZ gene is located between two I sites Not.

The PCR that Fig. 5 is Lm Δ actAplcB-lacZ identifies electrophoresis result figure, and in figure, 1 is with Lm Δ actAplcB-lacZ Genome is template amplification actA, and 2 is using Lm Δ actAplcB-lacZ genomes as template amplification orfBAldh, 3~4 for Lm Δ actAplcB-lacZ genome is template amplification Ery, and 5 be Ery gene magnification positive control, and 6 is using Lm genomes as mould Plate expands lacZ, and 7~8 is, using Lm Δ actAplcB-lacZ genome as template amplification lacZ, M are DNA ladder.

Fig. 6 is that blue hickie screens the successful positive restructuring bacterium of homologous recombination.After homologous recombination, bacterium becomes white from blue Color easily preliminary screening can go out positive bacteria by the identification of color (arrow meaning is one of them in figure).

Fig. 7 is the successful positive restructuring bacterium of antibiotic-screening homologous recombination.After homologous recombination, bacterium becomes from resistance to erythromycin Intolerant to erythromycin, scribing line has the plate of erythromycin and the plate without erythromycin to the same bacterium colony simultaneously, is only capable of in no erythromycin Growth on plate (arrow meaning is one of them in figure).

Fig. 8 is the homologous recombination process signal for preparing the recombinant attenuated Listeria monocytogenes that genome carries mesothelin gene Figure.

Fig. 9 is the survivorship curve that C57BL/6 mouse tail vein is inoculated with after Lm, Lm △ actAplcB-MESO.A is mouse tail Survivorship curve after the Lm of intravenous inoculation 3 dosage as shown in the figure, B are the Lm that mouse tail vein is inoculated with 3 dosage as shown in the figure Survivorship curve after △ actAplcB-MESO.

Figure 10 is Lm (dosage of inoculation：4×10⁴) and Lm △ actAplcB-MESO (dosage of inoculation Lm △ CFU actAplcB-MESO：7.8×10⁶) become by the pathology of mouse liver caused by the 3rd day and spleen after tail vein Mice Inoculated Change.A：Normal liver tissue (100 ×)；C：The hepatic tissue (100 ×) of mouse after Lm is inoculated with 3 days, it is seen that inflammation stove is in irregular shape, In shape is diffused, inflammatory cell infiltration is obvious；E：The hepatic tissue (200 ×) of mouse after Lm △ actAplcB-MESO is inoculated with 3 days, can See that the extensive balloon sample of liver cell becomes, cytoplasm is in empty balloon-shaped；B：Normal spleen tissue (200 ×)；D：Mouse after Lm is inoculated with 3 days Spleen tissue (40 ×), it is seen that serious, extensive tissue necrosis causes that acini lienalis is in irregular shape, edge blurry (arrow in figure It is shown), destroy the normal basic organizational structure of spleen；F：The spleen tissue of mouse after Lm △ actAplcB-MESO is inoculated with 3 days (200 ×), it is seen that spleen splenic corpuscle lymphocyte is loose, visible a small amount of neutrophil infiltration in red pulp.As seen from the figure, it is attenuated Caused pathology damage is lighter in the same time in the case where quantity of microorganism inoculated is bigger for bacterial strain, is demonstrated by lower toxicity.

Figure 11 is immune by Flow cytometry Lm Δ actAplcB-MESO and Li Δ actAplcB-MESO bacterial strain The cellullar immunologic response induced after mouse.Li Δ actAplcB-MESO is the recombination sheep Liszt for carrying mesothelin gene Bacterium, Lm Δ actAplcB-MESO and Li Δ actAplcB-MESO vaccine strains are small with the dosage tail vein inoculation of 0.1 × LD50 Mouse is immunized the specificity cellular immunity response that mouse induction generates using Flow cytometry, predominantly detects IFN-γ cell The secretion level of the factor evaluates vaccine candidate strain immune effect.A is the mouse that Li Δ actAplcB-MESO has been immunized Splenocyte is under the stimulation of GP33 peptide fragment, the secretion level of IFN-γ cell factor；B is that Lm Δ actAplcB-MESO has been immunized Mouse boosting cell is under the stimulation of GP33 peptide fragment, the secretion level of IFN-γ cell factor.As shown, carrying same antigen gene The amount of antigentic specificity IFN-γ of recombination Listeria monocytogenes induction improved more than 30 compared with recombinating sheep Listeria Times.

Specific embodiment

Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.

In the following embodiments of the present invention：Plasmid pCW203 from patent CN103074361B, plasmid pCW154 from Patent CN103074361B, Escherichia coli (Escherichia coli, E.coli) Top10 are century biology section purchased from Beijing health Skill Co., Ltd；Various molecular biology kits and biochemical reagents are purchased from Beijing CoWin Bioscience Co., Ltd.；Respectively Kind biology tool enzyme is purchased from New England Biolabs；Culture medium is purchased from Beijing Luqiao Technology Co., Ltd.； Primer synthesis and gene sequencing are completed by the Shanghai Invitrogen company.

Embodiment 1：The preparation of recombinant plasmid (pCW180)

(1) upstream carries the acquisition of the Lm mpl gene of Spe I, downstream carrying Not I

Lm mpl gene order is obtained from NCBI, I restriction enzyme site of Spe is added in upstream region of gene, I enzyme of Not is added in downstream Designed upstream is carried Spe I by enzyme site, the Lm mpl gene order of downstream carrying Not I hands over the Shanghai Invitrogen public Department synthesizes target DNA fragments with DNA synthesizer.

(2) it between I restriction enzyme site of Spe I and Not of Lm mpl genetic fragment insertion plasmid pCW154, obtains among first Recombinant plasmid pCW160

Respectively with I double digestion of Spe I and Not, double digestion system is 10 × buffer 3 by Lm mpl genetic fragment and pCW154 μ l, gene samples 15 μ l, Spe I 0.6 μ l, Not I 0.6 μ l, ddH₂O 10.8 μ l, 37 DEG C of water-bath 1h.Lm mpl gene piece Section double enzyme digestion product is recycled with universal DNA purification and recovery kit.Plasmid pCW154 makees through double digestion, and with alkaline phosphatase After dephosphorylation process, make 0.8% agarose gel electrophoresis, is recycled with quick Ago-Gel DNA QIAquick Gel Extraction Kit a length of The segment of 7325bp.Genetic fragment after the recovery and plasmid fragments T₄Ligase connection, obtains the first intermediate recombinant plasmid PCW160 (Fig. 1).Coupled reaction system and reaction condition are：10×T₄1 μ l of Lmgase reaction buffer, gene piece Section digestion regenerant 1 μ l, plasmid enzyme restriction regenerant 4 μ l, T₄Ligase 1 μ l, ddH₂3 μ l of O reacts at room temperature 2h.

Connect reaction solution transformed competence colibacillus E.coli Top10：5 μ l connection reaction solutions are inhaled in 50 μ l of pre-cooling with pre-cooling pipette tips In competence E.coli Top10, flicking tube bottom with finger is uniformly mixed liquid, sets ice bath 30min, 42 DEG C of water-bath 30s, ice Bathe 3min.The SOC culture medium of 37 DEG C of 250 μ l preheatings is added, cultivates 1h in 37 DEG C of 200r/min, takes 150 μ l bacterium solutions in containing 100 μ LB plate (hereinafter referred to as LB-Amp) center of g/ml ampicillin, is coated with entire plate for bacterium solution with sterile L shape glass rod, In 37 DEG C cultivate 18~for 24 hours, positive bacteria (E.coli/pCW160) grows up to white colony on the plate.It extracts and contains in positive bacteria Some recombinant plasmids are identified with Spe I and I double digestion of Not.

(3) upstream carries the acquisition of the Lm orfBAldh gene of Xba I, downstream carrying Not I

Lm orfBAldh gene order is obtained from NCBI, I restriction enzyme site of Xba is added in upstream region of gene, downstream is added Designed upstream is carried Xba I by I restriction enzyme site of Not, the Lm orfBAldh gene order of downstream carrying Not I is handed over The Shanghai Invitrogen company synthesizes target DNA fragments with DNA synthesizer.

(4) between I restriction enzyme site of Xba I and Not of Lm orfBAldh genetic fragment insertion plasmid pCW160, second is obtained Intermediate recombinant plasmid pCW170

Lm orfBAldh genetic fragment and pCW160 respectively with I double digestion of Xba I and Not, double digestion system is 10 × 3 μ l of buffer, gene samples 15 μ l, Xba I 0.6 μ l, Not I 0.6 μ l, ddH₂O 10.8 μ l, 37 DEG C of water-bath 1h.Lm OrfBAldh genetic fragment double enzyme digestion product is recycled with universal DNA purification and recovery kit.Plasmid pCW160 through double digestion, And after with alkaline phosphatase making dephosphorylation process, make 0.8% agarose gel electrophoresis, is recycled with quick Ago-Gel DNA Kit recycles the segment of a length of 6941bp.Genetic fragment after the recovery and plasmid fragments T₄Ligase connection, obtains second Intermediate recombinant plasmid pCW170 (Fig. 2).Coupled reaction system and the same step of reaction condition (2).

Connect the same step of screening (2) of reaction solution transformed competence colibacillus E.coli Top10 and positive bacteria.It extracts in positive bacteria The recombinant plasmid contained is identified with Xba I and I double digestion of Not.

(5) I restriction enzyme site of Not of one section of Not, the I digestion genetic fragment insertion plasmid pCW170 of pCW154, is recombinated Plasmid pCW180

For pCW154 and pCW170 respectively with I digestion of Not, digestion system is 10 × buffer, 3 μ l, 15 μ l of gene samples, Not I 0.6 μ l, ddH₂O 11.4 μ l, 37 DEG C of water-bath 1h.It is solidifying that pCW154 genetic fragment digestion products make 0.8% agarose Gel electrophoresis recycles the segment of a length of 1163bp with quick Ago-Gel DNA QIAquick Gel Extraction Kit；Plasmid pCW170 through digestion, and After making dephosphorylation process with alkaline phosphatase, recycled with universal DNA purification and recovery kit.Genetic fragment after the recovery and Plasmid fragments T₄Ligase connection, obtains recombinant plasmid pCW180 (Fig. 3).Coupled reaction system and the same step of reaction condition (2)。

Connect the same step of screening (2) of reaction solution transformed competence colibacillus E.coli Top10 and positive bacteria.It extracts in positive bacteria The recombinant plasmid contained is identified with I digestion of Not.

Embodiment 2：The preparation of recombinant bacterium Lm Δ actAplcB-lacZ

(1) acquisition of lacZ gene segment

The lacZ gene segment of double headed roller Not I restriction enzyme site is obtained as described in patent 201310044170.5.

(2) building prepares the target practice plasmid pCW190 of Lm Δ actAplcB-lacZ

The lacZ and 1 step of embodiment (4) obtained respectively to step (1) obtains pCW170 Not I digestion.Not I enzyme System and reaction condition are cut with step (5) in embodiment 1.LacZ gene segment digestion products are tried with universal DNA purification and recovery The recycling of agent box.Plasmid pCW190 is through digestion, and after making dephosphorylation process with alkaline phosphatase, with universal DNA purification and recovery Kit recycling.Genetic fragment after the recovery and plasmid fragments are by coupled reaction system and reaction condition in 1 step of embodiment (2) It is attached, connection product is pCW190 (Fig. 4).Reaction solution is connected by method transformed competence colibacillus in 1 step of embodiment (2) E.coli Top10.Positive bacteria (E.coli/pCW190) grows up to blue colonies on the LB-Amp plate that surface is coated with X-Gal. The recombinant plasmid contained in positive bacteria is extracted, is identified with Not I digestion.

(3) preparation of electrotransformation Lm competent cell

Lm is inoculated in the BHI fluid nutrient medium of 15ml sucrose containing 0.5mol/L, 37 DEG C of 220rpm cultivate 12~16h.It draws BHI fluid nutrient medium of the 12.5ml bacterium solution to 250ml sucrose containing 0.5mol/L, 37 DEG C of cultures to A₆₀₀For 0.4 or so (about 4h), PenicilLmn G (final concentration is added：12.5 μ g/ml), continue culture to A₆₀₀For 0.7 or so (about 2.5h).Shift bacterium solution in In clean, sterile, pre-cooling 250ml polypropylene centrifuge tube, ice bath 20min is set.4 DEG C of centrifugation 10000r/min 5min, in abandoning Clearly.The ice-cold 0.5mol/L sucrose liquid of 100ml is added, bacterium precipitating is resuspended, in 4 DEG C of centrifugation 10000r/min 5min, abandoning supernatant, then uses The ice-cold 0.5mol/L sucrose liquid of 100ml is washed 2 times.The ice-cold 0.5mol/L sucrose liquid of 2ml is added, bacterium precipitating is resuspended, packing pre-cooling is sterile EP manages (50 μ l/ pipe), saves backup immediately in -70 DEG C.

(4) plasmid pCW190 electrotransformation Lm

It takes out Lm cell prepared by 1 (3) step from -70 DEG C of refrigerators and is put in and melt on ice, on ice with the sucking of pre-cooling pipette tips The 5 μ l of recombinant plasmid pCW190 of pre-cooling, flicking tube bottom with finger is uniformly mixed liquid, sets ice bath 5min.It will with pre-cooling pipette tips Whole liquid suckings are put in electric shock cup (spacing 0.1cm) sample cell of ice bath pre-cooling in EP pipe, put 5min on ice.Take out electric shock Cup is placed in electric converter 1 time (the shock parameters 2KV, 5ms) that shock by electricity, and takes out electric shock cup immediately and puts ice bath 5min.750 μ l are added It is based in electric shock cup in the BHI Liquid Culture of the 0.5mol/L sucrose of 37 DEG C of pre-temperatures, gently with bacterium solution in pipette tips blowout sample cell And mix, whole liquid in electric shock cup are drawn in sterile EP tube, in 30 DEG C of 140rpm shaken cultivation 2h.By whole bacterium solutions drop in Containing erythromycin 1~5 μ g/ml, X-gal 20~60 μ g/ml BHI agar plate (hereinafter referred to as BHI-Ery-X-gal plate) Bacterium solution is coated with entire plate with sterile L shape glass rod by center, in 30 DEG C of 48~72h of culture, positive bacteria (Lm/pCW190) growth For blue colonies.

(5) the bis- homologous recombination hybridization cultures of Lm/pCW190

42 DEG C of culture 48h of Lm/pCW190 streak inoculation BHI-Ery-X-gal plate, the blue colonies grown are crossed again to be connect Kind BHI-Ery-X-gal plate, in 42 DEG C of culture 48h.Picking blue colonies are in 2ml BHI fluid nutrient medium, and 30 DEG C 220rpm shaken cultivation for 24 hours, draws 10 μ l bacterium solutions again in transferred species 2ml BHI fluid nutrient medium, 30 DEG C of 220rpm shaken cultivations For 24 hours, (transferred species) is walked in repetition 4 times.It takes 100 μ l bacterium solution PBS to make 10 times of dilutions, takes 10^-6It is flat to dilute bacterium solution coating BHI-X-gal Plate (the BHI agar plate of 20~60 μ g/ml containing X-gal) and BHI-Ery-X-gal plate, coating dilution bacterium on each plate 100 μ l of liquid.Positive bacterium colony Lm Δ actAplcB-lacZ grows up to blue colonies on BHI-X-gal plate, in BHI-Ery-X- It is not grown on gal plate.

(6) PCR identifies Lm Δ actAplcB-lacZ

Universal pillar genome extraction kit is used to extract Lm Δ actAplcB-lacZ genomic DNA as template, Wherein whether there is or not actA, orfBAldh, Ery and lacZ gene segment for PCR amplification identification, according to amplification to Lm Δ The genetic recombination situation of actAplcB-lacZ is identified.

Amplification actA reaction system be：10 × ultra pfu buffer 2.5 μ l, 4dNTPs (10mmol/L. kind) 0.5 μ l, as shown in SEQ ID NO.5 and SEQ ID NO.6, upstream primer (10 μm of ol/L) (Lm actA-f: 5'- GCTATAAATGAAGAG GCTTCAGG-3 ') 0.8 μ l, downstream primer (10 μm of ol/L) (Lm actA-r: 5'- CTCTTAAATCAGCT AGGCGATC-3 ') 0.8 μ l, 2.5 μ l, ultra pfu of bacterial genomic DNA samples, 0.25 μ l, ddH₂O 17.65μl.Reaction cycle condition is：94 DEG C of 5min → (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s) × 30 circulations → 72 DEG C of 10min → 4 DEG C, amplified production length about 950bp.

Amplification orfBAldh reaction system be：10 × ultra pfu buffer, 2.5 μ l, 4dNTPs (10mmol/L. Kind) 0.5 μ l, as shown in SEQ ID NO.7 and SEQ ID NO.8, upstream primer (10 μm of ol/L) (Lm orfBAldh-f:5'- CTTCGATGACAACAGCTGTACC-3 ') 0.8 μ l, downstream primer (10 μm of ol/L) (Lm orfBAldh-r:5'- AATCCTAAAGCATGCGCCTTCG-3 ') 0.8 μ l, 2.5 μ l, ultra pfu of bacterial genomic DNA samples, 0.25 μ l, ddH₂O 17.65μl.Reaction cycle condition is：94 DEG C of 5min → (94 DEG C of 1min, 57 DEG C of 30s, 72 DEG C of 2min) × 30 circulations → 72 DEG C of 10min → 4 DEG C, amplified production length about 1200bp.

Amplification Ery reaction system be：10 × ultra pfu buffer, 2.5 μ l, 4dNTPs (10mmol/L. kind) 0.5 μ l, as shown in SEQ ID NO.9 and SEQ ID NO.10, upstream primer (10 μm of ol/L) (Ery-f:5'- GTCGACGATTCACAAAAAATAGGC-3 ') 0.8 μ l, downstream primer (10 μm of ol/L) (Ery-r:5'- ACTAGTCCCGGGGCGAATTG-3 ') 0.8 μ l, 2.5 μ l, ultra pfu of bacterial genomic DNA samples 0.25 μ l, ddH₂O 17.65μl.Reaction cycle condition is：94 DEG C of 3min → (94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 2min) × 30 → 72 DEG C of circulations 10min → 4 DEG C, amplified production length about 1200bp.

The primer and reaction system and reaction cycle conditional synchronization of amplification lacZ is rapid (1), amplified production length about 4000bp.

The PCR of Lm Δ actAplcB-lacZ identifies positive findings figure such as Fig. 5.As seen from the figure, it is cultivated through double homologous hybridization, LacZ has been integrated into Lm genome, and bacterium does not carry Ery gene, and actA gene is knocked.

Embodiment 3：Using the recombinant plasmid and recombinant bacterium of above-mentioned preparation, genome foreign gene-carrying is prepared (with mesothelium For plain gene HuMeso) recombinant attenuated Listeria monocytogenes

(1) mesothelin gene segment synthesizes

Mesothelin protein gene is inquired from NCBI, and III restriction enzyme site of Hind is added in upstream, and I enzyme of Xho is added in downstream Then enzyme site is accordingly optimized according to Listeria codon, directly obtain DNA sequence dna (with company finally by synthesis The form for providing cloned plasmids pUC57-Meso obtains), sequence length 1781bp.

(2) target practice plasmid is constructed

1. the building of middle interstitial granules pCW203-Meso

Plasmid pUC57-Meso and pCW203 are extracted according to plasmid specification, with 30 μ L Elution Buffer elution.

PUC57-Meso and pCW203 I digestion of Hind III and Xho.According to 20 μ L systems：PUC57-Meso or pCW203 III 1 I 1 μ L, 10 × NEB Buffer of μ L, Xho of < 1ng, Hind 2 μ L, ddH₂System is supplied 20 μ L by O.37 DEG C of water-bath 1h enzymes Plasmid is cut, the segment after pCW203 digestion need to add 0.5 μ L CIAP, 37 DEG C of water-bath 30min dephosphorylations.By digestion mixture and 6 (pUC57-Meso is returned the segment of × Loading Buffer mixing, electrophoresis (1% agarose, 94V) and glue recycling corresponding length Small fragment, that is, mesothelin gene segment after receiving digestion, length is about 1781bp；PCW203 recycles the large fragment after digestion and carries Body segment, length is about>10Kb), with 30 μ L Elution Buffer elution.

It is returned after taking the mesothelin gene segment (Insert Fragment) and pCW203 digestion that recycle after pUC57-Meso digestion respectively The carrier segments of receipts, according to linked system：Carrier 50ng, Insert Fragment molal quantity：Carrier segments molal quantity=5:1, 1 μ L, 10Ligase Buffer of T4Ligase 5 μ L, ddH₂System is supplied 10 μ l, 22 DEG C of connection 1h by O.Connection product is pressed Volume 1:10 softly mix with the competent cell of bacillus coli DH 5 alpha, and ice bath 30min, 42 DEG C of thermal shock 45s, ice bath 3min add The 500 μ L of SOB meat soup for entering preheating, in 37 DEG C after mixing, 180rpm cultivates 1h, is coated on LA plate (LB-Amp plate：Containing 100 The LB plate of μ g/mL Amp), 37 DEG C of culture 16-20h.

PCR screening：The DNA of bacteria genome extracted using boiling method expands purpose band mesothelin, such as SEQ ID as template Shown in NO.11 and SEQ ID NO.12, upstream primer MESO-f：5 '-CCACAAGCTTTGTCCCGTACACTTG -3 ', downstream Primer MESO-r：5 '-TATACTCGAGAGCAAGTGTAGAAGC-3 ', reaction cycle condition are：94℃ 3min→(94℃ 1min, 47 DEG C of 30s, 72 DEG C of 1min) × → 72 DEG C of 10min → 4 DEG C of 30 circulations；Electrophoresis observes PCR result (deposition condition： 1% Ago-Gel；90V, applied sample amount：5 μ l PCR products), it is contemplated that result：Mesothelin segment about 1781bp.

Digestion verification：The positive bacteria of PCR screening verification extracts plasmid, according to above-mentioned digestion system with Hind III and Xho I into Digestion mixture is mixed with 6 × Loading Buffer and carries out electrophoresis (1% agarose, 94V) by row digestion.

Sequence verification：It extracts PCR and digestion verification meets expected plasmid and sequencing company is sent to be sequenced.It will be complete after sequence verification The Escherichia coli of the carrying positive plasmid of total correctness are stored in -80 DEG C.

2. the building of target practice plasmid pCW180-Meso

Plasmid pCW180 is extracted, with 30 μ L Elution Buffer elution.By plasmid pCW180 and middle interstitial granules PCW203-Meso carries out digestion with restriction enzyme BamH I and Xho I respectively.Digestion system is by taking 20 μ l as an example： pCW203- I 1 I 1 μ L, 10 × NEB Buffer of μ L, Xho of Meso or pCW180 < 1ng, BamH 2 μ L, ddH₂System is supplied 20 μ L by O.37 DEG C water-bath 1h digested plasmid, the segment after pCW180 digestion need to add 0.5 μ L CIAP, 37 DEG C of water-bath 30min dephosphorylations.By enzyme It cuts mixture to mix with 6 × Loading Buffer, the segment of electrophoresis (1% agarose, 94V) and glue recycling corresponding length (pCW203-Meso recycles the small fragment after digestion, length about 1840bp；PCW180 recycles the large fragment after digestion, and length is about 8621bp), with 30 μ L Elution Buffer elution.

It is recycled after the segment (Insert Fragment) and pCW180 digestion that are recycled after pCW203-Meso I digestion of BamH I and Xho Segment (carrier segments) according to linked system：Carrier 50ng, Insert Fragment molal quantity：Carrier segments molal quantity=5:1, 1 μ L, 10Ligase Buffer of T4Ligase 5 μ L, ddH₂System is supplied 10 μ l, 22 DEG C of connection 1h by O.Connection product presses (2) 1. method be transferred to bacillus coli DH 5 alpha, be coated on LA plate, PCR screening, BamH I and Xho I carried out to the single bacterium colony that grows Double digestion verifying and sequence verification, PCR screening technique same (2) is 1.；BamH I and I double digestion verification method of Xho are similar to (2) 1. the enzyme being used only and digestion system difference；Sequence verification method same (2) is 1..

(3) electrotransformation is with the preparation method of Lm Δ actAplcB-lacZ competent cell with embodiment 2 (3).

(4) target practice plasmid electrotransformation Lm Δ actAplcB-lacZ

It takes out Lm Δ actAplcB-lacZ competent cell prepared by 1 (3) step from -70 DEG C of refrigerators and is put in and melt on ice, Target practice plasmid pCW180-Meso prepared by (2) step is transferred to Lm Δ actAplcB-lacZ by the method for embodiment 2 (4).It will be complete Portion's bacterium solution drop in containing erythromycin 1~5 μ g/ml, X-gal 20~60 μ g/ml BHI agar plate (BHI-Ery-X-gal plate, Hereinafter referred to as BEXI plate) center, bacterium solution is coated with entire plate with sterile L shape glass rod, it is positive in 30 DEG C of 48~72h of culture Bacterium (Lm Δ actAplcB-lacZ/pCW180-Meso) is grown to blue colonies.In carrying out pure culture on BEXI plate, 30 DEG C Culture for 24 hours, carries out upgrading grain and PCR verifying.

Illustrate that (Listeria is gram positive bacteria to extraction plasmid, is needed before I use of Solution according to plasmid extraction kit Lysozyme is added, the final concentration of 20mg/L of lysozyme is resuspended, 37 DEG C of water-bath 45min after I-lysozyme of Solution is added), most Rear electrophoresis is eluted afterwards with 30 μ l Elution Buffer.Gel imaging, whether there is or not bands whether to judge recombinant plasmid importing for observation Success, plasmid size are 10455bp.

PCR verifying：All DNA expands purpose band mesothelin, such as SEQ ID as template in the bacterium extracted using boiling method Shown in NO.11 and SEQ ID NO.12, upstream primer MESO-f：5 '-CCACAAGCTTTGTCCCGTACACTTG -3 ', downstream Primer MESO-r：5 '-TATACTCGAGAGCAAGTGTAGAAGC-3 ', reaction cycle condition are：94℃ 3min→(94℃ 1min, 47 DEG C of 30s, 72 DEG C of 1min) × → 72 DEG C of 10min → 4 DEG C of 30 circulations；Electrophoresis observes PCR result (deposition condition：1% Ago-Gel；90V, applied sample amount：5 μ l PCR products), it is contemplated that result：Mesothelin segment about 1781bp.

(5) Lm Δ actAplcB-lacZ and target practice plasmid homologous recombination and screening

1. Lm Δ actAplcB-lacZ hybridizes culture with target practice plasmid homologous recombination

Electricity is transferred to the Lm Δ actAplcB-lacZ streak inoculation BEXI plate of target practice plasmid, 42 DEG C of secondary culture 2-3 generations (per generation incubation time is 48 hours), the single blue colonies inoculation 5ml BHI meat soup of picking, 30 DEG C, 6 generation of 200rpm secondary culture (per generation incubation time is 24 hours).In the 3rd generation of meat soup to be passed to, per generation, take 40 μ l cultures that 360 μ l BHI dilution is added 106 times, it is flat in BEXI and BXI (the BHI agar plate of 20~60 μ g/ml containing X-gal) to be respectively coated dilution 100 μ l of bacterium solution Plate, 30 DEG C of cultures are for 24 hours.Positive bacterium colony Lm Δ actAplcB-MESO grows up to white colony (see Fig. 6) on BXI plate, in BEXI It is not grown on plate (see Fig. 7).Target practice plasmid and Lm Δ actAplcB-lacZ homologous recombination process are shown in schematic diagram (Fig. 8).

2. PCR identifies Lm Δ actAplcB-MESO

Bacterial genomes DNA extraction kit extracts the genomic DNA of Lm Δ actAplcB-MESO as template, carries out The amplification of three groups of genetic fragments below：Anti- erythromycin gene, target antigen box gene and actA gene, according to amplification counterweight Group bacterium Lm Δ actAplcB-MESO is identified.Lm Δ actAplcB-MESO expands mesothelin, and primer and reaction condition are same (2) 1., the anti-erythromycin gene Ery of purpose band, about 1471bp, primer such as SEQ ID NO.13 and SEQ ID NO.14 are expanded It is shown, Ery-f/r (f:5 '-GATAAGTCGACGATTCACAAAAAATAG-3 ', r:5'-AAAACTAGTCCCGGG GCGAATTG-3 '), reaction cycle condition is：94 DEG C of 3min → (94 DEG C of 1min, 60 DEG C of 30s, 72 DEG C of 2min) × 30 circulations → 72℃10min→4℃；Amplification Lm actA gene, about 950bp, as shown in SEQ ID NO.5 and SEQ ID NO.6, primer Lm- actA-f/r(f:5 '-GCTATAAATGAAGAGGCTTCAGG-3 ', r:5 '-CTCTTAAATCAGCTAGGCGATC-3 '), instead The cycling condition is answered to be：94 DEG C of 3min → (94 DEG C of 1min, 50 DEG C of 30s, 72 DEG C of 1min) × → 72 DEG C of 10min → 4 of 30 circulations ℃.It is identified through PCR, Lm Δ actAplcB-MESO carries mesothelin gene, does not carry anti-erythromycin gene, and actA gene It is knocked.Qualification result coincide with expected, and Lm Δ actAplcB-MESO is successfully prepared.

3. gene sequencing is verified

Using bacterial genomes DNA as template after PCR identification, PCR amplification target antigen gene mesothelin surveys amplified production Sequence verifying carries out fungi preservation to correct bacterial strain is sequenced.

Embodiment 4：The recombinant attenuated Listeria monocytogenes pair of genome foreign gene-carrying (by taking mesothelin gene as an example) The toxicity test of animal

(1) recombinant attenuated Listeria monocytogenes are to the LD50 of mouse and the comparison of Listeria monocytogenes bacterium wild strain

The BHI fluid nutrient medium fresh cultured object of recombinant attenuated Listeria monocytogenes and Listeria monocytogenes bacterium wild strain, With normal saline, at different concentration, (wild strain is made into：5×10⁵、5×10⁶、5×10⁷CFU/ml, attenuated strain are made into：5 ×10⁸、 8×10⁸、1.2×10⁹CFU/ml).C57BL/6 mouse is grouped at random, cuts ear tag, and adaptive feeding 1 week； 75% alcohol disinfecting mouse tail skin, tail vein are slowly injected the 100 μ l of different bacteria suspensions of above-mentioned various concentration, are observed continuously With record dead mouse situation and weight 10d；Bacterial strain is calculated to the LD50 of C57BL/6 mouse using improvement karber's method, obtains open country LD50 after raw strain intravenous inoculation mouse is 4.0 × 10⁵CFU/ only, LD50 after attenuated strain intravenous inoculation mouse is 7.8 × 10⁷Only, the survivorship curve for being inoculated with the mouse of two plants of bacterium of various dose is shown in Fig. 9 to CFU/.Thus result as it can be seen that bacterium actA and The knockout of plcB gene causes its virulence to significantly reduce to get the effect for having arrived significant attenuation.

(2) pathology damage and Listeria monocytogenes bacterium wild strain of the recombinant attenuated Listeria monocytogenes to mouse vitals Comparison

The BHI fluid nutrient medium fresh cultured object of recombinant attenuated Listeria monocytogenes and Listeria monocytogenes bacterium wild strain, With normal saline, at different concentration, (wild strain is made into：4×10⁵CFU/ml, attenuated strain are made into：7.8×10⁷CFU/ml)。 C57BL/6 mouse is grouped at random, cuts ear tag, and adaptive feeding 1 week；75% alcohol disinfecting mouse tail skin, tail vein Above-mentioned 100 μ l of bacteria suspension is slowly injected, mouse ordinary circumstance is observed after inoculation, the 3rd day execution mouse after inoculation collects mouse Liver and spleen, which are soaked in 4% paraformaldehyde, to be fixed for 24 hours, and paraffin embedding and slice, production HE dye Pathologic specimen piece, will be sick Reason slice is placed under microscope, observation histotomy and acquisition image, the result is shown in Figure 10.As seen from Figure 10, attenuated strain is connecing In the case that kind of bacterium amount is bigger in the same time caused by pathology damage it is lighter, be demonstrated by lower toxicity.

Embodiment 5：The recombinant attenuated Listeria monocytogenes of genome foreign gene-carrying (by taking mesothelin gene as an example) lure The antigen-specific cellular immune response led and the comparison for carrying mutually isogenic recombinant attenuated sheep Listeria

(1) bacterial strain preparation and immune mouse

It is random to be grouped after the C57BL/6 female mice adaptable fed 3d of 6~8 week old, between the immune recombination of one group of tail vein carries Recombinant attenuated sheep Listeria (Li Δ actAplcB-MESO) (immunizing dose of skin plain gene：2×10⁷CFU), one group of tail Recombinant attenuated Listeria monocytogenes (Lm Δ actAplcB-MESO) (immunizing dose of intravenous immunisations carrying mesothelin gene： 7.8 ×10⁶CFU).The BHI fluid nutrient medium fresh cultured object of Lm Δ actAplcB-MESO and Li Δ actAplcB-MESO, with life At different concentration, (single increase is made into reason saline：7.8×10⁷CFU/ml, sheep are made into 2 × 10⁸CFU/ml).According to grouping Through 100 μ l bacterium solution of mouse tail vein injection, from inoculation, measured from mouse weight to inoculation daily the 9th day.

(2) CD4+ the and CD8+T lymphocyte ratio of Flow cytometry antigentic specificity secretion of gamma-IFN cell factor Rate

Splenocyte suspension preparation：The 9th day after mouse immune, cervical dislocation puts to death mouse, and sterile working takes spleen, is put in dress In the 5ml centrifuge tube for having 1ml RPMI Medium 1640 (dual anti-containing 1%), it is spare to set ice bath.Add 5ml RPMI Medium 1640 (dual anti-containing 1%), wherein putting a sterile 200 mesh nylon membrane, are put in a sterilizing flat board with Sterile ophthalmic tweezer spleen On nylon membrane, and spleen is wrapped up with nylon membrane, gently grinds spleen to its whiting (remaining quilt after splenocyte falls off with sterile pestle Film and do not reject clean connective tissue), with pipette tips by all splenocyte suspensions collect to sterilizing 15ml EP centrifuge tube.

Erythrocyte splitting：To be put into refrigerated centrifuge equipped with the centrifuge tube of splenocyte suspension, 1300rpm/min, 4 DEG C from Heart 5min, discards supernatant, and shakes centrifuge tube, makes the cell dispersion for being deposited in centrifugation bottom of the tube, 4ml is added into centrifuge tube and passes through The erythrocyte cracked liquid for filtering out bacterium, turn upside down centrifuge tube, comes into full contact with cell with erythrocyte cracked liquid, after standing 5min, 4ml RPMI 1640Medium (dual anti-containing 1%) is added immediately and terminates and splits red reaction, 4 DEG C of centrifugation 5min of 1300rpm/min are abandoned Supernatant is removed, centrifuge tube is shaken, 5ml RPMI 1640Medium (dual anti-containing 1%) is added into centrifuge tube, turn upside down centrifuge tube To wash away remaining erythrocyte cracked liquid, 1300rpm/min, 4 DEG C of centrifugation 5min are discarded supernatant, and shake centrifuge tube, repeated washing Once.After shaking centrifuge tube, 2ml RPMI 1640Medium (contain 10% fetal calf serum) is added into centrifuge tube and is resuspended, it will be from Heart pipe is placed on ice, spare.

Stimulant is prepared：1.gp33-41 stock solution：1ml DMSO is added in the peptide fragment of 1mg, is configured to the deposit of 1mg/ml Liquid, 10 μ l/ branch dispense EP pipe, are stored in -20 DEG C of refrigerators, avoid multigelation；2.gp61-80 stock solution：In the peptide fragment of 1mg 1ml DMSO is added, is configured to the stock solution of 1mg/ml, 10 μ l/ branch dispense EP pipe, are stored in -20 DEG C of refrigerators, avoid repeatedly Freeze thawing；3. streaming PMA stock solution：1ml DMSO is added in the PMA of 0.1mg, is configured to the storage of 0.1mg/ml with liquid, 10 μ l/ branch EP pipe is dispensed, is stored in -20 DEG C of refrigerators, avoids multigelation；4. streaming Ionomycine stock solution：1mg's 1ml DMSO is added in Ionomycine, is configured to the stock solution of 1mg/ml, 10 μ l/ branch dispense EP pipe, are stored in -20 DEG C of ice In case, multigelation is avoided；5. streaming positive stimulus mixed liquor：100 μ l RPMI 1640Medium are drawn (containing 10% tire ox Serum), 0.2 μ l Golgi Stop, 0.2 μ l Ionomycine stock solution and 0.2 μ l PMA stock solution managed in EP, blown with pipette tips It beats and is uniformly mixed；6. streaming experiment stimulation mixed liquor：Draw 100 μ l RPMI 1640Medium (containing 10% fetal calf serum), 0.2 μ l gp33-41 stock solution, 0.2 μ l gp61-80 stock solution and 0.2 μ l Golgi Stop are blown and beaten with pipette tips and are mixed in EP pipe Uniformly.

Stimulation：Each sample does 2 holes of negative control (not stimulating) and experiment process (GP33, GP61 peptide fragment) simultaneously；Respectively Group randomly selects 2 sample positive stimulus object (PMA+Ionomycine) stimulations as positive quality control.In 96 orifice plates, every hole 50 μ l splenocyte suspensions and the corresponding stimulant of 50 μ l, 5%CO is added₂37 DEG C of stimulation 5h of incubator.

Surface antibody dyeing：After stimulation, 96 orifice plates are taken out, 4 DEG C of 1300rpm/min are centrifuged 5min, abandon supernatant, will 96 orifice plates, which are placed in whirlpool concussion instrument, to be shaken, so that the cell dispersion of board bottom is deposited in, in being protected from light place for surface antibody Anti- Mouse CD8a PE、Anti-Mouse TCR beta PerCP-Cyanine5.5、Anti-Human/Mouse CD44FITC、 Anti-Mouse IFN gamma APC is with PBS (2% fetal calf serum containing volumetric concentration) respectively according to volume in sterilizing EP pipe It is diluted to 1:200 (V:V).Every hole is added 50 μ l surface antibody mixed liquors and is resuspended, after 4 DEG C are protected from light and are incubated for 30min, in be protected from light to 150 μ l PBS (2% fetal calf serum containing volumetric concentration) is added in every hole cell and terminates incubation, 4 DEG C of 1300rpm/min centrifugations 5min gets rid of abandoning supernatant, shakes 96 orifice plates, adds 200 μ l PBS (containing 2% fetal calf serum) to be resuspended, washes away remaining antibody, 1300rpm/ 4 DEG C of centrifugation 5min of min get rid of abandoning supernatant, shake 96 orifice plates.

Rupture of membranes：Every hole is added 100 μ l Cytofix/Cytoperm and is resuspended, 4 DEG C be protected from light stand 20min after be protected from light addition 100 μ l Buffer Perm/Wash is terminated, 4 DEG C of centrifugation 5min of 1300rpm/min, gets rid of abandoning supernatant, shakes 96 orifice plates, every hole adds 200 Washing is resuspended in μ l Buffer Perm/Wash, and 4 DEG C of centrifugation 5min of 1300rpm/min get rid of abandoning supernatant, shakes 96 orifice plates.

Intracellular antibody dyeing：In be protected from light place with 1 × perm/wash buffer in the EP pipe that sterilizes by antibody A nti- Mouse IFN gamma APC presses 1:The dilution of 200 volume ratios.Every hole is separately added into 50 μ l of mixed antibody resuspension, and 4 DEG C are protected from light incubation After 45min, 150 μ 1 × perm/wash of l buffer termination incubation, 1300rpm/min 4 are added into every hole cell in being protected from light DEG C centrifugation 5min, get rid of abandon supernatant shake 96 orifice plates, add 200 μ 1 × perm/wash of l buffer be resuspended, wash away remaining antibody, 4 DEG C of centrifugation 5min of 1300rpm/min get rid of abandoning supernatant, and every hole cell is added the resuspension of 200 μ l, 2% paraformaldehyde solution and collects, and 4 The upper machine of preparation DEG C is kept in dark place.

Upper machine testing result：Carry out Performance QC with Research Beads to correct optical path and flow path, go from Sub- water is sample wash streaming instrument about 10min, until Threshold Rate is in 20evts/sec or less.Debug set factors, choosing Used fluorescence is taken, determines sample collection number (10⁶Events), adjusted and tested with blank well (cell without any processing) Voltage makes FSC A-FSC H scatter plot, cell mass and axis of abscissas are each in about 45 ° of angles in FSC A-SSC A scatter plot Go out peak value (negative peak-to-peak value) of the kind dyestuff in crest figure is respectively less than 10²；Measurement sample is gone forward side by side a step section voltage, makes to analyze When the cell that certain fluorescent dye is incubated for, the bimodal peak value (negative peak-to-peak value, positive peak-to-peak value) of the fluorescent dye is with 10²For Boundary, remaining fluorescent dye peak value is less than 10².Result is analyzed with Flowjo.The result is shown in Figure 11.

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Sequence table

<110>Nanjing Song Yue Biotechnology Co., Ltd

<120>A kind of the attenuation recombination Listeria monocytogenes and preparation method of genome foreign gene-carrying

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9285

<212> DNA

<213>Manually (Artificial)

<400> 1

gaattcccga ttatgtcttt tgcgcactcg gcttaaacca gttttcgctg gtgcgaaaaa 60

agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt 120

tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa 180

tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat 240

aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc 300

atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg 360

atcgtccatg tgaagcatat atctcacaag acctttcaca cttcctgcaa tctgcggaat 420

agtcgcattc aattcttctg ttattatttt tatctgttca taagatttat taccctcata 480

catcactaga atatgataat gctctttttt catcctacct tctgtatcag tatccctatc 540

atgtaatgga gcactacaaa ttgaatgtgt aactctttta aatactctaa ccactcggct 600

ttgctgattc tggatataaa acaaatgtcc aattacgtcc tcttgaattt ttcttgtttt 660

cagtttcttt tattacattt tcgctcatga tataataacg gtgctaatac acttaacaaa 720

atttagtcat agataggcag catgccagtg ctgtctatct ttttttgttt aaaatgcacc 780

gtattcctcc tttgcatatt tttttattag aataccggtt gcatctgatt tgctaatatt 840

atatttttct ttgattctat ttaatatctc attttcttct gttgtaagtc ttaaagtaac 900

agcaactttt ttctcttctt ttctatctac aactatcact gtacctccca acatctgttt 960

ttttcacttt aacataaaaa acaacctttt aacattaaaa acccaatatt tatttatttg 1020

tttggacaat ggacaatgga cacctagggg ggaggtcgta gtacccccct atgttttctc 1080

ccctaaataa ccccaaaaat ctaagaaaaa aagacctcaa aaaggtcttt aattaacatc 1140

tcaaatttcg catttattcc aatttccttt ttgcgtgtga tgcgctgcgt ccattaaaaa 1200

tcctagagct ttgcaaccga aagttaatag ctgtcgctac tactttcgct tacgctctaa 1260

gtatatttta aggactgtca cacgcaaaaa gttttctcgg cataaaagta cctctacatc 1320

tctaaatcgt ctgtacgctg tttctcacgc tttctatcga tcccgcaaga ggcccggcag 1380

taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat 1440

gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa 1500

actaccgcat taaagcttgt cgacgattca caaaaaatag gcacacgaaa aacaagttaa 1560

gggatgcagt ttatgcatcc cttaacttac ttattaaata atttatagct attgacaaga 1620

gataagaatt gttcaaagct aatattgttt aaatcgtcaa ttcctgcatg ttttaaggaa 1680

ttgttaaatt gattttttgt aaatattttc ttgtattctt tgttaaccca tttcagaacg 1740

aaataattat acttttgttt atctttgtgt gatattcttg atttttttct acttaatctg 1800

ataagtgagc tattcacttt aggtttagga tgaaaatatt ctcttggaac catacttaat 1860

atagaaatat caacttctgc cattaaaagt aatgccaatg agcgttttgt atttaataat 1920

cttttagcaa acccgtattc cacgattaaa taaatctcat tagctatact atcaagaaca 1980

attttgcgta ttatatccgt acttatgtta taaggtatat taccatatat tttataggat 2040

tggtttttag gaaatttaaa ctgcaatata tccttgttta aaacttggaa attatcgtga 2100

tcaacaagtt tattttctgt agttttgcat aatttatggt ctatttcaat ggcagttacg 2160

aaattacacc tctttactaa ttcaagggta aaatggcctt ttcctgagcc gatttcaaag 2220

atattatcat gttcatttaa tcttatattt gtcattattt tatctatatt atgttttgaa 2280

gtaataaagt tttgactgtg ttttatattt ttctcgttca ttataaccct ctttaatttg 2340

gttatatgaa ttttgcttat taacgattca ttataaccac ttattttttg tttggttgat 2400

aatgaactgt gctgattaca aaaatactaa aaatgcccat attttttcct ccttataaaa 2460

ttagtataat tatagcacga gctctgataa atatgaacat gatgagtgat cgttaaattt 2520

atactgcaat cggatgcgat tattgaataa aagatatgag agatttatct aatttctttt 2580

ttcttgtaaa aaaagaaagt tcttaaaggt tttatagttt tggtcgtaga gcacacggtt 2640

taacgactta attacgaagt aaataagtct agtgtgttag actttatgaa atctttatac 2700

gtttatatat atttattatc cggaggtgta gcatgtctca ttcaattttg agggttgcca 2760

gagttaaagg atcaagtaat acaaacggga tacaaagaca taatcaaaga gagaataaaa 2820

actataataa taaagacata aatcatgagg aaacatataa aaattatgat ttgattaacg 2880

cacaaaatat aaagtataaa gataaaattg atgaaacgat tgatgagaat tattcaggga 2940

aacgtaaaat tcggtcagat gcaattcgcc ccgggactag ttaaaggtgg agaaattgat 3000

tcgtttgtcc attatggctt gaattgcaat aatgcctttt gggatggcca agaaattctt 3060

tatggagatg gggacaaaaa gaatttcaaa ccattttcat gcgccaaaac tattgttggt 3120

catgaactaa cgcatgcagt tatccagtat tcggcgggat tggaatacga agggcaatca 3180

ggtgcgctaa acgagtcgtt cgccgatgtt tttggttatt ttattgcgcc aaatcattgg 3240

ttgattggtg aggatgtctg tgtgcgtggg tcgcgagatg ggcgaataag aagcattaaa 3300

gatcctgaca aatataatca agcggctcat atgaaggatt acgaatcgct tccaatcaca 3360

gaggaaggcg actggggcgg agttcattat aatagtggta tcccgaataa agcagcctat 3420

aatactatca ctaaacttgg aaaagaaaaa acagaacagc gacgatttcg cgccttaaag 3480

tactatttaa cgaaaaaagc ccagtttacc gatgcgaaaa aagcgcttca acaagcagcg 3540

aaagatttat atggtgaaga tgcttctaaa aaagttgctg aagcttggga agcggtagga 3600

gttaactgag cggccgccag tgtgatggat atctgcagaa ttaattcggc tttctagagt 3660

gacttttatg ttgaggcatt aacatttgtt aacgacgata aagggacagc aggactagaa 3720

taaagctata aagcaagcat ataatattgc gtttcatctt tagaagcgaa tttcgccaat 3780

attataatta tcaaaagaga ggggtggcaa acggtatttg gcattattag gttaaaaaat 3840

gtagaaggag agtgaaaccc atgaaaaaaa taatgctagt ttttattaca cttatattag 3900

ttagtctacc aattgcgcaa caaactgaag caaaggatgc atcggatctt aaagctgttt 3960

ataattttgc tactatgaag gatccatatc catatgatgt tccagattat gcatcattga 4020

attcaatgag aagtgaaaga ccacaagctt tgttggggaa gtgcttgacc gcgtgctgtt 4080

gctcgcgatt gctttttttg tggtgtatcg tgccgttcta tcttgctgtg ctcgtcaacg 4140

ccagcaacaa caacagctct catattcagt tgatttataa cttaacgcta tgtgagctga 4200

atggcacaga ttggctggcg caaaaatttg actgggcagt ggagactttt gtcatcttcc 4260

ccgtgttgac tcacattgtt tcctatgggg cactcaccac cagccatttc cttgacacag 4320

ttggtctggc cactgtgtcc accgccggat attatcacgg gcggtatgtc ttgagtagca 4380

tttacgcagt ctgtgctctg gctgcgttga tttgctttgt cattaggctt gcgaagaact 4440

gcatgtcctg gcgctactct tgtaccagat ataccaactt ccttctggac actaagggca 4500

gactctatcg ttggcggtcg cccgtcattg tggagaaagg gggtaaggtt gaggtcgaag 4560

gtcacctgat cgacctcaag agagttgtgc ttgatggttc cgcggcaacc cctttaacca 4620

gagtttcagc ggaacaatgg ggtcgtctcc tcgagggatt aaatggacca gatatttata 4680

aaggagttta tcaatttaaa agtgttgaat ttgatgtcga gtatacagat attgaaatga 4740

atagattagg aaaatgataa gtcggcggcc gctaataatc ttgcgcttcg atgacaacag 4800

ctgtaccaga tgcagtgacc attagcattc cgttatcagc tccaagcact tcataatcaa 4860

tatcaacacc gataacggca tttgcgccaa tatctttcgc acgttgttcc atttcacgaa 4920

ttgcttcctc gcgagcatta ataagttcat cttcatagcc ttgcgaacgg cccccgaaga 4980

aatttcgaag tccagcccca atatctttca taaagttaac gccagtgatg acttcgccga 5040

aaacgatttt tttatattcg ataatttgtt tgccttcaat atttggtgaa gttgttacaa 5100

tcatgagtta tccctacagt ttttctttta tcatacctct tagtactttt tctagtcaaa 5160

ggatatccgg ttatttcgta cgatttcgcg ctttttctat ataagaaata gcatctggaa 5220

ctttacaagc tgtatttcca aggtttacat gaactttccc gactgatttc gcggcttcca 5280

tcgctttttc gtgcaaatcg tctttgtaaa ttccacaagt gataataaaa ccgttcattg 5340

aatagcgggt tctattggtt tctgtttgca gcgtttcttt cgcgcgttga agtaatgtct 5400

ctgcttcagg caaattgtta atatgttcgc tgtaaatctg ccagcccatg gatttagtta 5460

aatcataatc actttcaatc catgttttgg cgaaaaggag tgcatcatca cgtttgctta 5520

caatcgatgc tagtccaaaa gttagttgtg accatgtttc gcgaaaagcg atattccatt 5580

tttcgatttg ttcggtggtt attttgtttg gattaattgc tagcaaaccg agataaatca 5640

agtcactatt atttgattca atcagtttta cggcgagttc gtgatttttt gtcagttttt 5700

ctcggcggat gattttcttt aaatcaccaa tttttagtcc gtaaagatct aatgaatccg 5760

gacaaccgtg attacgaaaa attttgatcg tattggggtt ttctaaggct tgtagctcgg 5820

tgtcaagttg gtcaaaagta atcatacgcg tcactcctct cgaataaagt aagtataaca 5880

aaaaaagcat gcgaaggcgc atgctttagg atttaagaat attagtctat ttgtttcatt 5940

gcgtcgttct agaacttttt taagtgtatc tgcggagttt ttcatttgtt ctttttcttt 6000

gtcatttaag ttcatttcaa caatatggcg aacgccttga cggttaacga ctgctggtgc 6060

acctatataa atatcgttca taccgtaatg gccatctaaa taaacagaaa gtggcaaaat 6120

cgcattttcg ttatttagaa ttgcttttgt aatacgagca agagctgcag caacgccgta 6180

gaatgtagcg ccttttttat taataatttc ataagctgca tcacgaacac ttacgaaaat 6240

agtatccatt gcaccttgtt catcttcgct aatccattca gtaattggaa ggccgccgac 6300

agttgtgtgg ctccatgctg ggaattcttg aagacgaaag ggcctcgtga tacgcctatt 6360

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 6420

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 6480

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 6540

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 6600

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 6660

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 6720

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 6780

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 6840

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 6900

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 6960

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 7020

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc 7080

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 7140

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 7200

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 7260

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 7320

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 7380

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 7440

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 7500

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 7560

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 7620

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 7680

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 7740

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 7800

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 7860

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 7920

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 7980

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 8040

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 8100

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 8160

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 8220

tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 8280

tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 8340

gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 8400

cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 8460

gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 8520

tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 8580

cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg 8640

tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc 8700

tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc 8760

tgtttggtca cttgatgcct ccgtgtaagg gggaatttct gttcatgggg gtaatgatac 8820

cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 8880

tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 8940

ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 9000

agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 9060

gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 9120

tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 9180

ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg 9240

gccaggaccc aacgctgccc gacgatgata agctgtcaaa catga 9285

<210> 2

<211> 7967

<212> DNA

<213>Manually (Artificial)

<400> 2

gaattcccga ttatgtcttt tgcgcactcg gcttaaacca gttttcgctg gtgcgaaaaa 60

agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt 120

tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa 180

tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat 240

aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc 300

atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg 360

atcgtccatg tgaagcatat atctcacaag acctttcaca cttcctgcaa tctgcggaat 420

agtcgcattc aattcttctg ttattatttt tatctgttca taagatttat taccctcata 480

catcactaga atatgataat gctctttttt catcctacct tctgtatcag tatccctatc 540

atgtaatgga gcactacaaa ttgaatgtgt aactctttta aatactctaa ccactcggct 600

ttgctgattc tggatataaa acaaatgtcc aattacgtcc tcttgaattt ttcttgtttt 660

cagtttcttt tattacattt tcgctcatga tataataacg gtgctaatac acttaacaaa 720

atttagtcat agataggcag catgccagtg ctgtctatct ttttttgttt aaaatgcacc 780

gtattcctcc tttgcatatt tttttattag aataccggtt gcatctgatt tgctaatatt 840

atatttttct ttgattctat ttaatatctc attttcttct gttgtaagtc ttaaagtaac 900

agcaactttt ttctcttctt ttctatctac aactatcact gtacctccca acatctgttt 960

ttttcacttt aacataaaaa acaacctttt aacattaaaa acccaatatt tatttatttg 1020

tttggacaat ggacaatgga cacctagggg ggaggtcgta gtacccccct atgttttctc 1080

ccctaaataa ccccaaaaat ctaagaaaaa aagacctcaa aaaggtcttt aattaacatc 1140

tcaaatttcg catttattcc aatttccttt ttgcgtgtga tgcgctgcgt ccattaaaaa 1200

tcctagagct ttgcaaccga aagttaatag ctgtcgctac tactttcgct tacgctctaa 1260

gtatatttta aggactgtca cacgcaaaaa gttttctcgg cataaaagta cctctacatc 1320

tctaaatcgt ctgtacgctg tttctcacgc tttctatcga tcccgcaaga ggcccggcag 1380

taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat 1440

gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa 1500

actaccgcat taaagcttgt cgacgattca caaaaaatag gcacacgaaa aacaagttaa 1560

gggatgcagt ttatgcatcc cttaacttac ttattaaata atttatagct attgacaaga 1620

gataagaatt gttcaaagct aatattgttt aaatcgtcaa ttcctgcatg ttttaaggaa 1680

ttgttaaatt gattttttgt aaatattttc ttgtattctt tgttaaccca tttcagaacg 1740

aaataattat acttttgttt atctttgtgt gatattcttg atttttttct acttaatctg 1800

ataagtgagc tattcacttt aggtttagga tgaaaatatt ctcttggaac catacttaat 1860

atagaaatat caacttctgc cattaaaagt aatgccaatg agcgttttgt atttaataat 1920

cttttagcaa acccgtattc cacgattaaa taaatctcat tagctatact atcaagaaca 1980

attttgcgta ttatatccgt acttatgtta taaggtatat taccatatat tttataggat 2040

tggtttttag gaaatttaaa ctgcaatata tccttgttta aaacttggaa attatcgtga 2100

tcaacaagtt tattttctgt agttttgcat aatttatggt ctatttcaat ggcagttacg 2160

aaattacacc tctttactaa ttcaagggta aaatggcctt ttcctgagcc gatttcaaag 2220

atattatcat gttcatttaa tcttatattt gtcattattt tatctatatt atgttttgaa 2280

gtaataaagt tttgactgtg ttttatattt ttctcgttca ttataaccct ctttaatttg 2340

gttatatgaa ttttgcttat taacgattca ttataaccac ttattttttg tttggttgat 2400

aatgaactgt gctgattaca aaaatactaa aaatgcccat attttttcct ccttataaaa 2460

ttagtataat tatagcacga gctctgataa atatgaacat gatgagtgat cgttaaattt 2520

atactgcaat cggatgcgat tattgaataa aagatatgag agatttatct aatttctttt 2580

ttcttgtaaa aaaagaaagt tcttaaaggt tttatagttt tggtcgtaga gcacacggtt 2640

taacgactta attacgaagt aaataagtct agtgtgttag actttatgaa atctttatac 2700

gtttatatat atttattatc cggaggtgta gcatgtctca ttcaattttg agggttgcca 2760

gagttaaagg atcaagtaat acaaacggga tacaaagaca taatcaaaga gagaataaaa 2820

actataataa taaagacata aatcatgagg aaacatataa aaattatgat ttgattaacg 2880

cacaaaatat aaagtataaa gataaaattg atgaaacgat tgatgagaat tattcaggga 2940

aacgtaaaat tcggtcagat gcaattcgcc ccgggactag ttaaaggtgg agaaattgat 3000

tcgtttgtcc attatggctt gaattgcaat aatgcctttt gggatggcca agaaattctt 3060

tatggagatg gggacaaaaa gaatttcaaa ccattttcat gcgccaaaac tattgttggt 3120

catgaactaa cgcatgcagt tatccagtat tcggcgggat tggaatacga agggcaatca 3180

ggtgcgctaa acgagtcgtt cgccgatgtt tttggttatt ttattgcgcc aaatcattgg 3240

ttgattggtg aggatgtctg tgtgcgtggg tcgcgagatg ggcgaataag aagcattaaa 3300

gatcctgaca aatataatca agcggctcat atgaaggatt acgaatcgct tccaatcaca 3360

gaggaaggcg actggggcgg agttcattat aatagtggta tcccgaataa agcagcctat 3420

aatactatca ctaaacttgg aaaagaaaaa acagaacagc gacgatttcg cgccttaaag 3480

tactatttaa cgaaaaaagc ccagtttacc gatgcgaaaa aagcgcttca acaagcagcg 3540

aaagatttat atggtgaaga tgcttctaaa aaagttgctg aagcttggga agcggtagga 3600

gttaactgag cggccgcaca aaaaacggaa atcagttagt aaaactggtt tccgtttttt 3660

attaatagtc ttgagcctca ataacaacag cagtgccaga tgctgtaacc attaacatac 3720

cattatccgc tccaagtact tcataatcga tatccacacc aattacggca tttgctccga 3780

tatctttggc gcgctgttcc atctctttaa tagcttcctc acgtgcgtta attaactcgt 3840

cttcataacc ttgtgatcgt ccaccgaaaa agtttctgag gccagctcca atgtctttca 3900

taaaattaac accagtaatt acttctccga aaacgatttt tttatattcg ataatttgct 3960

tgccttcaat atttggtgag gtagttacaa tcattattaa atccctccag ttttctttta 4020

tcatacctct aagttgtttt tttagtcaaa ggatagctgg ttatttttta cgggcgtggg 4080

ctttttcgat ataagtaatc gcatctggga ctttgcaagc agtttttcct aaatctacat 4140

atactttacc tattgattct gctgcttcca atgcttttgt gtaaagttcc tctttataaa 4200

ttccgcaagc gatgatataa ctattcatag agtaacgagt tctgttcgtt tcagattgta 4260

aagattcttt tgcgagttcc agtaattcct cggcttcagg caaagtcgcg atgtgttctg 4320

tgaaaatttg ccagcccatt gatttagtta aatcagcatc acttttaatc catttcctag 4380

caaaggtgag tgcgtcatca cgtttactta caacggaggc tagcccaaaa gttaacatcg 4440

tccaagcatc tcgaaaagct atattccatt tctcgatttg tgaaacagct acttttttag 4500

ggttcacggc aagtaaacca aggtaaatta agtcgctatt attagactcg attaattgga 4560

gtgctaattc gtgattttta ttcaattttt cacggcgaat aattttcttt aaatcaccaa 4620

tctttaatct ctagaacttt tttaagtgta tctgcggagt ttttcatttg ttctttttct 4680

ttgtcattta agttcatttc aacaatatgg cgaacgcctt gacggttaac gactgctggt 4740

gcacctatat aaatatcgtt cataccgtaa tggccatcta aataaacaga aagtggcaaa 4800

atcgcatttt cgttatttag aattgctttt gtaatacgag caagagctgc agcaacgccg 4860

tagaatgtag cgcctttttt attaataatt tcataagctg catcacgaac acttacgaaa 4920

atagtatcca ttgcaccttg ttcatcttcg ctaatccatt cagtaattgg aaggccgccg 4980

acagttgtgt ggctccatgc tgggaattct tgaagacgaa agggcctcgt gatacgccta 5040

tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5100

ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5160

ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5220

attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5280

gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5340

ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5400

cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt 5460

gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5520

tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5580

gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5640

ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5700

tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca 5760

gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5820

caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5880

cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5940

atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6000

gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6060

attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6120

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6180

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6240

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6300

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6360

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6420

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6480

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6540

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6600

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6660

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6720

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6780

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6840

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6900

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6960

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 7020

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 7080

ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 7140

gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 7200

cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 7260

gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 7320

cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 7380

tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 7440

cctgtttggt cacttgatgc ctccgtgtaa gggggaattt ctgttcatgg gggtaatgat 7500

accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt 7560

actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat 7620

cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca 7680

gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc 7740

cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt 7800

tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt 7860

aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg 7920

tggccaggac ccaacgctgc ccgacgatga taagctgtca aacatga 7967

<210> 3

<211> 8130

<212> DNA

<213>Manually (Artificial)

<400> 3

gaattcccga ttatgtcttt tgcgcactcg gcttaaacca gttttcgctg gtgcgaaaaa 60

agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt 120

tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa 180

tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat 240

aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc 300

atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg 360

atcgtccatg tgaagcatat atctcacaag acctttcaca cttcctgcaa tctgcggaat 420

agtcgcattc aattcttctg ttattatttt tatctgttca taagatttat taccctcata 480

catcactaga atatgataat gctctttttt catcctacct tctgtatcag tatccctatc 540

atgtaatgga gcactacaaa ttgaatgtgt aactctttta aatactctaa ccactcggct 600

ttgctgattc tggatataaa acaaatgtcc aattacgtcc tcttgaattt ttcttgtttt 660

cagtttcttt tattacattt tcgctcatga tataataacg gtgctaatac acttaacaaa 720

atttagtcat agataggcag catgccagtg ctgtctatct ttttttgttt aaaatgcacc 780

gtattcctcc tttgcatatt tttttattag aataccggtt gcatctgatt tgctaatatt 840

atatttttct ttgattctat ttaatatctc attttcttct gttgtaagtc ttaaagtaac 900

agcaactttt ttctcttctt ttctatctac aactatcact gtacctccca acatctgttt 960

ttttcacttt aacataaaaa acaacctttt aacattaaaa acccaatatt tatttatttg 1020

tttggacaat ggacaatgga cacctagggg ggaggtcgta gtacccccct atgttttctc 1080

ccctaaataa ccccaaaaat ctaagaaaaa aagacctcaa aaaggtcttt aattaacatc 1140

tcaaatttcg catttattcc aatttccttt ttgcgtgtga tgcgctgcgt ccattaaaaa 1200

tcctagagct ttgcaaccga aagttaatag ctgtcgctac tactttcgct tacgctctaa 1260

gtatatttta aggactgtca cacgcaaaaa gttttctcgg cataaaagta cctctacatc 1320

tctaaatcgt ctgtacgctg tttctcacgc tttctatcga tcccgcaaga ggcccggcag 1380

taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat 1440

gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa 1500

actaccgcat taaagcttgt cgacgattca caaaaaatag gcacacgaaa aacaagttaa 1560

gggatgcagt ttatgcatcc cttaacttac ttattaaata atttatagct attgacaaga 1620

gataagaatt gttcaaagct aatattgttt aaatcgtcaa ttcctgcatg ttttaaggaa 1680

ttgttaaatt gattttttgt aaatattttc ttgtattctt tgttaaccca tttcagaacg 1740

aaataattat acttttgttt atctttgtgt gatattcttg atttttttct acttaatctg 1800

ataagtgagc tattcacttt aggtttagga tgaaaatatt ctcttggaac catacttaat 1860

atagaaatat caacttctgc cattaaaagt aatgccaatg agcgttttgt atttaataat 1920

cttttagcaa acccgtattc cacgattaaa taaatctcat tagctatact atcaagaaca 1980

attttgcgta ttatatccgt acttatgtta taaggtatat taccatatat tttataggat 2040

tggtttttag gaaatttaaa ctgcaatata tccttgttta aaacttggaa attatcgtga 2100

tcaacaagtt tattttctgt agttttgcat aatttatggt ctatttcaat ggcagttacg 2160

aaattacacc tctttactaa ttcaagggta aaatggcctt ttcctgagcc gatttcaaag 2220

atattatcat gttcatttaa tcttatattt gtcattattt tatctatatt atgttttgaa 2280

gtaataaagt tttgactgtg ttttatattt ttctcgttca ttataaccct ctttaatttg 2340

gttatatgaa ttttgcttat taacgattca ttataaccac ttattttttg tttggttgat 2400

aatgaactgt gctgattaca aaaatactaa aaatgcccat attttttcct ccttataaaa 2460

ttagtataat tatagcacga gctctgataa atatgaacat gatgagtgat cgttaaattt 2520

atactgcaat cggatgcgat tattgaataa aagatatgag agatttatct aatttctttt 2580

ttcttgtaaa aaaagaaagt tcttaaaggt tttatagttt tggtcgtaga gcacacggtt 2640

taacgactta attacgaagt aaataagtct agtgtgttag actttatgaa atctttatac 2700

gtttatatat atttattatc cggaggtgta gcatgtctca ttcaattttg agggttgcca 2760

gagttaaagg atcaagtaat acaaacggga tacaaagaca taatcaaaga gagaataaaa 2820

actataataa taaagacata aatcatgagg aaacatataa aaattatgat ttgattaacg 2880

cacaaaatat aaagtataaa gataaaattg atgaaacgat tgatgagaat tattcaggga 2940

aacgtaaaat tcggtcagat gcaattcgcc ccgggactag ttaaaggtgg agaaattgat 3000

tcgtttgtcc attatggctt gaattgcaat aatgcctttt gggatggcca agaaattctt 3060

tatggagatg gggacaaaaa gaatttcaaa ccattttcat gcgccaaaac tattgttggt 3120

catgaactaa cgcatgcagt tatccagtat tcggcgggat tggaatacga agggcaatca 3180

ggtgcgctaa acgagtcgtt cgccgatgtt tttggttatt ttattgcgcc aaatcattgg 3240

ttgattggtg aggatgtctg tgtgcgtggg tcgcgagatg ggcgaataag aagcattaaa 3300

gatcctgaca aatataatca agcggctcat atgaaggatt acgaatcgct tccaatcaca 3360

gaggaaggcg actggggcgg agttcattat aatagtggta tcccgaataa agcagcctat 3420

aatactatca ctaaacttgg aaaagaaaaa acagaacagc gacgatttcg cgccttaaag 3480

tactatttaa cgaaaaaagc ccagtttacc gatgcgaaaa aagcgcttca acaagcagcg 3540

aaagatttat atggtgaaga tgcttctaaa aaagttgctg aagcttggga agcggtagga 3600

gttaactgag cggccgctaa taatcttgcg cttcgatgac aacagctgta ccagatgcag 3660

tgaccattag cattccgtta tcagctccaa gcacttcata atcaatatca acaccgataa 3720

cggcatttgc gccaatatct ttcgcacgtt gttccatttc acgaattgct tcctcgcgag 3780

cattaataag ttcatcttca tagccttgcg aacggccccc gaagaaattt cgaagtccag 3840

ccccaatatc tttcataaag ttaacgccag tgatgacttc gccgaaaacg atttttttat 3900

attcgataat ttgtttgcct tcaatatttg gtgaagttgt tacaatcatg agttatccct 3960

acagtttttc ttttatcata cctcttagta ctttttctag tcaaaggata tccggttatt 4020

tcgtacgatt tcgcgctttt tctatataag aaatagcatc tggaacttta caagctgtat 4080

ttccaaggtt tacatgaact ttcccgactg atttcgcggc ttccatcgct ttttcgtgca 4140

aatcgtcttt gtaaattcca caagtgataa taaaaccgtt cattgaatag cgggttctat 4200

tggtttctgt ttgcagcgtt tctttcgcgc gttgaagtaa tgtctctgct tcaggcaaat 4260

tgttaatatg ttcgctgtaa atctgccagc ccatggattt agttaaatca taatcacttt 4320

caatccatgt tttggcgaaa aggagtgcat catcacgttt gcttacaatc gatgctagtc 4380

caaaagttag ttgtgaccat gtttcgcgaa aagcgatatt ccatttttcg atttgttcgg 4440

tggttatttt gtttggatta attgctagca aaccgagata aatcaagtca ctattatttg 4500

attcaatcag ttttacggcg agttcgtgat tttttgtcag tttttctcgg cggatgattt 4560

tctttaaatc accaattttt agtccgtaaa gatctaatga atccggacaa ccgtgattac 4620

gaaaaatttt gatcgtattg gggttttcta aggcttgtag ctcggtgtca agttggtcaa 4680

aagtaatcat acgcgtcact cctctcgaat aaagtaagta taacaaaaaa agcatgcgaa 4740

ggcgcatgct ttaggattta agaatattag tctatttgtt tcattgcgtc gttctagaac 4800

ttttttaagt gtatctgcgg agtttttcat ttgttctttt tctttgtcat ttaagttcat 4860

ttcaacaata tggcgaacgc cttgacggtt aacgactgct ggtgcaccta tataaatatc 4920

gttcataccg taatggccat ctaaataaac agaaagtggc aaaatcgcat tttcgttatt 4980

tagaattgct tttgtaatac gagcaagagc tgcagcaacg ccgtagaatg tagcgccttt 5040

tttattaata atttcataag ctgcatcacg aacacttacg aaaatagtat ccattgcacc 5100

ttgttcatct tcgctaatcc attcagtaat tggaaggccg ccgacagttg tgtggctcca 5160

tgctgggaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 5220

catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 5280

ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 5340

ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 5400

cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 5460

ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 5520

tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 5580

cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca 5640

actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 5700

aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 5760

tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 5820

ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 5880

tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt 5940

gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 6000

gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 6060

tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg 6120

gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 6180

ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 6240

gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 6300

aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 6360

ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 6420

ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 6480

tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 6540

gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 6600

agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 6660

taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 6720

gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 6780

gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 6840

caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 6900

aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 6960

tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7020

acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 7080

ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 7140

gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct 7200

ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc 7260

tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct 7320

gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 7380

tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 7440

tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat 7500

tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat 7560

gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcacttga 7620

tgcctccgtg taagggggaa tttctgttca tgggggtaat gataccgatg aaacgagaga 7680

ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg 7740

gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc 7800

agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc 7860

agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac 7920

ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc 7980

ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc 8040

ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc 8100

tgcccgacga tgataagctg tcaaacatga 8130

<210> 4

<211> 12118

<212> DNA

<213>Manually (Artificial)

<400> 4

gaattcccga ttatgtcttt tgcgcactcg gcttaaacca gttttcgctg gtgcgaaaaa 60

agagtgtctt gtgacaccta aattcaaaat ctatcggtca gatttatacc gatttgattt 120

tatatattct tgaataacat acgccgagtt atcacataaa agcgggaacc aatcatcaaa 180

tttaaacttc attgcataat ccattaaact cttaaattct acgattcctt gttcatcaat 240

aaactcaatc atttctttaa ttaatttata tctatctgtt gttgttttct ttaataattc 300

atcaacatct acaccgccat aaactatcat atcttctttt tgatatttaa atttattagg 360

atcgtccatg tgaagcatat atctcacaag acctttcaca cttcctgcaa tctgcggaat 420

agtcgcattc aattcttctg ttattatttt tatctgttca taagatttat taccctcata 480

catcactaga atatgataat gctctttttt catcctacct tctgtatcag tatccctatc 540

atgtaatgga gcactacaaa ttgaatgtgt aactctttta aatactctaa ccactcggct 600

ttgctgattc tggatataaa acaaatgtcc aattacgtcc tcttgaattt ttcttgtttt 660

cagtttcttt tattacattt tcgctcatga tataataacg gtgctaatac acttaacaaa 720

atttagtcat agataggcag catgccagtg ctgtctatct ttttttgttt aaaatgcacc 780

gtattcctcc tttgcatatt tttttattag aataccggtt gcatctgatt tgctaatatt 840

atatttttct ttgattctat ttaatatctc attttcttct gttgtaagtc ttaaagtaac 900

agcaactttt ttctcttctt ttctatctac aactatcact gtacctccca acatctgttt 960

ttttcacttt aacataaaaa acaacctttt aacattaaaa acccaatatt tatttatttg 1020

tttggacaat ggacaatgga cacctagggg ggaggtcgta gtacccccct atgttttctc 1080

ccctaaataa ccccaaaaat ctaagaaaaa aagacctcaa aaaggtcttt aattaacatc 1140

tcaaatttcg catttattcc aatttccttt ttgcgtgtga tgcgctgcgt ccattaaaaa 1200

tcctagagct ttgcaaccga aagttaatag ctgtcgctac tactttcgct tacgctctaa 1260

gtatatttta aggactgtca cacgcaaaaa gttttctcgg cataaaagta cctctacatc 1320

tctaaatcgt ctgtacgctg tttctcacgc tttctatcga tcccgcaaga ggcccggcag 1380

taccggcata accaagccta tgcctacagc atccagggtg acggtgccga ggatgacgat 1440

gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta actgtgataa 1500

actaccgcat taaagcttgt cgacgattca caaaaaatag gcacacgaaa aacaagttaa 1560

gggatgcagt ttatgcatcc cttaacttac ttattaaata atttatagct attgacaaga 1620

gataagaatt gttcaaagct aatattgttt aaatcgtcaa ttcctgcatg ttttaaggaa 1680

ttgttaaatt gattttttgt aaatattttc ttgtattctt tgttaaccca tttcagaacg 1740

aaataattat acttttgttt atctttgtgt gatattcttg atttttttct acttaatctg 1800

ataagtgagc tattcacttt aggtttagga tgaaaatatt ctcttggaac catacttaat 1860

atagaaatat caacttctgc cattaaaagt aatgccaatg agcgttttgt atttaataat 1920

cttttagcaa acccgtattc cacgattaaa taaatctcat tagctatact atcaagaaca 1980

attttgcgta ttatatccgt acttatgtta taaggtatat taccatatat tttataggat 2040

tggtttttag gaaatttaaa ctgcaatata tccttgttta aaacttggaa attatcgtga 2100

tcaacaagtt tattttctgt agttttgcat aatttatggt ctatttcaat ggcagttacg 2160

aaattacacc tctttactaa ttcaagggta aaatggcctt ttcctgagcc gatttcaaag 2220

atattatcat gttcatttaa tcttatattt gtcattattt tatctatatt atgttttgaa 2280

gtaataaagt tttgactgtg ttttatattt ttctcgttca ttataaccct ctttaatttg 2340

gttatatgaa ttttgcttat taacgattca ttataaccac ttattttttg tttggttgat 2400

aatgaactgt gctgattaca aaaatactaa aaatgcccat attttttcct ccttataaaa 2460

ttagtataat tatagcacga gctctgataa atatgaacat gatgagtgat cgttaaattt 2520

atactgcaat cggatgcgat tattgaataa aagatatgag agatttatct aatttctttt 2580

ttcttgtaaa aaaagaaagt tcttaaaggt tttatagttt tggtcgtaga gcacacggtt 2640

taacgactta attacgaagt aaataagtct agtgtgttag actttatgaa atctttatac 2700

gtttatatat atttattatc cggaggtgta gcatgtctca ttcaattttg agggttgcca 2760

gagttaaagg atcaagtaat acaaacggga tacaaagaca taatcaaaga gagaataaaa 2820

actataataa taaagacata aatcatgagg aaacatataa aaattatgat ttgattaacg 2880

cacaaaatat aaagtataaa gataaaattg atgaaacgat tgatgagaat tattcaggga 2940

aacgtaaaat tcggtcagat gcaattcgcc ccgggactag ttaaaggtgg agaaattgat 3000

tcgtttgtcc attatggctt gaattgcaat aatgcctttt gggatggcca agaaattctt 3060

tatggagatg gggacaaaaa gaatttcaaa ccattttcat gcgccaaaac tattgttggt 3120

catgaactaa cgcatgcagt tatccagtat tcggcgggat tggaatacga agggcaatca 3180

ggtgcgctaa acgagtcgtt cgccgatgtt tttggttatt ttattgcgcc aaatcattgg 3240

ttgattggtg aggatgtctg tgtgcgtggg tcgcgagatg ggcgaataag aagcattaaa 3300

gatcctgaca aatataatca agcggctcat atgaaggatt acgaatcgct tccaatcaca 3360

gaggaaggcg actggggcgg agttcattat aatagtggta tcccgaataa agcagcctat 3420

aatactatca ctaaacttgg aaaagaaaaa acagaacagc gacgatttcg cgccttaaag 3480

tactatttaa cgaaaaaagc ccagtttacc gatgcgaaaa aagcgcttca acaagcagcg 3540

aaagatttat atggtgaaga tgcttctaaa aaagttgctg aagcttggga agcggtagga 3600

gttaactgag cggccgctag ctttaaggct aaatgccgaa tggttggcac ctaccgcatt 3660

ggcaaccgtg gcagaagagg gcgcatccgt tttggcgaaa aagagtaaaa cggcgaggat 3720

gagtgcacag ccagagccca gccagaaaac aaactgatta ttgatggtga acatgatgcc 3780

gacaatcgag gcacacagcg cccagccaac acagccaaac atccgcgcgc gaccaaattc 3840

gaaattactg cgacggctga ctttctcaat aaatgcctct actgctggcg caccggcgtt 3900

aaaacaaaag cctagataaa taccaccaac aatcgatcct actaaaatgt tgtattgtaa 3960

cagtggcccg aagataaaaa taaagaacgg cgcaaacatc actaacatgc cggtaataat 4020

ccacagcagg tatttgcgca gcccgagttt gtcagaaagc agaccaaaca gcggttggaa 4080

taatagcgag aacagagaaa tagcggcaaa aataataccc gtatcacttt tgctgatatg 4140

gttgatgtca tgtagccaaa tcgggaaaaa cgggaagtag gctcccatga taaaaaagta 4200

aaagaaaaag aataaaccga acatccaaaa gtttgtgttt tttaaatagt acataatgga 4260

tttccttacg cgaaatacgg gcagacatgg cctgcccggt tattattatt tttgacacca 4320

gaccaactgg taatggtagc gaccggcgct cagctggaat taattccgcc gatactgacg 4380

ggctccagga gtcgtcgcca ccaatcccca tatggaaacc gtcgatattc agccatgtgc 4440

cttcttccgc gtgcagcaga tggcgatggc tggtttccat cagttgctgt tgactgtagc 4500

ggctgatgtt gaactggaag tcgccgcgcc actggtgtgg gccataattc aattcgcgcg 4560

tcccgcagcg cagaccgttt tcgctcggga agacgtacgg ggtatacatg tctgacaatg 4620

gcagatccca gcggtcaaaa caggcggcag taaggcggtc gggatagttt tcttgcggcc 4680

ctaatccgag ccagtttacc cgctctgcta cctgcgccag ctggcagttc aggccaatcc 4740

gcgccggatg cggtgtatcg ctcgccactt caacatcaac ggtaatcgcc atttgaccac 4800

taccatcaat ccggtaggtt ttccggctga taaataaggt tttcccctga tgctgccacg 4860

cgtgagcggt cgtaatcagc accgcatcag caagtgtatc tgccgtgcac tgcaacaacg 4920

ctgcttcggc ctggtaatgg cccgccgcct tccagcgttc gacccaggcg ttagggtcaa 4980

tgcgggtcgc ttcacttacg ccaatgtcgt tatccagcgg tgcacgggtg aactgatcgc 5040

gcagcggcgt cagcagttgt tttttatcgc caatccacat ctgtgaaaga aagcctgact 5100

ggcggttaaa ttgccaacgc ttattaccca gctcgatgca aaaatccatt tcgctggtgg 5160

tcagatgcgg gatggcgtgg gacgcggcgg ggagcgtcac actgaggttt tccgccagac 5220

gccactgctg ccaggcgctg atgtgcccgg cttctgacca tgcggtcgcg ttcggttgca 5280

ctacgcgtac tgtgagccag agttgcccgg cgctctccgg ctgcggtagt tcaggcagtt 5340

caatcaactg tttaccttgt ggagcgacat ccagaggcac ttcaccgctt gccagcggct 5400

taccatccag cgccaccatc cagtgcagga gctcgttatc gctatgacgg aacaggtatt 5460

cgctggtcac ttcgatggtt tgcccggata aacggaactg gaaaaactgc tgctggtgtt 5520

ttgcttccgt cagcgctgga tgcggcgtgc ggtcggcaaa gaccagaccg ttcatacaga 5580

actggcgatc gttcggcgta tcgccaaaat caccgccgta agccgaccac gggttgccgt 5640

tttcatcata tttaatcagc gactgatcca cccagtccca gacgaagccg ccctgtaaac 5700

ggggatactg acgaaacgcc tgccagtatt tagcgaaacc gccaagactg ttacccatcg 5760

cgtgggcgta ttcgcaaagg atcagcgggc gcgtctctcc aggtagcgaa agccattttt 5820

tgatggacca tttcggcaca gccgggaagg gctggtcttc atccacgcgc gcgtacatcg 5880

ggcaaataat atcggtggcc gtggtgtcgg ctccgccgcc ttcatactgc accgggcggg 5940

aaggatcgac agatttgatc cagcgataca gcgcgtcgtg attagcgccg tggcctgatt 6000

cattccccag cgaccagatg atcacactcg ggtgattacg atcgcgctgc accattcgcg 6060

ttacgcgttc gctcatcgcc ggtagccagc gcggatcatc ggtcagacga ttcattggca 6120

ccatgccgtg ggtttcaata ttggcttcat ccaccacata caggccgtag cggtcgcaca 6180

gcgtgtacca cagcggatgg ttcggataat gcgaacagcg cacggcgtta aagttgttct 6240

gcttcatcag caggatatcc tgcaccatcg tctgctcatc catgacctga ccatgcagag 6300

gatgatgctc gtgacggtta acgcctcgaa tcagcaacgg cttgccgttc agcagcagca 6360

gaccattttc aatccgcacc tcgcggaaac cgacatcgca ggcttctgct tcaatcagcg 6420

tgccgtcggc ggtgtgcagt tcaaccaccg cacgatagag attcgggatt tcggcgctcc 6480

acagtttcgg gttttcgacg ttcagacgta gtgtgacgcg atcggcataa ccaccacgct 6540

catcgataat ttcaccgccg aaaggcgcgg tgccgctggc gacctgcgtt tcaccctgcc 6600

ataaagaaac tgttacccgt aggtagtcac gcaactcgcc gcacatctga acttcagcct 6660

ccagtacagc gcggctgaaa tcatcattaa agcgagtggc aacatggaaa tcgctgattt 6720

gtgtagtcgg tttatgcagc aacgagacgt cacggaaaat gccgctcatc cgccacatat 6780

cctgatcttc cagataactg ccgtcactcc aacgcagcac catcaccgcg aggcggtttt 6840

ctccggcgcg taaaaatgcg ctcaggtcaa attcagacgg caaacgactg tcctggccgt 6900

aaccgaccca gcgcccgttg caccacagat gaaacgccga gttaacgcca tcaaaaataa 6960

ttcgcgtctg gccttcctgt agccagcttt catcaacatt aaatgtgagc gagtaacaac 7020

ccgtcggatt ctccgtggga acaaacggcg gattgaccgt aatgggatag gttacgttgg 7080

tgtagatggg cgcatcgtaa ccgtgcatct gccagtttga ggggacgacg acagtatcgg 7140

cctcaggaag atcgcactcc agccagcttt ccggcaccgc ttctggtgcc ggaaaccagg 7200

caaagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 7260

cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 7320

cgccagggtt ttcccagtca cgacgttgta aaacgacggg atccataaaa actagcatta 7380

tttttttcat gggtttcact ctccttctac attttttaac ctaataatgc caaataccgt 7440

ttgccacccc tctcttttga taattataat attggcgaaa ttcgcttcta aagatgaaac 7500

gcaatattat atgcttgctt tatagcttta ttctagtcct gctgtccctt tatcgtcgtt 7560

aacaaatgtt aatgcctcaa cataaaagtc actctaggcg gccgctaata atcttgcgct 7620

tcgatgacaa cagctgtacc agatgcagtg accattagca ttccgttatc agctccaagc 7680

acttcataat caatatcaac accgataacg gcatttgcgc caatatcttt cgcacgttgt 7740

tccatttcac gaattgcttc ctcgcgagca ttaataagtt catcttcata gccttgcgaa 7800

cggcccccga agaaatttcg aagtccagcc ccaatatctt tcataaagtt aacgccagtg 7860

atgacttcgc cgaaaacgat ttttttatat tcgataattt gtttgccttc aatatttggt 7920

gaagttgtta caatcatgag ttatccctac agtttttctt ttatcatacc tcttagtact 7980

ttttctagtc aaaggatatc cggttatttc gtacgatttc gcgctttttc tatataagaa 8040

atagcatctg gaactttaca agctgtattt ccaaggttta catgaacttt cccgactgat 8100

ttcgcggctt ccatcgcttt ttcgtgcaaa tcgtctttgt aaattccaca agtgataata 8160

aaaccgttca ttgaatagcg ggttctattg gtttctgttt gcagcgtttc tttcgcgcgt 8220

tgaagtaatg tctctgcttc aggcaaattg ttaatatgtt cgctgtaaat ctgccagccc 8280

atggatttag ttaaatcata atcactttca atccatgttt tggcgaaaag gagtgcatca 8340

tcacgtttgc ttacaatcga tgctagtcca aaagttagtt gtgaccatgt ttcgcgaaaa 8400

gcgatattcc atttttcgat ttgttcggtg gttattttgt ttggattaat tgctagcaaa 8460

ccgagataaa tcaagtcact attatttgat tcaatcagtt ttacggcgag ttcgtgattt 8520

tttgtcagtt tttctcggcg gatgattttc tttaaatcac caatttttag tccgtaaaga 8580

tctaatgaat ccggacaacc gtgattacga aaaattttga tcgtattggg gttttctaag 8640

gcttgtagct cggtgtcaag ttggtcaaaa gtaatcatac gcgtcactcc tctcgaataa 8700

agtaagtata acaaaaaaag catgcgaagg cgcatgcttt aggatttaag aatattagtc 8760

tatttgtttc attgcgtcgt tctagaactt ttttaagtgt atctgcggag tttttcattt 8820

gttctttttc tttgtcattt aagttcattt caacaatatg gcgaacgcct tgacggttaa 8880

cgactgctgg tgcacctata taaatatcgt tcataccgta atggccatct aaataaacag 8940

aaagtggcaa aatcgcattt tcgttattta gaattgcttt tgtaatacga gcaagagctg 9000

cagcaacgcc gtagaatgta gcgccttttt tattaataat ttcataagct gcatcacgaa 9060

cacttacgaa aatagtatcc attgcacctt gttcatcttc gctaatccat tcagtaattg 9120

gaaggccgcc gacagttgtg tggctccatg ctgggaattc ttgaagacga aagggcctcg 9180

tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 9240

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 9300

atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 9360

agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 9420

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 9480

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 9540

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 9600

tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 9660

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 9720

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 9780

cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 9840

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 9900

cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 9960

tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 10020

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 10080

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 10140

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 10200

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 10260

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 10320

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 10380

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 10440

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 10500

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 10560

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 10620

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 10680

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 10740

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 10800

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 10860

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 10920

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 10980

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 11040

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 11100

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 11160

cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 11220

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 11280

gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 11340

gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 11400

gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta 11460

aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag 11520

ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag 11580

ggcggttttt tcctgtttgg tcacttgatg cctccgtgta agggggaatt tctgttcatg 11640

ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa 11700

catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac 11760

cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca 11820

cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac 11880

ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag 11940

gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc 12000

tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc 12060

atgcgcaccc gtggccagga cccaacgctg cccgacgatg ataagctgtc aaacatga 12118

<210> 5

<211> 23

<212> DNA

<213>Manually (Artificial)

<400> 5

gctataaatg aagaggcttc agg 23

<210> 6

<211> 22

<212> DNA

<213>Manually (Artificial)

<400> 6

ctcttaaatc agctaggcga tc 22

<210> 7

<211> 22

<212> DNA

<213>Manually (Artificial)

<400> 7

cttcgatgac aacagctgta cc 22

<210> 8

<211> 22

<212> DNA

<213>Manually (Artificial)

<400> 8

aatcctaaag catgcgcctt cg 22

<210> 9

<211> 24

<212> DNA

<213>Manually (Artificial)

<400> 9

gtcgacgatt cacaaaaaat aggc 24

<210> 10

<211> 20

<212> DNA

<213>Manually (Artificial)

<400> 10

actagtcccg gggcgaattg 20

<210> 11

<211> 25

<212> DNA

<213>Manually (Artificial)

<400> 11

ccacaagctt tgtcccgtac acttg 25

<210> 12

<211> 25

<212> DNA

<213>Manually (Artificial)

<400> 12

tatactcgag agcaagtgta gaagc 25

<210> 13

<211> 27

<212> DNA

<213>Manually (Artificial)

<400> 13

gataagtcga cgattcacaa aaaatag 27

<210> 14

<211> 23

<212> DNA

<213>Manually (Artificial)

<400> 14

aaaactagtc ccggggcgaa ttg 23

Claims (9)

1. a kind of attenuation of genome foreign gene-carrying recombinates Listeria monocytogenes, which is characterized in that the attenuation singly increases Lee This special bacterium is Listeria monocytogenes completely knock out the two virulence genes of actA and plcB and obtain.

2. a kind of attenuation of genome foreign gene-carrying according to claim 1 recombinates Listeria monocytogenes, feature It is, III restriction enzyme site of upstream belt Hind, I restriction enzyme site of downstream belt Xho of the foreign gene.

3. a kind of preparation method of the attenuation recombination Listeria monocytogenes of genome foreign gene-carrying as claimed in claim 1 or 2, It is characterized in that, be to be attenuated Listeria monocytogenes as carrier, foreign gene-carrying and obtain.

4. preparation method according to claim 3, which is characterized in that specific step is as follows：

(1) foreign gene of quasi- integration is inserted between the Hind III digestion site and Xho I restriction enzyme site of plasmid pCW203, Obtain intermediate recombinant plasmid；

(2) segment between the BamH I restriction enzyme site for the intermediate recombinant plasmid that step (1) obtains and Xho I restriction enzyme site is cut It is inserted between the BamH I restriction enzyme site and Xho I restriction enzyme site of recombinant plasmid pCW180, obtains target practice plasmid, the recombination matter The gene order of grain pCW180 is shown in SEQ ID NO.1 in sequence table；

(3) the target practice plasmid electrotransformation for obtaining step (2) recombinates Listeria monocytogenes Lm Δ actAplcB-lacZ, then will It converts obtained bacterium and hybridizes culture through single, double homologous recombination, obtain object bacteria.

5. the preparation method according to claim 4, which is characterized in that in step (1), the preparation method of the foreign gene It is：It is obtained from biological genome template or gene clone plasmid library PCR amplification or DNA synthesizer directly synthesizes.

6. the preparation method according to claim 4, which is characterized in that in step (2), the preparation side of recombinant plasmid pCW180 Method is as follows：

Upstream is carried I enzyme of Spe I and Not of the Lm mpl gene insertion plasmid pCW154 of Spe I, downstream carrying Not I by (2-1) Between enzyme site, the first intermediate recombinant plasmid pCW160 is obtained, the gene order of the first intermediate recombinant plasmid pCW160 is In sequence table shown in SEQ ID NO.2；

(2-2) obtains Lm orfBAldh gene insertion above-mentioned steps (2-1) that upstream carries Xba I, downstream carrying Not I Between I restriction enzyme site of Xba I and Not of first intermediate recombinant plasmid pCW160, the second intermediate recombinant plasmid pCW170, institute are obtained The gene order of the second intermediate recombinant plasmid pCW170 is stated as shown in SEQ ID NO.3 in sequence table；

(2-3) uses I digested plasmid pCW154 of Not, obtains one section of gene that both ends are I restriction enzyme site of Not, is inserted into above-mentioned steps I restriction enzyme site of Not for the second intermediate recombinant plasmid pCW170 that (2-2) is obtained, obtains recombinant plasmid pCW180.

7. the preparation method according to claim 4, which is characterized in that in step (3), recombinate Listeria monocytogenes Lm Δ The preparation method of actAplcB-lacZ is as follows：

(3-1) the lacZ gene segment with Not I restriction enzyme site is inserted into the second intermediate recombinant plasmid pCW170 respectively by upstream and downstream Not I restriction enzyme site between, obtain target practice plasmid a pCW190, the plasmid pCW190 gene order be sequence table in Shown in SEQ ID NO.4；

(3-2) by target practice plasmid pCW190 electrotransformation Listeria monocytogenes obtained by step (3-1), the bacterium for then obtaining conversion is passed through Single, double homologous recombination hybridization culture to get.

8. the preparation method according to claim 4, which is characterized in that in step (3), recombinate Listeria monocytogenes Lm Δ The genome orfBAldh upstream region of gene of actAplcB-lacZ has been integrated into beta-galactosidase gene, can express beta galactose Glycosides enzyme grows up to blue colonies on the culture medium of 4 chloro- 3- indoles-β-D- galactoside bromo- containing 5-.

9. the preparation method according to claim 4, which is characterized in that in step (3), in the genome of the object bacteria OrfBAldh upstream region of gene incorporates foreign gene.