基于lux报告系统的猪链球菌发光菌及其构建方法

Streptococcus suis luminescent bacterium based on lux report system and construction method thereof

文档序号：3245 发布日期：2021-09-17 浏览：50次中文

1. A Streptococcus suis luminescent bacterium is obtained by inserting a luciferase reporter system knock-in fragment into a genome of Streptococcus suis at a fixed point between an SSU05_0518 gene and an SSU05_0519 gene;

the luciferase reporter knock-in fragment sequentially comprises an expression cassette 1, an expression cassette 2 and an expression cassette 3 from a 5 'end to a 3' end; the expression cassette 1 is used for expressing a gene cluster luxAB of a luciferase report system; the expression cassette 2 is used for expressing a gene cluster luxcDE of a luciferase reporter system; the expression cassette 3 is used for expressing a resistance marker gene.

2. The Streptococcus suis luminophore of claim 1, wherein: in the expression cassette 2, the promoter for promoting the transcription of the gene cluster luxcDE is CP25G promoter; the CP25G promoter is a promoter with an AGGAGG sequence obtained by modifying a CP25 promoter;

further, the nucleotide sequence of the CP25G promoter is shown in position 3437-3573 of SEQ ID No. 1.

3. The Streptococcus suis luminophore according to claim 1 or 2, wherein: in the expression cassette 1, the promoter for starting transcription of the gene cluster luxAB is a 147AGGAGG promoter or a 1647 promoter;

the 147AGGAG promoter is a promoter which is obtained by modifying SSU05_0147 gene promoter in the genome of streptococcus suis and has an AGGAGG sequence; the 1647AGGAGG promoter is a promoter which is obtained by modifying SSU05_1647 gene promoter in a streptococcus suis genome and has an AGGAGG sequence; the 1647 promoter is an SSU05_1647 gene promoter in a streptococcus suis genome;

further, the nucleotide sequence of the 147AGGAGG promoter is shown in the 1171-1351 position of SEQ ID No. 1; the 1647AGGAGG promoter has a nucleotide sequence shown in the 1171-1282 position of SEQ ID No. 2; the 1647 promoter has a nucleotide sequence shown in the 1171-1271 position of SEQ ID No. 3.

4. The Streptococcus suis luminophore according to any one of claims 1-3, wherein: the luciferase reporter knock-in fragment replaces position 1350-1424 of the sequence shown in SEQ ID No.4 in the genome of Streptococcus suis.

5. The Streptococcus suis luminophore according to any one of claims 1-4, wherein: the streptococcus suis luminescent bacterium is prepared by the following steps: and (3) utilizing homologous recombination to insert the knocked-in fragment of the luciferase reporter system into the position between the SSU05_0518 gene and the SSU05_0519 gene of the streptococcus suis, thus obtaining the streptococcus suis luminescent bacterium.

6. The Streptococcus suis luminophore of claim 5, wherein: when the homologous recombination is carried out, the sequence of the upstream homology arm is shown as 1-1170 of SEQ ID No.1, and the sequence of the downstream homology arm is shown as 8147-9379 of SEQ ID No. 1.

7. The Streptococcus suis luminophore of claim 6, wherein: the streptococcus suis luminescent bacterium is prepared by a method comprising any one of the following steps:

p1, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.1 into streptococcus suis, and realizing fixed-point insertion of a knock-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis luminescent bacteria;

p2, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.2 into streptococcus suis, and realizing fixed-point insertion of a knock-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis luminescent bacteria;

p3, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.3 into streptococcus suis, and realizing fixed-point insertion of the knocked-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis luminescent bacteria.

8. A method of constructing S.suis luminophores comprising the steps of any one of claims 5-7.

9. The Streptococcus suis luminophore according to any one of claims 1-7 or the method of claim 8, wherein: the streptococcus suis is streptococcus suis type 2;

further, the streptococcus suis type 2 is streptococcus suis 05ZYH 33.

10. Use of a Streptococcus suis bacterium according to any one of claims 1 to 7 for non-disease diagnostic imaging of animals in vivo.

Background

Streptococcus suis is an important zoonotic pathogen, which causes huge losses to the pig breeding industry every year, in recent years, cases of human infection with Streptococcus suis are reported every year, in southeast Asia regions, especially Thailand and Vietnam, the Streptococcus suis infection becomes the primary cause of Vietnam adult meningitis, and in Thailand the second place is the cause of the Vietnam adult meningitis, which attracts more and more attention, and the infection route of the Streptococcus suis and related virulence factors are the key points in the research of the Streptococcus suis. Although the tracer observation of streptococcus suis is reported to be realized based on green fluorescent protein, the fluorescent tracer can only be used in vitro experiments, and cannot be used for real-time observation in animals.

The core elements of the lux reporter system derived from the luminescent bacteria comprise five genes of luxA, B, C, D and E, wherein the luxA and the luxB respectively code alpha subunit and beta subunit of the luciferase, and the luxC, the luxD and the luxE respectively code fatty acid reductase, acyltransferase and ATP synthetase which depend on NADPH, and the three jointly form a fatty acid reductase complex to generate long-chain fatty aldehyde as an electron donor of luminescent reaction. Because of instantaneity and high sensitivity, the method has potential application value in molecular biology, clinical microorganism and biochemical detection. Pathogenic bacteria such as streptococcus pneumoniae, streptococcus pyogenes and staphylococcus aureus are reported to be luminescent bacteria constructed based on the system, and play an important role in the research of the infection process and pathogenic mechanism of bacteria.

Disclosure of Invention

In order to effectively solve the technical problems, the invention aims to provide the streptococcus suis luminescent bacterium based on the lux report system and a construction method thereof, wherein the streptococcus suis luminescent bacterium can be observed in real time in an animal body.

In a first aspect, the invention claims a streptococcus suis luminophor.

The streptococcus suis luminescent bacteria claimed by the invention are obtained by inserting the luciferase reporter system knock-in fragment into the genome of streptococcus suis at a fixed point between an SSU05_0518 gene and an SSU05_0519 gene. Wherein the SSU05_0518 gene and the SSU05_0519 gene are used as gene annotations of the 05ZYH33 strain in the Refseq database of NCBI. Wherein the "site-directed insertion of the luciferase reporter knock-in fragment between the SSU05_0518 gene and the SSU05_0519 gene in the genome of Streptococcus suis" may comprise substituting the luciferase reporter knock-in fragment for a partial sequence between the SSU05_0518 gene and the SSU05_0519 gene in the genome of Streptococcus suis.

The luciferase reporter knock-in fragment sequentially comprises an expression cassette 1, an expression cassette 2 and an expression cassette 3 from a 5 'end to a 3' end; the expression cassette 1 is used for expressing a gene cluster luxAB of a luciferase report system; the expression cassette 2 is used for expressing a gene cluster luxcDE of a luciferase reporter system; the expression cassette 3 is used for expressing a resistance marker gene.

Further, in the expression cassette 2, the promoter that initiates transcription of the gene cluster luxCDE may be a CP25G promoter; the CP25G promoter is a promoter with an AGGAGG sequence obtained by modifying a CP25 promoter.

Further, the nucleotide sequence of the CP25G promoter may be as shown in positions 3437-3573 of SEQ ID No. 1.

Further, in the expression cassette 1, the promoter for initiating transcription of the gene cluster luxAB may be a 147AGGAGG promoter or a 1647 promoter.

The 147AGGAGG promoter is a promoter with an AGGAGG sequence obtained by modifying SSU05_0147 gene promoter in a streptococcus suis genome. The 1647AGGAGG promoter is a promoter which is obtained by modifying SSU05_1647 gene promoter in a streptococcus suis genome and has an AGGAGG sequence. The 1647 promoter is an SSU05_1647 gene promoter in a streptococcus suis genome.

Further, the nucleotide sequence of the 147AGGAGG promoter can be shown in the 1171-1351 position of SEQ ID No. 1; the 1647AGGAGG promoter nucleotide sequence can be shown as the 1171-1282 position of SEQ ID No. 2; the nucleotide sequence of the 1647 promoter can be shown as the position 1171-1271 of SEQ ID No. 3.

Further, in the expression cassette 3, the resistance marker gene may be a chloramphenicol resistance gene.

In one embodiment of the invention, the nucleotide sequence of the expression cassette 1 is shown in SEQ ID No.1 at positions 1171-3436; the nucleotide sequence of the expression cassette 2 is shown as 3437-7090 of SEQ ID No. 1; the nucleotide sequence of the expression cassette 3 is shown as 7091-8146 site of SEQ ID No. 1. Accordingly, the nucleotide sequence of the knock-in fragment of the luciferase reporter system is shown in positions 1171-8146 of SEQ ID No.1 (corresponding to combination 7).

In another embodiment of the invention, the nucleotide sequence of the expression cassette 1 is as shown in SEQ ID No.2 at positions 1171-3367; the nucleotide sequence of the expression cassette 2 is shown as the 3368-7021 site of SEQ ID No. 2; the nucleotide sequence of the expression cassette 3 is shown in 7022-8077 of SEQ ID No. 2. Accordingly, the nucleotide sequence of the knock-in fragment of the luciferase reporter system is shown in SEQ ID No.1 at position 1171-8077 (corresponding to combination 5).

In yet another embodiment of the present invention, the nucleotide sequence of the expression cassette 1 is shown in SEQ ID No.3 at positions 1171-3356; the nucleotide sequence of the expression cassette 2 is shown as 3357-7010 of SEQ ID No. 3; the nucleotide sequence of the expression cassette 3 is shown in 7011-8066 of SEQ ID No. 3. Accordingly, the nucleotide sequence of the knock-in fragment of the luciferase reporter system is shown in SEQ ID No.1 at position 1171-8066 (corresponding to combination 3).

In a particular embodiment of the invention, the luciferase reporter knock-in fragment specifically replaces position 1350-1424 of the sequence shown in SEQ ID No.4 in the genome of Streptococcus suis.

Further, the streptococcus suis luminescent bacteria can be prepared according to a method comprising the following steps: and (3) utilizing homologous recombination to insert the knocked-in fragment of the luciferase reporter system into the position between the SSU05_0518 gene and the SSU05_0519 gene of the streptococcus suis, thus obtaining the streptococcus suis luminescent bacterium.

When the homologous recombination is carried out, the sequence of the upstream homology arm is shown as 1-1170 (i.e. 1-1170 of SEQ ID No.2 or 1-1170 of SEQ ID No. 3) of SEQ ID No.1, and the sequence of the downstream homology arm is shown as 8147-9379 (i.e. 8078-9310 of SEQ ID No.2 or 8067-9299 of SEQ ID No. 3) of SEQ ID No. 1.

Furthermore, the streptococcus suis luminescent bacteria can be prepared by a method comprising any one of the following steps:

p1, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.1 into streptococcus suis through natural transformation, and realizing site-specific insertion of the knocked-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis luminescent bacteria (corresponding to the combination 7);

p2, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.2 into streptococcus suis through natural transformation, and realizing site-specific insertion of the knocked-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis luminescent bacteria (corresponding to the combination 5);

p3, introducing a DNA fragment with a nucleotide sequence shown as SEQ ID No.3 into streptococcus suis through natural transformation, and realizing site-specific insertion of the knocked-in fragment of the luciferase reporter system between an SSU05_0518 gene and an SSU05_0519 gene of the streptococcus suis through homologous recombination to obtain the streptococcus suis photobacterium (corresponding to combination 3).

Wherein, the natural transformation process comprises adding the polypeptide ComS into the system_13-21The step (2). The polypeptide canThe nucleic acid around the bacteria is collected by stimulating the streptococcus suis, so that the knock-in fragment is absorbed into the bacteria to generate homologous recombination.

In a second aspect, the invention claims a method for constructing streptococcus suis luminescent bacteria.

The method for constructing S.suis P.suis P.sp.as claimed in the present invention may comprise the relevant steps as described in the first aspect above.

In the first or second aspect hereinbefore, the streptococcus suis may be streptococcus suis type 2.

In a particular embodiment of the invention, said streptococcus suis is in particular streptococcus suis 05ZYH 33.

In a third aspect, the invention claims the non-disease diagnostic use of the Streptococcus suis luminophores as described in the first aspect above in vivo imaging of animals.

Wherein the animal may be a mouse.

According to the invention, by designing 7 construction strategies of the streptococcus suis luminescent bacteria and optimizing and screening different promoters, the construction method of the streptococcus suis luminescent bacteria based on the lux report system is finally established, the bacteria can be observed in a mouse body in real time, and the method has a certain application prospect in the research of the infection route and pathogenic mechanism of the streptococcus suis.

Drawings

FIG. 1 is a schematic diagram of the location of the lux gene cluster insertion.

FIG. 2 is a schematic diagram of 7 construction strategies according to the present invention. Note: 1647 is the endogenous SSU05_1647 gene promoter of Streptococcus suis; 1647AGGAGG is the SSU05_1647 gene promoter with AGGAGG sequence; 147AGGAGG is the SSU05_0147 gene promoter with AGGAGG sequence; CP25 is CP25 promoter; CP25G is based on CP25 promoter, with a G base added to form the AGGAGG sequence promoter.

Fig. 3 shows knock-in fragments in different combinations.

FIG. 4 shows the images of the luminescent bacteria in different combinations.

FIG. 5 shows fluorescence values of the luminescent bacteria. WT represents the non-engineered S.suis wild strain 05ZYH 33.

FIG. 6 is a mouse in vivo imaging observation. Note: the left-most side is a wild-infected mouse, and the last four are luminophorous-infected mice.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction of Streptococcus suis luminophores based on lux reporter System

First, experimental material

1. Experimental strains and plasmids

Streptococcus suis wild strain 05ZYH 33: a streptococcus suis type 2 virulent strain which is preserved in a laboratory. Described in "Wenhua Huang, et al, LytR plate a roll in normal section formation and controls to full virus in Streptococcus suis. The applicant can obtain the said product, and can only use it for repeating the experiment of the invention, and has no other use.

Plasmid pSET 1: temperature sensitive suicide plasmid, chloramphenicol resistance, laboratory preservation. Described in "Wenhua Huang, et al, LytR plate a roll in normal section formation and controls to full virus in Streptococcus suis. The applicant can obtain the said product, and can only use it for repeating the experiment of the invention, and has no other use.

Plasmid pXen 5: coli-gram-positive shuttle plasmid, lux reporter system, product of caliper, USA.

2. Primer and polypeptide sequence

Specifically, the results are shown in Table 1.

TABLE 1 primer and polypeptide sequences according to the invention

3. Experimental reagent

As shown in table 2.

TABLE 2 Experimental reagents according to the invention

4. Experimental equipment

As shown in table 3.

TABLE 3 Experimental facilities related to the present invention

Second, Experimental methods

1. Knock-in fragment design

There are two main types of lux reporter systems expressed in prokaryotes: a genome-based lux reporter system and a plasmid-based lux reporter system. Plasmid-based lux reporter systems require the use of antibiotics during culture to prevent plasmid loss, which limits the use of reporter strains in live animals. Therefore, the invention is constructed by adopting a method of inserting the genome of the streptococcus suis. In previous researches, the lux reporter system is generally randomly inserted into a genome through a transposon, the number of the insertions cannot be controlled, and the insertions can be inserted into some key genes, such as some important virulence factors and the like, the knock-in system can realize the directional insertion into the bacterial genome, the insertion positions are controllable, and the insertion between an SSU05_0518 gene and an SSU05_0519 gene is finally selected by looking at the gene locus of 05ZYH33 so as to prevent the insertion of the lux gene cluster from influencing the transcription of upstream and downstream genes, and the distribution of the genes near the insertion positions is shown in FIG. 1. In particular to the replacement of the 1350-1422 th position of the sequence shown in SEQ ID No.4 in the genome of the streptococcus suis 05ZYH 33.

The lux reporter system is a large gene cluster, divided into two parts, luxAB and luxCDE, and requires a strong promoter for transcription and translation. The present invention separately transcribes the two parts luxAB and luxCDE to increase the transcription level of the system. The invention finally selects the promoters of two genes of SSU05_1647 and SSU05_0147 endogenous in Streptococcus suis and the artificially synthesized CP25 promoter as candidate strong promoters. The pXen5 plasmid contains an optimized lux reporter system for gram-positive bacteria, and an AGGAGG sequence is added in front of each gene transcription initiation site to improve the transcription level of the genes, so the AGGAGG sequence or a base G is added behind three promoters to form AGGAGG, and the following seven combinations are tried, as shown in FIG. 2.

2. Construction of knock-in fragments of lux reporter system containing different promoters

Amplifying an upstream homology arm, a downstream homology arm, an SSU05_0147 gene promoter (including the promoter with AGGAGG and the promoter without AGGAGG) and an SSU05_1647 gene promoter (including the promoter with AGGAGG and the promoter without AGGAGG) by using a genome of a wild streptococcus suis strain 05ZYH33 as a template; using the CP25 gene synthesized by the marine engineering bioengineering company Limited as a template to amplify the CP25 promoter (including G-bearing and G-free); amplifying to obtain a chloramphenicol resistance gene cassette by taking pSET1 plasmid as a template; the plasmid pXen5 was used as a template to amplify to obtain the luxAB and luxcDNA fragments (the sequences of the primers are shown in Table 1). After obtaining each fragment, ligation was performed by using overlapping PCR in order (7 combinations shown in FIG. 2). The PCR reagent used was PrimeSTAR Max DNA Polymerase, and the PCR reaction system and reaction program were as follows:

and (3) PCR reaction system: PrimeSTAR Max DNA Polymerase 25. mu.L; 1 μ L of upstream primer (10 μ M); 1 μ L of downstream primer (10 μ M); 2 mu L of template; 21 μ L of water.

PCR reaction procedure: 10s at 98 ℃, 5s at 55 ℃, 5s/kb at 72 ℃ for 32 cycles.

The final PCR products corresponding to the 7 combinations in FIG. 2 were sequenced to confirm the correct sequence.

Wherein the final PCR product sequence corresponding to "combination 7" in FIG. 2 is shown in SEQ ID No. 1. The 1 st-1170 position of SEQ ID No.1 is an upstream homology arm sequence, the 1171 st-1351 st-1352 st-3436 st-luraB gene cluster sequence, the 3437 st-3573 st-CP 25G promoter sequence, the 3574 st-7090 st-luraXDE gene cluster sequence, the 7091 st-8146 th-chloramphenicol resistance gene cassette sequence, and the 8147 st-9379 th-downstream homology arm sequence.

The final PCR product sequence corresponding to "combination 5" in FIG. 2 is shown in SEQ ID No. 2. The 1 st-1170 position of SEQ ID No.2 is an upstream homology arm sequence, the 1171 st-1282 th-1647 AGGAGG promoter sequence, the 1283 st-3367 th-position of gene cluster luxAB, the 3368 st-3504 th-position of CP25G promoter sequence, the 3505 st-7021 st-position of gene cluster luxCDE sequence, the 7022 st-8077 th-position of chloramphenicol resistance gene cassette sequence, and the 8078 st-9310 th-position of downstream homology arm sequence.

The final PCR product sequence corresponding to "combination 3" in FIG. 2 is shown in SEQ ID No. 2. The 1 st-1170 site of SEQ ID No.2 is an upstream homology arm sequence, the 1171 st-1271 st-hugh 1271 site is a 1647 promoter sequence, the 1272 nd-hugh 3356 site is a gene cluster luxAB sequence, the 3357 nd-hugh 3493 site is a CP25G promoter sequence, the 3494 st-hugh 7010 site is a gene cluster luxDE sequence, the 7011 st-hugh 8066 site is a chloramphenicol resistance gene cassette sequence, and the 8067 th-hugh 9299 site is a downstream homology arm sequence.

3. Knock-in of the Lux reporter System into the genome of Streptococcus suis 05ZYH33 by Natural transformation

Inoculating Streptococcus suis wild strain 05ZYH33 in 5mL THY medium, and culturing to OD₆₀₀When the concentration is 0.6, 100. mu.L of the bacterial solution is transferred to 5mL of THY medium, the culture is performed in an incubator at 37 ℃ for static culture, after 1 hour, 100. mu.L of the bacterial solution is taken to a sterile 1.5mL centrifuge tube, and 1.2. mu.g of the knock-in fragment containing the lux luminescence system obtained by the PCR in the step 2 and 10. mu.l of 2.5mM polypeptide ComS are added_13-21And after being uniformly mixed, the mixture is kept stand and incubated for 2h at 37 ℃, then the mixture is coated on a THY plate containing 5 mu g/mL chloramphenicol, the mixture is kept stand and incubated for 12h at 37 ℃, and the bacteria growing on the resistant plate are the luminescent bacteria to be screened.

4. Evaluation of the luminescence intensity under different promoters

(1) Imaging the bacteria on the resistant plate by using a ChemiDocXRS + high-sensitivity chemiluminescence imager of the American bio-rad company, evaluating whether the bacteria can emit light under different combinations, and screening luminescent bacteria which are successfully recombined;

(2) selecting bacteria on the resistant plate, recovering the bacteria in a THY chloramphenicol resistant culture medium, culturing to the late logarithmic phase, taking 500 mu L of bacteria liquid of each bacteria, centrifugally enriching, discarding supernatant, cleaning the bacteria once by PBS, and uniformly adjusting OD₆₀₀The amount of each strain was adjusted to 0.4. mu.L and 200. mu.L of the adjusted liquid was applied to a 96-well ELISA plate and usedThe 96Microplate Luminometer measures the luminescence intensity of each well.

5. Mouse living body imaging observation of luminous effect of constructed strain

After the evaluation of luminous intensity, the strain with the combination 7 (corresponding to fig. 2) with the highest luminous intensity is finally determined, the strain is inoculated into the THY liquid culture medium, and 1 × 10 resuspended in 1mL of the THY culture medium is used when the strain is cultured to the late logarithmic phase⁸CFU of bacteria were intraperitoneally infected with ball/c female mice at 6 weeks after hair removal treatment and observed using IVIS Spectrum in vivo imager.

Third, experimental results

1. Construction of knock-in fragments of lux reporter system containing different promoters

The total length of the knock-in fragments in different combinations is about 9300bp, and the final amplification result is shown in FIG. 3. Each combination has a hybrid band at 3000bp, which cannot be eliminated after several PCR condition optimizations. However, if the length of the lux luminescence system is as long as 5600bp, the hybridization band is recombined with the genome, and no luminescence is generated. Luminescence can then be used to further screen for recombinant correct strains.

2. Evaluation of the luminescence intensity under different promoters

(1) The plates in the different combinations were imaged on a bio-rad chemiDocXRS + high sensitivity chemiluminescence imager, and the results are shown in FIG. 4, where it can be seen that the strains in the other combinations, except combination 1, emit light.

(2) In order to more accurately compare the luminous efficiency of the strains under different combinations, the invention cultures each strain to the late logarithmic phase, uses PBS to adjust the bacterial quantity to be the same, and usesThe 96Microplate Luminometer detects the luminous intensity, and the result is shown in fig. 5, and it can be seen that compared with a single promoter, the luminous intensity can be obviously enhanced by separately transcribing the two parts; under the condition that the luxAB promoter is the same, the luminous efficiency of the strain with the CP25G promoter is obviously higher than that of the strain with the CP25 promoter; the different promoters of luxAB have little influence on the luminous efficiency of the strain, and the luminous efficiency of the strain with 147AGGAG promoter is slightly higher.

3. Mouse live imaging

The luminescent bacteria constructed according to the combination 7 are used for infecting mice, the mice are subjected to in vivo imaging after infection, and the result is shown in fig. 6, and the luminescent bacteria can be detected in the mice (the mice infected by wild strains serving as the left-most side as a control have no luminescent signals), so that the luminescent bacteria has potential application prospect in the research of pathogenic mechanisms of streptococcus suis.

Combining the above results, it can be seen that: the invention establishes and optimizes a construction method of streptococcus suis luminescent bacteria based on a lux report system, can observe bacteria in a mouse body in real time, and has a certain application prospect in the research of infection ways and pathogenic mechanisms of streptococcus suis.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> military medical research institute of military science institute of people's liberation force of China

<120> streptococcus suis luminescent bacterium based on lux report system and construction method thereof

<130> GNCLN211422

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 9379

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<213> Artificial sequence

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tcttaaagaa aaacaacgtt cgctattata ttaagtcgag gaggagaaag aaatgaaatt 2460

tggattgttc ttccttaact tcatcaattc aacaactgtt caagaacaaa gtatagttcg 2520

catgcaggaa ataacggagt atgttgataa gttgaatttt gaacagattt tagtgtatga 2580

aaatcatttt tcagataatg gtgttgtcgg cgctcctctg actgtttctg gttttctgct 2640

cggtttaaca gagaaaatta aaattggttc attaaatcac atcattacaa ctcatcatcc 2700

tgtcgccata gcggaggaag cttgcttatt ggatcagtta agtgaaggga gatttatttt 2760

agggtttagt gattgcgaaa aaaaagatga aatgcatttt tttaatcgcc cggttgaata 2820

tcaacagcaa ctatttgaag agtgttatga aatcattaac gatgctttaa caacaggcta 2880

ttgtaatcca gataacgatt tttatagctt ccctaaaata tctgtaaatc cccatgctta 2940

tacgccaggc ggacctcgga aatatgtaac agcaaccagt catcatattg ttgagtgggc 3000

ggccaaaaaa ggtattcctc tcatctttaa gtgggatgat tctaatgatg ttagatatga 3060

atatgctgaa agatataaag ccgttgcgga taaatatgac gttgacctat cagagataga 3120

ccatcagtta atgatattag ttaactataa cgaagatagt aataaagcta aacaagagac 3180

gcgtgcattt attagtgatt atgttcttga aatgcaccct aatgaaaatt tcgaaaataa 3240

acttgaagaa ataattgcag aaaacgctgt cggaaattat acggagtgta taactgcggc 3300

taagttggca attgaaaagt gtggtgcgaa aagtgtattg ctgtcctttg aaccaatgaa 3360

tgatttgatg agccaaaaaa atgtaatcaa tattgttgat gataatatta agaagtacca 3420

catggaatat acctaaatcg attcgagatc tgcaggatcc cattatgctt tggcagttta 3480

ttcttgacat gtagtgaggg ggctggtata atcacatagt actgttcggg atccttaaga 3540

atgggtctag aattaaagag gaggaaatta agcatgacta aaaaaatttc attcattatt 3600

aacggccagg ttgaaatctt tcccgaaagt gatgatttag tgcaatccat taattttggt 3660

gataatagtg tttacctgcc aatattgaat gactctcatg taaaaaacat tattgattgt 3720

aatggaaata acgaattacg gttgcataac attgtcaatt ttctctatac ggtagggcaa 3780

agatggaaaa atgaagaata ctcaagacgc aggacataca ttcgtgactt aaaaaaatat 3840

atgggatatt cagaagaaat ggctaagcta gaggccaatt ggatatctat gattttatgt 3900

tctaaaggcg gcctttatga tgttgtagaa aatgaacttg gttctcgcca tatcatggat 3960

gaatggctac ctcaggatga aagttatgtt cgggcttttc cgaaaggtaa atctgtacat 4020

ctgttggcag gtaatgttcc attatctggg atcatgtcta tattacgcgc aattttaact 4080

aagaatcagt gtattataaa aacatcgtca accgatcctt ttaccgctaa tgcattagcg 4140

ttaagtttta ttgatgtaga ccctaatcat ccgataacgc gctctttatc tgttatatat 4200

tggccccacc aaggtgatac atcactcgca aaagaaatta tgcgacatgc ggatgttatt 4260

gtcgcttggg gagggccaga tgcgattaat tgggcggtag agcatgcgcc atcttatgct 4320

gatgtgatta aatttggttc taaaaagagt ctttgcatta tcgataatcc tgttgatttg 4380

acgtccgcag cgacaggtgc ggctcatgat gtttgttttt acgatcagcg agcttgtttt 4440

tctgcccaaa acatatatta catgggaaat cattatgagg aatttaagtt agcgttgata 4500

gaaaaactta atctatatgc gcatatatta ccgaatgcca aaaaagattt tgatgaaaag 4560

gcggcctatt ctttagttca aaaagaaagc ttgtttgctg gattaaaagt agaggtggat 4620

attcatcaac gttggatgat tattgagtca aatgcaggtg tggaatttaa tcaaccactt 4680

ggcagatgtg tgtaccttca tcacgtcgat aatattgagc aaatattgcc ttatgttcaa 4740

aaaaataaga cgcaaaccat atctattttt ccttgggagt catcatttaa atatcgagat 4800

gcgttagcat taaaaggtgc ggaaaggatt gtagaagcag gaatgaataa catatttcga 4860

gttggtggat ctcatgacgg aatgagaccg ttgcaacgat tagtgacata tatttctcat 4920

gaaaggccat ctaactatac ggctaaggat gttgcggttg aaatagaaca gactcgattc 4980

ctggaagaag ataagttcct tgtatttgtc ccataagtcg aggaggagta aaagtatgga 5040

aaatgaatca aaatataaaa ccatcgacca cgttatttgt gttgaaggaa ataaaaaaat 5100

tcatgtttgg gaaacgctgc cagaagaaaa cagcccaaag agaaagaatg ccattattat 5160

tgcgtctggt tttgcccgca ggatggatca ttttgctggt ctggcggaat atttatcgcg 5220

gaatggattt catgtgatcc gctatgattc gcttcaccac gttggattga gttcagggac 5280

aattgatgaa tttacaatgt ctataggaaa gcagagcttg ttagcagtgg ttgattggtt 5340

aactacacga aaaataaata acttcggtat gttggcttca agcttatctg cgcggatagc 5400

ttatgcaagc ctatctgaaa tcaatgcttc gtttttaatc accgcagtcg gtgttgttaa 5460

cttaagatat tctcttgaaa gagctttagg gtttgattat ctcagtctac ccattaatga 5520

attgccggat aatctagatt ttgaaggcca taaattgggt gctgaagtct ttgcgagaga 5580

ttgtcttgat tttggttggg aagatttagc ttctacaatt aataacatga tgtatcttga 5640

tataccgttt attgctttta ctgcaaataa cgataattgg gtcaagcaag atgaagttat 5700

cacattgtta tcaaatattc gtagtaatcg atgcaagata tattctttgt taggaagttc 5760

gcatgacttg agtgaaaatt tagtggtcct gcgcaatttt tatcaatcgg ttacgaaagc 5820

cgctatcgcg atggataatg atcatctgga tattgatgtt gatattactg aaccgtcatt 5880

tgaacattta actattgcga cagtcaatga acgccgaatg agaattgaga ttgaaaatca 5940

agcaatttct ctgtcttaag tcgaggagga aaacaggtat gacttcatat gttgataaac 6000

aagaaattac agcaagctca gaaattgatg atttgatttt ttcgagcgat ccattagtgt 6060

ggtcttacga cgagcaggaa aaaatcagaa agaaacttgt gcttgatgca tttcgtaatc 6120

attataaaca ttgtcgagaa tatcgtcact actgtcaggc acacaaagta gatgacaata 6180

ttacggaaat tgatgacata cctgtattcc caacatcggt ttttaagttt actcgcttat 6240

taacttctca ggaaaacgag attgaaagtt ggtttaccag tagcggcacg aatggtttaa 6300

aaagtcaggt ggcgcgtgac agattaagta ttgagagact cttaggctct gtgagttatg 6360

gcatgaaata tgttggtagt tggtttgatc atcaaataga attagtcaat ttgggaccag 6420

atagatttaa tgctcataat atttggttta aatatgttat gagtttggtg gaattgttat 6480

atcctacgac atttaccgta acagaagaac gaatagattt tgttaaaaca ttgaatagtc 6540

ttgaacgaat aaaaaatcaa gggaaagatc tttgtcttat tggttcgcca tactttattt 6600

atttactctg ccattatatg aaagataaaa aaatctcatt ttctggagat aaaagccttt 6660

atatcataac cggaggcggc tggaaaagtt acgaaaaaga atctctgaaa cgtgatgatt 6720

tcaatcatct tttatttgat actttcaatc tcagtgatat tagtcagatc cgagatatat 6780

ttaatcaagt tgaactcaac acttgtttct ttgaggatga aatgcagcgt aaacatgttc 6840

cgccgtgggt atatgcgcga gcgcttgatc ctgaaacgtt gaaacctgta cctgatggaa 6900

cgccggggtt gatgagttat atggatgcgt cagcaaccag ttatccagca tttattgtta 6960

ccgatgatgt cgggataatt agcagagaat atggtaagta tcccggcgtg ctcgttgaaa 7020

ttttacgtcg cgtcaatacg aggacgcaga aagggtgtgc tttaagctta accgaagcgt 7080

ttgatagtta taattcgatg ggttccgagg ctcaacgtca ataaagcaat tggaataaag 7140

aagcgaaaaa ggagaagtcg gttcagaaaa agaaggatat ggatctggag ctgtaatata 7200

aaaaccttct tcaactaacg gggcaggtta gtgacattag aaaaccgact gtaaaaagta 7260

cagtcggcat tatctcatat tataaaagcc agtcattagg cctatctgac aattcctgaa 7320

tagagttcat aaacaatcct gcatgataac catcacaaac agaatgatgt acctgtaaag 7380

atagcggtaa atatattgaa ttacctttat taatgaattt tcctgctgta ataatgggta 7440

gaaggtaatt actattatta ttgatattta agttaaaccc agtaaatgaa gtccatggaa 7500

taatagaaag agaaaaagca ttttcaggta taggtgtttt gggaaacaat ttccccgaac 7560

cattatattt ctctacatca gaaaggtata aatcataaaa ctctttgaag tcattcttta 7620

caggagtcca aataccagag aatgttttag atacaccatc aaaaattgta taaagtggct 7680

ctaacttatc ccaataacct aactctccgt cgctattgta accagttcta aaagctgtat 7740

ttgagtttat cacccttgtc actaagaaaa taaatgcagg gtaaaattta tatccttctt 7800

gttttatgtt tcggtataaa acactaatat caatttctgt ggttatacta aaagtcgttt 7860

gttggttcaa ataatgatta aatatctctt ttctcttcca attgtctaaa tcaattttat 7920

taaagttcat ttgatatgcc tcctaaattt ttatctaaag tgaatttagg aggcttactt 7980

gtctgctttc ttcattagaa tcaatccttt tttaaaagtc aatattactg taacataaat 8040

atatatttta aaaatatccc actttatcca attttcgttt gttgaaccat tatatcacat 8100

tatccattaa aaatcaaaca aattttcatc aagctctagt tcggtggaaa tatgaacttg 8160

ttgacgaact ctctatgatt atttgagttc tttcccactc ccaatcctaa atatttttca 8220

taatctcctg aaaaacctga ccatgtggtt gggatttttg aatgcaaaaa cactcttgga 8280

atttccaaga gtgttttacg taacggtgtt aaacagtttg taatttttat ttcatcgtcg 8340

ggagtgagaa ggtctggtag accttttcag ccaatcgcca agaaactgga gagcgatttg 8400

aacatcctag tgaaaacaaa cctagcaagc tactcttgct tacttcatag tagggaatag 8460

caacacatcc cgaatagtag ttgtatctgt caagagcatg cagaggcggt cgataccgat 8520

tccaagtcca cctgttggtg gcataccgta ttcgagggct tcgatgtagt cgtagtcaac 8580

gcctgtcgct tcgtcgtcac cgagttcttt agccttggct tgggcttcaa agcgttccaa 8640

ttggtcgatt gggtcgttca attctgtaaa ggcatttccg tattccttgg tcatgatgaa 8700

gagctcgaag cgatctgtga agcgtgggtc ttcatcgttt ttcttagcca gtggagatac 8760

ggctactggg tgaccataga caaaggttgg ttggataaga gttgcttcaa cgtattcttc 8820

aaagaagctg ttgatgattt gaccaacttc tgtatggtgt ttttccacag ggaccttgtg 8880

ctcagctgca agggcttttg cttcctcgaa gctcatctct tgccagaagt ccacgccagt 8940

ttgttcctta atagcgtcaa ccatgtggat acgcttgaat ggctcgtgga tagcaatttc 9000

agtgccctga taagtcaccg gaccatcacc gacaacagcc ttggcagtgt gctggataat 9060

accttcggtc aagtccatga tgtcttggta gtctgcataa gcttggtaaa cctcaatgga 9120

agtgaactca gggttgtggg tcgcatccat accttcgtta cggaagatgc ggccgatttc 9180

ataaacgcgt tccataccgc cgacaatcag gcgtttgagg tggagttctg tcgcgatacg 9240

aaggaccatg tcaatgtttt gggcattgtg gtgggtgatg aatggacgag ctgcagcacc 9300

accagcttcg ttgtgaagaa ctggtgtttc cacttccaag aaaccaagtc cgtcaaggta 9360

acggcggatt tctgagata 9379

<210> 2

<211> 9310

<212> DNA

<213> Artificial sequence

<400> 2