CN115044599B - Streptococcus agalactiae strain delta ess C and construction method and application thereof - Google Patents

Abstract

The invention relates to a streptococcus agalactiae strain delta ess with knock-out ess gene, a construction method and application thereof, wherein the ess gene sequence is shown as SEQ ID NO.1, or is a sequence which is completely complementary and paired with SEQ ID NO.1, or is a nucleotide sequence which is shown as SEQ ID NO.1, is substituted, deleted and/or added with one or more nucleotides, can code the same functional protein, or is a nucleotide sequence which codes an amino acid sequence shown as SEQ ID NO. 2. It has been found that the deletion of the ess c gene results in secretion of the immunogenic extracellular protein ESAT6 to produce a secretory disorder, thereby reducing the toxicity of the streptococcus agalactiae strain. Meanwhile, the delta ess C of the streptococcus agalactiae gene knockout strain constructed by the invention is basically consistent with the growth rate of a wild strain, has obvious attenuation characteristics, provides a theoretical basis for researching a type VII secretion system of streptococcus agalactiae, and provides a key clue for searching an action target point of a streptococcus agalactiae gene protein medicine.

Description

Streptococcus agalactiae strain delta ess C and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a streptococcus agalactiae strain delta ess C, a construction method and application thereof.

Background

Streptococcus agalactiae (Streptococcus agalactiae) belongs to Group B Streptococcus (GBS), has a wide host range, can infect hosts such as human beings, mammals, reptiles, amphibians, fishes and the like, and causes great harm to global public health and human health. The disease incidence and the death rate of the tilapia are the first of the tilapia infectious diseases, and the epidemic situation is characterized by three high (high disease incidence, high death rate and high drug resistance rate) so as to form national control pressure.

In recent years, it has been found that a novel secretory system, type VII secretory system (Type 7 secretion system,T7SS), exists in gram-positive bacteria such as Actinobacillus and Thick-walled bacteria. The VII type secretion system is composed of multiple components, and secretes two immunogenic extracellular proteins, ESAT6 and CFP-10, which are closely related to strain virulence. Streptococcus agalactiae studies have shown that ESAT6 protein functions as a homodimer. The individual genes of the VII type secretion system respectively code proteins with specific functions, determine ESAT6/CFP10 and virulence protein secretion and transportation thereof, and have important connection with bacterial virulence and pathogenesis, so that the research on the VII type secretion system is helpful for understanding the virulence protein secretion mechanism of streptococcus agalactiae, is helpful for elucidating the molecular basis of survival, proliferation and diffusion of streptococcus agalactiae, and provides a key clue for searching the action target point of the streptococcus agalactiae gene protein medicine, thereby providing possibility for fundamentally controlling the streptococcus agalactiae diseases.

Disclosure of Invention

Based on the above, one of the purposes of the present invention is to provide an application of the ess gene and the protein in the toxicity control of streptococcus agalactiae, and researches show that the deficiency of the ess gene in the streptococcus agalactiae causes secretion of immunogenic extracellular protein ESAT6 to produce secretion disorder, thereby reducing the toxicity of the strain.

The technical scheme for achieving the purpose is as follows:

the application of the ess C gene in toxicity regulation of streptococcus agalactiae is characterized in that the sequence of the ess C gene is shown as SEQ ID NO.1, or is a sequence which is completely complementary and paired with the SEQ ID NO.1, or is a nucleotide sequence which is shown as SEQ ID NO.1, is substituted, deleted and/or added with one or more nucleotides, can code the same functional protein, or is a nucleotide sequence which is shown as SEQ ID NO. 2.

The application of the ess C protein in toxicity regulation of streptococcus agalactiae is that the amino acid sequence of the ess C protein is shown as SEQ ID NO.2, or the amino acid sequence shown as SEQ ID NO.2 is substituted, deleted and/or added with one or more amino acids, but the protein activity is the same.

In some of these embodiments, the toxicity is manifested by secretion of an immunogenic extracellular protein, preferably a secreted ESAT6 protein.

In some of these embodiments, the toxicity modulation is positive, preferably the expression of ESAT6 protein of Streptococcus agalactiae is down-regulated by the gene knockout of the ess C.

The invention also aims at providing an ess gene recombination knockout vector, which is characterized in that a fusion fragment of a chloramphenicol resistance gene CAT for replacing the ess gene is inserted into the recombination vector, and the fusion fragment consists of an upstream fragment, a downstream fragment and a CAT gene fragment of the ess gene.

In some of these embodiments, the upstream fragment of the ess gene is its upstream 632bp homology arm and the downstream fragment of the ess gene is its downstream 571bp homology arm.

The invention also aims at providing a construction method of the ess C gene recombination knockout vector.

The technical scheme for achieving the purpose is as follows:

(1) PCR amplifying the upstream fragment of the ess gene, namely the ess cup, by taking streptococcus agalactiae genome DNA as a template, taking SEQ ID NO.3-SEQ ID NO.4 as a primer pair, and taking SEQ ID NO.5-SEQ ID NO.6 as a primer pair; PCR amplifying a chloramphenicol resistance gene fragment CAT by using a plasmid pSET5S as a template and SEQ ID NO.7-SEQ ID NO.8 as a primer pair;

(2) Purifying the three fragments obtained in the step (1) and then carrying out fusion PCR amplification;

(3) Performing PCR amplification verification by taking the fusion PCR product obtained in the step (2) as a template and taking SEQ ID NO.3-SEQ ID NO.6 as a primer pair;

(4) And (3) connecting the fusion enzyme obtained in the step (3) after verification to a linearization vector pSET4s, and carrying out identification and screening after conversion of a connecting product.

The invention also aims to provide an application of the ess C gene recombination knockout vector in regulating and controlling the toxicity of streptococcus agalactiae.

In some of these embodiments, the toxicity modulation is the downregulation of ESAT6 protein expression in Streptococcus agalactiae by the knock-out of the ess C gene.

It is also an object of the present invention to provide an ess gene knock-out streptococcus agalactiae strain Δessc.

The specific technical scheme for achieving the purposes is as follows:

a delta ess streptococcus agalactiae strain with a knocked-out ess gene is delta ess streptococcus agalactiae, wherein delta ess is deletion of the ess gene in streptococcus agalactiae, the sequence of the deleted ess gene is shown as SEQ ID NO.1, or the sequence of the ess gene is completely complementary and paired with SEQ ID NO.1, or the nucleotide sequence shown as SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides, and the nucleotide sequence can code the same functional protein, or the nucleotide sequence with the coded amino acid sequence shown as SEQ ID NO. 2.

Compared with the prior art, the invention has the following beneficial effects:

the inventor of the invention discovers an important membrane protein ess C affecting a VII type secretion system in streptococcus agalactiae, and discovers that the deficiency of the ess C gene causes secretion of immunogenic extracellular protein ESAT6 to generate secretion disorder, thereby reducing the toxicity of the streptococcus agalactiae strain. Meanwhile, the stress C gene knockout streptococcus agalactiae strain delta stress C constructed by the invention has basically consistent growth rate with the wild strain of streptococcus agalactiae, has obvious attenuation characteristics, provides a theoretical basis for researching a type VII secretion system of streptococcus agalactiae, and provides a key clue for searching an action target point of a streptococcus agalactiae gene protein medicine.

Drawings

FIG. 1 is a graph showing the results of the ess C homology arm fusion PCR in example 1.

FIG. 2 is a graph showing PCR identification of the delta essC DNA level of the knockout strain in example 1.

FIG. 3 is a graph showing PCR identification of the delta essC RNA level of the knockout strain in example 1.

FIG. 4 is a graph showing the results of measurement of the delta essC growth curve of the knockout strain in example 2.

FIG. 5 is a graph showing the toxicity test results of the knockout strain ΔessC and the wild strain in example 2.

FIG. 6 is a graph showing the results of qPCR detection of ESAT6 genes of the knockout strain ΔessC and the wild strain in example 3, wherein a is a graph showing the analysis result of amplification efficiency; b is a dissolution curve analysis result graph; c is a graph of agarose gel detection results of amplified products.

FIG. 7 shows the effect of the knockout strain ΔessC and the wild strain on the expression level of the substrate protein ESAT6 in example 3.

Detailed Description

In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The inventor amplifies to obtain the full-length gene of streptococcus agalactiae ess C, and the nucleotide sequence of the ess C gene is shown as SEQ ID NO.1, and the amino acid sequence of the ess C protein is shown as SEQ ID NO. 2.

EXAMPLE 1 construction of the delta ess C knockout Strain of Streptococcus agalactiae

1.1 Experimental plasmids

Wild streptococcus agalactiae virulent strain ZX1 (Wild type, serotype Ia), temperature-sensitive suicide plasmids pSET4S and pSET5S were given a doctor benefit from japan animal health institute Daisuke Takamatsu.

1.2 primer design and Synthesis

Primers were designed using oligo7.0 and Primer 6.0 software, and the sequences of the primers used in the experiments are shown in Table 1, and were synthesized by Beijing qing Biotech Co.

Table 1 primers for test

Note that: restriction enzyme sites are underlined.

1.3 construction of recombinant vector for Gene knockout

(1) Amplification of 632bp homologous arm upstream of the ess C gene

The streptococcus agalactiae genome DNA is used as a template, the stress cup-FSali (SEQ ID NO. 3)/stress cup-R (SEQ ID NO. 4) is used for amplifying a 632bp homologous arm at the upstream of a stress C gene, and the reaction procedure is as follows: pre-denaturation at 98 ℃ for 5min;98℃10s,58℃10s,72℃15s,35 cycles; extending at 72℃for 5min. The reaction system is as follows: 25. Mu.L of 2 XPFU enzyme, 1. Mu.L of forward/reverse primer each, 1. Mu.L of genomic DNA, and 22. Mu.L of ddH 2O.

(2) Homologous arm amplification of 571bp downstream of the ess C gene

The primer pair of the ess Cdown-F (SEQ ID NO. 5)/ess Cdown-REcoRI (SEQ ID NO. 6) is used for amplifying 571bp homologous arms at the downstream of the ess C gene by taking the streptococcus agalactiae genome DNA sequence as a template. The reaction procedure is: pre-denaturation at 98 ℃ for 5min;98℃10s,58℃10s,72℃15s,35 cycles; extending at 72℃for 5min. The reaction system is as follows: 25. Mu.L of 2 XPFU enzyme, 1. Mu.L of forward/reverse primer each, 1. Mu.L of genomic DNA, and 22. Mu.L of ddH 2O.

(3) cat gene amplification

The target fragment of 1056bp of chloramphenicol expression cassette is amplified by using pSET5S plasmid as a template and Cat (ess C) -F (SEQ ID NO. 7)/Cat-R (SEQ ID NO. 8) primer pair. The reaction procedure is: pre-denaturing at 98deg.C for 5min;98 ℃ for 10s,56 ℃ for 10s,72 ℃ for 30s,35 cycles; extending at 72℃for 5min. The reaction system is as follows: 25. Mu.L of 2 XPFU enzyme, 1. Mu.L of forward/reverse primer each, 1. Mu.L of genomic DNA, and 22. Mu.L of ddH 2O.

(4) Fusion PCR amplification

Subjecting the upstream homology arm of the ess, the cat gene and the downstream homology arm of the ess to agarose gel electrophoresis, cutting and purifying, mixing equal volumes (8 mu L), taking the ess cup-FSali (SEQ ID NO. 3)/the ess Cdown-REcoRI (SEQ ID NO. 6) as forward and reverse primers, carrying out fusion PCR amplification, and carrying out a reaction program by adopting pre-denaturation at 98 ℃ for 5min;98℃15s,60℃15s,72℃90s,35 cycles; the PCR products were subjected to agarose gel electrophoresis analysis after extension at 72℃for 5min. As shown in FIG. 1, the detection result shows that the homologous arm sequence of 632bp at the upstream of the ess C gene, the homologous arm sequence of 571bp at the downstream of the ess C gene and the fragment of 1056bp of the chloramphenicol expression cassette are obtained, and the fusion fragment of 2259bp is obtained through fusion PCR.

And then cutting and purifying the fused target fragment, cutting the fused fragment by SalI and EcoRI, respectively carrying out T4 DNase connection with a linearized vector pSET4s after double cutting, converting the connection product into E.coli DH5 alpha competent cells, coating the culture on an LB solid plate containing chloramphenicol (25 mug/mL) and spectinomycin (50 mug/mL), screening positive clones and carrying out colony PCR identification. The identified positive plasmid was sent to Beijing qingke biosciences, inc. for sequencing. The gene knockout vector was obtained and designated pSET4S- ΔessC plasmid.

1.4 construction of Gene deletion mutant

Referring to the method of Takamatsu, wild-type electrotransformation competent cells were prepared. Adding 5 mu L of pSET 4S-delta-ess C plasmid into 100 mu L of ZX1 electrotransformation competent cells, slightly mixing, adding the mixture into a 0.2cm electrorotating cup, and carrying out ice bath for 10min, and carrying out electric shock under the parameters of 2.25kV/cm,200 omega and 25 mu F; after electric shock, 1mL of SOC culture medium is rapidly added, and shaking is carried out for 3-4 hours at 30 ℃ and 160 rpm; mu.L of the recovered bacterial liquid was spread on THB Spc+Cm+ plates, and cultured in a 30℃incubator for 48 hours. Microcolonies are picked up to Cm+THB culture medium, cultured for 6-7 h at 30 ℃ and cultured for 24h at 37 ℃. The colonies were transferred to cm+ resistant and spc+ resistant THB medium, respectively, and continued to culture. Wherein the suspected positive colonies which can grow in Cm+ resistant medium but cannot grow in Spc+ resistant medium continue to be stably passaged, PCR identified and sequenced, and the strains are kept in a refrigerator at-80 ℃.

As shown in FIG. 2, the ZX1 strain is amplified to a target fragment of 1263bp by using an ess-C verification primer, and the suspected ess-knock-out strain is amplified to a target fragment of 2077bp, so that the target fragment is obtained to be subjected to sequencing verification, the cat gene sequence is inserted into the ess gene, the success of the knock-out is proved, and the knock-out strain is named as streptococcus agalactiae delta-ess.

On the basis, the strain with resistance to chloramphenicol screened from the 9 th generation strain is extracted, the RNA of the strain is reversely transcribed into cDNA, and the RT-PCR technology is used for verifying the gene deletion mutant strain delta ess C at the RNA level. The detection result is shown in figure 3, mRNA level verification is carried out on the delta ess knockout strain by using the primer pair of the ess-F (SEQ ID NO. 11)/the ess-R (SEQ ID NO. 12), and the result is not amplified to a target strip, so that the expression deletion of the ess protein is proved, namely the construction of the delta ess of the deletion strain is successful.

EXAMPLE 2 analysis of the biological Properties of the Gene deletion mutant Deltaess C

2.1 growth curve analysis

Streaking and inoculating the frozen streptococcus agalactiae ZX1 strain and the DeltaessC strain at the temperature of-80 ℃ to THB solid culture medium, and culturing for 24 hours at the temperature of 30 ℃. Single colonies were picked to 5mL THB broth and shaken at 30℃for 24h. The following day was inoculated at a 1:100 ratio to 100mL THB broth and shaken at 30 ℃. Sampling 2 every 2 hoursmL, OD determination ₆₀₀ Read, 3 replicates of each of the four strains, OD ₆₀₀ The values were statistically analyzed using SPSS software.

By measuring the OD of bacterial liquid at different times ₆₀₀ The difference of growth rates of the Deltaess C strain and the ZX1 strain is compared, the result is shown in figure 4, the growth rate of the Deltaess C knockout strain in the early stage is reduced compared with that of the ZX1 strain, but the growth rate of the Deltaess C knockout strain in the later stage is basically consistent, and the OD of the Deltaess C strain is compared with that of the ZX1 strain through statistical detection ₆₀₀ There was no significant difference in the read values.

2.2 toxicity test

Tilapia mossambica for test is purchased from a tilapia fingerland in Guangdong province, the weight is about 10g, the tilapia mossambica is temporarily cultured in an indoor glass jar for 2 weeks before the test, 5 tilapia mossambica is randomly selected and inspected, and the tilapia mossambica is used for pathogen separation test, so that the tilapia for test does not carry streptococcus agalactiae pathogen. The animals were randomly divided into 7 groups (6 challenge groups and 1 control group, 20 each). Dissolved oxygen (> 5.0 mg/L) was maintained. Changing water every day, and maintaining water quality. 2 satiety per day before the test.

Taking frozen streptococcus agalactiae ZX1 strain and DeltaessC strain at-80 ℃, streaking and inoculating THB solid culture medium, culturing for 24 hours at 30 ℃, picking up single colony, inoculating THB liquid culture medium, culturing for 24 hours at 30 ℃, moderately diluting, taking 100 mu L of the mixture, coating the mixture on a THB flat plate, culturing for 24 hours at 30 ℃, and counting. Are all diluted to 5 multiplied by 10 ⁸ cfu/mL、5×10 ⁷ cfu/mL、5×10 ⁶ cfu/mL three doses, artificially infecting tilapia, injecting healthy tilapia into 0.1 mL/tail abdominal cavity, and injecting equivalent physiological saline into a control group. The test fish is placed in an aquarium with the length of 40cm multiplied by 30cm multiplied by 40cm, the aquarium is continuously inflated, the water temperature is maintained at 27-30 ℃, the death condition of the tilapia is observed every day and recorded, the death tilapia is decomposed, and the tilapia is streaked again to be inoculated with pathogen for separation. Survival curves were plotted using GraphPad Prism software and significance analysis was performed.

As shown in FIG. 5, the detection result was 5X 10 ⁸ The cfu/mL dose is 0.1 mL/tail injection for toxin counteracting, the cumulative death rate of the delta ess knockout strain is 70 percent, and the cumulative death rate of the ZX1 strain is 100 percent; at 5X 10 ⁷ The cfu/mL dose is 0.1 mL/tail injection for toxin counteracting, the cumulative death rate of the delta ess knockout strain is 60 percent, and the cumulative death rate of the ZX1 strain is 100 percent; at 5X 10 ⁶ cfu/mL dose 0.1 mL/tail injection for detoxification, 30% of accumulated death rate of delta ess C knockout strain, 100% of accumulated death rate of ZX1 strain, 5X 10 by survival rate curve analysis ⁶ When cfu/mL dose attacks toxin, the delta ess knockout strain ZX1 has obvious difference (P<0.01). The dead tilapia is subjected to cross-sectional decomposition, typical symptoms of streptococcus agalactiae infection such as liver congestion, cerebral hemorrhage, ascites, and white and prominent eyeballs of the fish body are visible, blood agar plates are inoculated by streaking and inoculation, gray microcolonies with beta hemolysis and needle tip size are inoculated, and the streptococcus agalactiae infection is identified by gram staining and PCR (polymerase chain reaction) by using the primer pair of the ess C-F (SEQ ID NO. 11)/the ess C-R (SEQ ID NO. 12), so that regression tests are proved to be successful. PBS groups did not find death during the challenge trial period.

Example 3 Effect of knockout strain ΔessC on expression level of substrate protein ESAT6

Establishment of 3.1ESAT6 gene qPCR detection method

Taking the constructed ESAT6-pMD18T plasmid as a template, carrying out 10-time gradient dilution, and respectively taking stock solution and 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Preparing a qPCR reaction system by taking 7 doses as templates, and performing qPCR amplification by a three-step method to obtain an ESAT6 gene qPCR amplification curve, wherein the amplification efficiency analysis result is shown in FIG. 6a, and the amplification efficiency is 95.1% and meets the requirements; the analysis of the dissolution curve is shown in FIG. 6b, and the dissolution curve shows a single peak at about 75 ℃ to prove that the amplified product is single; the agarose gel detection result of the amplified product is shown in FIG. 6c, and the amplified product is subjected to agarose gel electrophoresis, so that a specific band of 88bp is obtained. The ESAT6 gene qPCR detection method is successfully established, namely the specific steps are as follows: ESAT6-F (SEQ ID NO. 13) and ESAT6-R (SEQ ID NO. 14) are used as primers, and a PCR reaction system is as follows: 2X qPCR SYBR Green Master Mix. Mu.L, 1. Mu.L of ESAT6-F primer, 1. Mu.L of ESAT6-R primer, 1. Mu.L of plasmid DNA, and 7. Mu.L of ddH 2O; the PCR reaction procedure was: 95 ℃ for 5min;95 ℃ for 10s,60 ℃ for 30s,40 cycles; the dissolution profile was analyzed at 95℃for 15s,60℃for 60s, and 95℃for 15 s.

3.2 Effect of ESAT6 protein expression in ΔessC

The amplification primers 16S-rRNA-F (SEQ ID NO. 15) and 16S-rRNA-R (SEQ ID NO. 16) are designed by taking the streptococcus agalactiae 16S rRNA gene as an internal reference gene, and the expression quantity difference of the delta ess C knockout strain and the ZX1 strain ESAT6 is compared when the time is 4h, 8h, 12h and 24h. As shown in fig. 7, the expression level of ESAT6 was significantly reduced (P < 0.01) in the case of the Δessc knockout strain compared to the case of the ZX1 strain at 4h, 8h, and 12h, but the expression level of ESAT6 was not significantly different from that of the ZX1 strain at 24h. The result shows that ESAT6 expression is influenced by the deletion of the ess C gene in the early, logarithmic and stationary phases of streptococcus agalactiae expression, so that the ESAT6 expression level is obviously reduced, and the expression of the substrate protein ESAT6 is not influenced by the deletion of the ess C gene in the 24-hour decay phase. Therefore, analysis of the secretion influence of ESAT6 protein shows that the delta ess knock-out strain has a significantly lower expression than ZX1 strain in the analysis influence of ESAT6 protein, and the ess gene deletion causes secretion disorder of secreted protein ESAT 6.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

Sequence listing

<110> animal health institute of academy of agricultural sciences in Guangdong province

Guangdong Shun An Feng Tai aquatic technologies Co., ltd

<120> a streptococcus agalactiae strain delta ess C, its construction method and application

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4410

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aatttatttg aaggtgatat ttatttgaat aatcatcttc aaaaagtagt gcagcagtta 420

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cgcattgatt cctatcatga ctatttgtct gataagtcaa ttgaacttca gaagttagct 900

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gagcgttgga taagccatgc tccgtataaa agcttagctg ttccacttgg cttacgtggt 1920

caggatgata ttgtttatct aaacctacat gaaaaagctc atggtcctca tggattagtt 1980

gcaggaacaa cgggatcagg gaagtcagaa attatccagt cctatatcct ttctttagcg 2040

gttaatttcc atccacacga tgtagctttc cttctcatag actacaaagg tggggggatg 2100

gcaaatctct tcaaggacct gccacactta cttggcacca tcaccaactt agatggtgcc 2160

caatcgatgc gtgctttggt ttctattaat gctgaattga aacgtcgtca acgtttattt 2220

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caacctgagt ttatgaaaga gcttgtctca acagcgcgaa ttggtcgttc cctagggatt 2400

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gaccaaggga ttgaagacca taccatttat tccatcaacg accttggcca atacgaaatc 2700

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ttggatgcca tcgttgaaaa cattcaagct ttaaccaaag aaatgggcat ctcagactta 2820

cctcaaccgt ggctaccacc tctaagcaat cagattgcag taaccgatct ccgtaaggaa 2880

gaatcagtag acctctggag taaagctcca tcttacaaag ctgttctggg ctttatggat 2940

ataccaagcc aacaagcaca agaagtagct tggcacgatt ttgaagacga tgggcatttg 3000

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ctagcacgtc acaatagtcc agaatttcta aacctttatc tctttgattt tggtaccaat 3120

ggcctcttac cattaagacg tttgccacat gtagcagatt tctttaccat tgatgatgac 3180

gaaaaaatag ccaagtttat tgcccgtatc aaagttgaaa tgtctgatcg taagaaagct 3240

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caagtggccc tcccaacaga aggcactgaa acctttgaca tcatcagtaa catccaaacc 3660

gaatctgacg ccatgaacga caaatggact ggaccaagac caaaagcaat accaattgtt 3720

ccagaagaat taacctttga cgactttatg gctacagata gtgttcaagc agatctatca 3780

gctaatcgtt taccacttgg gcttgaaatg gttgacgttg aaagctttag cctaccatta 3840

acgaaattta agcacctctt attcctatca gattcagatg aaggtttaga aaatctaggc 3900

agtcatttac ttaaaacgtt gattaaagtg ccagaataca gcaccatgat cattgattcc 3960

ttaggcgaac atgaagccta ccaaggacaa gtgagaacct atgtcggtgc tgatatgatt 4020

agtgatatgg cagaacaact caattatgag ctaggaaaac gtcaagaaca aaacgctttc 4080

gatagatggt ttatcttaat tccagatttt gaaagtttcg tcagcaaaac aaaccttagc 4140

ctagagcaaa tccaaaatct cttagataat ggacccaaag tcggcttaca cctgatcatt 4200

ggctcagcct ttagctttgt tggttcaaaa cttgaccccg ttaacaaata cgtcaaaacc 4260

aatagccaat atgttatgct aggcatgcgc ctaatggacc aaacattcct agagaaagtg 4320

tataatagta aagaagcacg tctggaacgc gacgaagcct acattcacga tagaaaaaac 4380

tatcaaaaac taaaactcag catagactag 4410

<210> 2

<211> 1469

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Leu Val Ile Cys Tyr Tyr Gln Ser Leu Arg Tyr Glu Phe Asn Ile

1 5 10 15

Glu Glu Glu Lys Ser Phe Leu Ile Ser Ser Asn Gly Lys Ser Pro Ile

20 25 30

Pro Val Ser Glu Leu Glu Asn Asp Ile Thr Leu Lys Asn Met Gln Gly

35 40 45

Gln Leu Val Tyr Ile Ile Asp Gln Lys Glu Lys Glu Leu Thr Asn Gly

50 55 60

Val Glu Ile Ser Gly Ile Val Phe Tyr Leu Ala Asn Asn Gln Lys Glu

65 70 75 80

Ile Tyr Thr Pro Leu Asp Tyr Glu Asp Ile Leu Ile Gly Asp Lys Glu

85 90 95

Gly Tyr His Ile Arg Phe Lys Glu Gly Ala Pro Asn Leu Leu Leu Lys

100 105 110

Lys Ile Glu Ser Asn Trp Gln Leu Asn Leu Phe Glu Gly Asp Ile Tyr

115 120 125

Leu Asn Asn His Leu Gln Lys Val Val Gln Gln Leu Pro Leu Ser Leu

130 135 140

Gly Asp Glu Ile Ser Phe Gln Gly Ile Ile Ile Lys Leu Phe Pro Asp

145 150 155 160

Glu Ile Gln Ile Trp Gly Gly Thr Asp Tyr Glu Thr Ser Leu Thr Lys

165 170 175

Lys Val Met Ser Ala Tyr Gln Phe Tyr Ala Gly Tyr Pro Asp Phe His

180 185 190

Arg Ser Pro Arg Ile Ile Tyr Arg Ser Ser Glu Asp Lys Ile Thr Val

195 200 205

Asn Ala Pro Gly Asn Glu Pro Asn Lys Ser Lys Asp Glu Leu Leu Lys

210 215 220

Leu Ile Val Pro Pro Leu Val Met Ile Gly Val Ser Ile Leu Ile Ser

225 230 235 240

Ile Phe Arg Pro Arg Gly Ile Tyr Ile Ile Ala Thr Met Ser Met Ala

245 250 255

Leu Val Thr Met Ile Phe Ser Ile Thr Gly Tyr Phe Lys Asn Arg Lys

260 265 270

Gln Tyr Lys Gln Asp Leu Gln Glu Arg Ile Asp Ser Tyr His Asp Tyr

275 280 285

Leu Ser Asp Lys Ser Ile Glu Leu Gln Lys Leu Ala Lys Glu Gln Lys

290 295 300

Arg Gly Gln His Tyr His Tyr Pro Thr Ile Glu Gly Leu Gln Glu Met

305 310 315 320

Ala Asp Thr Tyr His His Arg Ile Tyr Glu Lys Thr Pro Leu His Phe

325 330 335

Asp Phe Leu Tyr Tyr Arg Leu Gly Leu Gly Glu Val Pro Thr Ser Tyr

340 345 350

Asn Ile His Tyr Ser Gln Pro Glu Arg Ser Gly Lys Lys Asp Pro Leu

355 360 365

Glu Asn Glu Gly Tyr Asn Leu Tyr Phe Asn Asn Arg Tyr Ile Lys Asn

370 375 380

Met Pro Ile Val Ala Asn Leu Ser His Gly Pro Val Gly Tyr Ile Gly

385 390 395 400

Pro Arg Gly Leu Val Leu Glu Gln Leu Gln Leu Met Val Asn Gln Leu

405 410 415

Ala Phe Phe His Ser Tyr His Asp Val Gln Phe Ile Thr Ile Val Pro

420 425 430

Glu Glu Glu Met Asp Lys Trp Ser Trp Met Arg Trp Leu Pro His Ala

435 440 445

Thr Leu Gln Asp Val Asn Val Arg Gly Phe Val Tyr Asn Gln Arg Ser

450 455 460

Arg Asp Gln Val Leu Asn Ser Leu Asn Gln Ile Leu Lys Leu Arg Arg

465 470 475 480

Thr Gln Arg Glu Asp Lys Ser Ala Lys Glu Gly Thr Leu Phe Ser Pro

485 490 495

His Tyr Val Val Ile Val Thr Asp Glu Lys Leu Ile Leu Asp His Val

500 505 510

Ile Met Glu Phe Phe Thr Glu Asp Pro Thr Glu Leu Gly Cys Ser Leu

515 520 525

Ile Phe Val Gln Asp Val Met Ser Ser Leu Ser Glu Asn Ile Lys Thr

530 535 540

Ile Ile Asn Ile Lys Asp Arg Asn Thr Gly Gln Leu Val Ile Glu Glu

545 550 555 560

Gly Glu Leu Lys Glu Thr Asp Phe Glu Leu Asp His Phe Leu Glu Asp

565 570 575

Tyr Asp Lys Glu Asn Ile Ser Arg Arg Leu Ala Pro Leu Asn His Leu

580 585 590

Gln Asn Leu Lys Ser Ser Ile Pro Glu Ala Val Thr Phe Met Glu Met

595 600 605

Tyr Gln Ala Glu Glu Phe Glu Asp Leu His Val Gln Glu Arg Trp Ile

610 615 620

Ser His Ala Pro Tyr Lys Ser Leu Ala Val Pro Leu Gly Leu Arg Gly

625 630 635 640

Gln Asp Asp Ile Val Tyr Leu Asn Leu His Glu Lys Ala His Gly Pro

645 650 655

His Gly Leu Val Ala Gly Thr Thr Gly Ser Gly Lys Ser Glu Ile Ile

660 665 670

Gln Ser Tyr Ile Leu Ser Leu Ala Val Asn Phe His Pro His Asp Val

675 680 685

Ala Phe Leu Leu Ile Asp Tyr Lys Gly Gly Gly Met Ala Asn Leu Phe

690 695 700

Lys Asp Leu Pro His Leu Leu Gly Thr Ile Thr Asn Leu Asp Gly Ala

705 710 715 720

Gln Ser Met Arg Ala Leu Val Ser Ile Asn Ala Glu Leu Lys Arg Arg

725 730 735

Gln Arg Leu Phe Ala Lys Ala Asp Val Asn His Ile Asn Gln Tyr Gln

740 745 750

Lys Lys Tyr Lys Leu Gly Glu Val Ser Glu Pro Met Pro His Leu Phe

755 760 765

Leu Ile Ser Asp Glu Phe Ala Glu Leu Lys Ser Asn Gln Pro Glu Phe

770 775 780

Met Lys Glu Leu Val Ser Thr Ala Arg Ile Gly Arg Ser Leu Gly Ile

785 790 795 800

His Leu Ile Leu Ala Thr Gln Lys Pro Ser Gly Val Val Asp Asp Gln

805 810 815

Ile Trp Ser Asn Ser Arg Phe Lys Leu Ala Leu Lys Val Ala Asp Arg

820 825 830

Gly Asp Ser Ile Glu Met Leu His Thr Pro Asp Ala Ala Glu Ile Thr

835 840 845

Gln Ala Gly Arg Ala Tyr Phe Gln Val Gly Asn Asn Glu Val Tyr Glu

850 855 860

Leu Phe Gln Ser Ala Trp Ser Gly Ala Asp Tyr Gln Pro Glu Lys Asp

865 870 875 880

Asp Gln Gly Ile Glu Asp His Thr Ile Tyr Ser Ile Asn Asp Leu Gly

885 890 895

Gln Tyr Glu Ile Leu Asn Asp Asp Leu Ser Gly Leu Asp Gln Ala Glu

900 905 910

Asn Ile Lys Glu Val Pro Thr Glu Leu Asp Ala Ile Val Glu Asn Ile

915 920 925

Gln Ala Leu Thr Lys Glu Met Gly Ile Ser Asp Leu Pro Gln Pro Trp

930 935 940

Leu Pro Pro Leu Ser Asn Gln Ile Ala Val Thr Asp Leu Arg Lys Glu

945 950 955 960

Glu Ser Val Asp Leu Trp Ser Lys Ala Pro Ser Tyr Lys Ala Val Leu

965 970 975

Gly Phe Met Asp Ile Pro Ser Gln Gln Ala Gln Glu Val Ala Trp His

980 985 990

Asp Phe Glu Asp Asp Gly His Leu Ser Ile Phe Ala Gly Pro Ser Met

995 1000 1005

Gly Lys Ser Thr Ala Leu Gln Thr Val Thr Met Asp Leu Ala Arg His

1010 1015 1020

Asn Ser Pro Glu Phe Leu Asn Leu Tyr Leu Phe Asp Phe Gly Thr Asn

1025 1030 1035 1040

Gly Leu Leu Pro Leu Arg Arg Leu Pro His Val Ala Asp Phe Phe Thr

1045 1050 1055

Ile Asp Asp Asp Glu Lys Ile Ala Lys Phe Ile Ala Arg Ile Lys Val

1060 1065 1070

Glu Met Ser Asp Arg Lys Lys Ala Leu Ser Arg Tyr Asn Val Ala Thr

1075 1080 1085

Ala Lys Leu Tyr Arg Gln Val Ser Gly Glu Thr Met Pro Gln Ile Leu

1090 1095 1100

Ile Val Ile Asp Ser Tyr Glu Gly Leu Arg Glu Ala Gln Thr Leu Thr

1105 1110 1115 1120

Asn Leu Glu Ala Cys Phe Gln Asn Ile Ser Arg Asp Gly Ser Ser Leu

1125 1130 1135

Gly Ile Ser Leu Val Ile Ser Ala Gly Arg Met Ala Ala Leu Arg Ser

1140 1145 1150

Ser Leu Met Ala Asn Leu Lys Glu Arg Ile Ala Leu Lys Leu Thr Asp

1155 1160 1165

Asp Ser Glu Ser Arg Thr Leu Val Gly Arg His Gln His Ile Met Glu

1170 1175 1180

Asp Ile Pro Gly Arg Gly Leu Ile Lys Arg Asp Asp Ile Glu Val Leu

1185 1190 1195 1200

Gln Val Ala Leu Pro Thr Glu Gly Thr Glu Thr Phe Asp Ile Ile Ser

1205 1210 1215

Asn Ile Gln Thr Glu Ser Asp Ala Met Asn Asp Lys Trp Thr Gly Pro

1220 1225 1230

Arg Pro Lys Ala Ile Pro Ile Val Pro Glu Glu Leu Thr Phe Asp Asp

1235 1240 1245

Phe Met Ala Thr Asp Ser Val Gln Ala Asp Leu Ser Ala Asn Arg Leu

1250 1255 1260

Pro Leu Gly Leu Glu Met Val Asp Val Glu Ser Phe Ser Leu Pro Leu

1265 1270 1275 1280

Thr Lys Phe Lys His Leu Leu Phe Leu Ser Asp Ser Asp Glu Gly Leu

1285 1290 1295

Glu Asn Leu Gly Ser His Leu Leu Lys Thr Leu Ile Lys Val Pro Glu

1300 1305 1310

Tyr Ser Thr Met Ile Ile Asp Ser Leu Gly Glu His Glu Ala Tyr Gln

1315 1320 1325

Gly Gln Val Arg Thr Tyr Val Gly Ala Asp Met Ile Ser Asp Met Ala

1330 1335 1340

Glu Gln Leu Asn Tyr Glu Leu Gly Lys Arg Gln Glu Gln Asn Ala Phe

1345 1350 1355 1360

Asp Arg Trp Phe Ile Leu Ile Pro Asp Phe Glu Ser Phe Val Ser Lys

1365 1370 1375

Thr Asn Leu Ser Leu Glu Gln Ile Gln Asn Leu Leu Asp Asn Gly Pro

1380 1385 1390

Lys Val Gly Leu His Leu Ile Ile Gly Ser Ala Phe Ser Phe Val Gly

1395 1400 1405

Ser Lys Leu Asp Pro Val Asn Lys Tyr Val Lys Thr Asn Ser Gln Tyr

1410 1415 1420

Val Met Leu Gly Met Arg Leu Met Asp Gln Thr Phe Leu Glu Lys Val

1425 1430 1435 1440

Tyr Asn Ser Lys Glu Ala Arg Leu Glu Arg Asp Glu Ala Tyr Ile His

1445 1450 1455

Asp Arg Lys Asn Tyr Gln Lys Leu Lys Leu Ser Ile Asp

1460 1465

<210> 3

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gcgtcgacaa acggcaagtc gccaatacca g 31

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tgctgactcc aatcatcact ag 22

<210> 5

<211> 53

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gacgttgagc ctcggaaccc atcgaattac aggaaatggc tgatacttat cac 53

<210> 6

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gcgaattctt aacttttcat cagttacaat 30

<210> 7

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cctcctctag tgatgattgg agtcagcaca ccgaactaga gcttgatgaa 50

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

taattcgatg ggttccgagg ctc 23

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cgtcctcgtg gtatctatat c 21

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

caatatcatc ctgaccacgt aag 23

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tcgtggtatc tatatcattg caac 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gataaacaat atcatcctga ccac 24

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

atactgctgg ttctcaacaa 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gtcaataact gcttgctctt 20

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cgacgataca tagccgacc 19

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

ccgtcacttg gtagattttc c 21

Claims (4)

1. The method comprises the following steps ofessCUse of a gene for toxicity control of streptococcus agalactiae, characterized in thatessCThe gene sequence is shown as SEQ ID NO.1 or the nucleotide sequence of the coded amino acid sequence shown as SEQ ID NO.2, the toxicity regulation is forward regulation,essCthe gene knockout down regulates the expression of streptococcus agalactiae ESAT6 protein.

2.essCUse of a gene recombination knockout vector for regulating toxicity of streptococcus agalactiae, wherein the toxicity is regulated byessCThe gene knockout down regulates the ESAT6 protein expression of streptococcus agalactiae; the saidessCGene recombination knockout vector is inserted with chloramphenicol resistance gene on recombination vectorCATInstead ofessCFusion fragment of a gene consisting of the gene as set forth in claim 1essCUpstream and downstream fragments of the GeneCATGene fragment composition.

3. The use according to claim 2, characterized in that theessCThe upstream fragment of the gene is an upstream 632bp homologous arm thereof, theessCThe downstream fragment of the gene isDownstream 571bp homology arm.

4. The use according to claim 3, wherein,essCthe construction method of the gene recombination knockout vector comprises the following steps:

(1) PCR amplification with Streptococcus agalactiae genomic DNA as template and SEQ ID NO.3-SEQ ID NO.4 as primer pairessCGene upstream fragmentessCup, PCR amplification with SEQ ID NO.5-SEQ ID NO.6 as primer pairessCDownstream fragment of GeneessCdown; PCR amplifying a chloramphenicol resistance gene fragment CAT by using a plasmid pSET5S as a template and SEQ ID NO.7-SEQ ID NO.8 as a primer pair;

(2) Purifying the three fragments obtained in the step (1) and then carrying out fusion PCR amplification;

(3) Performing PCR amplification verification by taking the fusion PCR product obtained in the step (2) as a template and taking SEQ ID NO.3-SEQ ID NO.6 as a primer pair;

(4) And (3) connecting the fusion enzyme obtained in the step (3) after verification to a linearization vector pSET4s, and carrying out identification and screening after conversion of a connecting product.