CN115161333B - Reverse screening marker of streptococcus suis, streptococcus suis containing reverse screening marker and application of streptococcus suis - Google Patents
Reverse screening marker of streptococcus suis, streptococcus suis containing reverse screening marker and application of streptococcus suis Download PDFInfo
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Abstract
The invention discloses a reverse screening marker of streptococcus suis, streptococcus suis containing the reverse screening marker and application thereof. The reverse screening marker is a mutated pheS gene for encoding phenylalanine tRNA synthetase alpha subunit of streptococcus suis, the mutated pheS gene leads to T261S and A315G double substitution mutation of the encoded phenylalanine tRNA synthetase alpha subunit of streptococcus suis, and the PheS mutant is used for competing with wild PheS protein of the strain, p-Cl-Phe is erroneously doped into synthesized protein in the translation process, so that the strain expressing the PheS mutant dies in the presence of the p-Cl-Phe, and the strain not expressing the PheS mutant survives. By utilizing the efficient reverse screening marker, the invention also establishes a high-efficiency streptococcus suis traceless gene operation method, which is used for realizing traceless gene operation such as traceless gene deletion, gene fusion, gene mutation and the like of streptococcus suis, and has good application prospects in the aspects of researching physiological and pathological mechanisms of streptococcus suis and preparing vaccine strains.
Description
Technical Field
The invention relates to a reverse screening marker of streptococcus suis, and also relates to streptococcus suis containing the reverse screening marker and application thereof. The invention belongs to the field of biotechnology.
Background
Streptococcus suis is a gram positive bacterium that causes meningitis, arthritis, sepsis and death in pigs. In addition, it can be transmitted to humans by contact with diseased pigs, leading to human meningitis, toxic shock-like syndrome in streptococci and death. Thus, streptococcus suis is an important zoonotic pathogen, which severely threatens the pig industry and human public health. Streptococcus suis has received increasing attention over the last two decades, and important advances have been made in both physiological and pathological mechanism studies and in vaccine preparation and production. Gene manipulation techniques play a key role in this process. However, conventional genetic manipulation techniques have either introduced a resistance gene marker or used a non-resistance gene manipulation, but the efficiency was low, and it was difficult to perform a complicated, multi-gene manipulation. However, whether further intensive, complex and objective physiological and pathological studies are performed, or vaccine strains are developed which cannot carry resistance gene markers and require polygenic manipulation, efficient traceless deletion, fusion or mutation of the relevant genes is required. Therefore, the lack of an efficient traceless gene operating system has become a bottleneck limiting streptococcus suis research and vaccine preparation, and an efficient reverse screening marker is a key to achieving efficient traceless gene operation of bacteria.
There are two presently reported reverse screening markers for creating Streptococcus suis, namely, bacillus subtilis sucrase SacB and Vibrio parahaemolyticus toxin YoeB. In the report of SacB as a streptococcus suis reverse screening marker, the natural sacB gene of bacillus subtilis and the promoter thereof are adopted as the reverse screening marker in a simple sentence, and no specific method exists, and the efficiency is not mentioned. We used the Bacillus subtilis native sacB gene and its promoter in the report to screen hundreds of colonies tolerant to sucrose, and did not screen the clone of interest, indicating that its screening efficiency was less than 1%. In the report of using YeoB as a reverse screening marker, the induction of YeoB does not completely inhibit the growth of Streptococcus suis, which fundamentally limits the screening efficiency. Therefore, this method requires not only enrichment of the desired clone by multiple passages and addition of an inducer, but also the highest screening efficiency is less than 100%, in most cases only about 50%. Therefore, the presently reported streptococcus suis reverse screening markers are all relatively inefficient, and there is a need to develop more efficient reverse screening markers for streptococcus suis traceless gene manipulation.
In recent years, mutants of the pheS gene encoding the alpha subunit of phenylalanine tRNA synthetase have been found to be useful as reverse screening markers for bacteria. A294G single amino acid substitution mutants (amino acid positions referenced to E.coli PheS protein) were developed earlier and have found use in a variety of bacteria. However, this single-substitution mutant screening was inefficient, requiring high concentrations of p-Cl-phe (> 5 mM) and the use of nutrient limiting media. Further studies found that the T251S/A294G double-substituted mutant had a higher p-Cl-phe incorporation efficiency, and therefore it was speculated that it could produce a higher screening efficiency, which was demonstrated in Bacillus subtilis and Streptococcus mutans, with a screening efficiency of 100%.
PheS mutants by competing with the wild PheS protein of the strain itself, the phenylalanine analog p-Cl-phe is erroneously incorporated into the synthesized protein during translation, so that the strain expressing the PheS mutant dies in the presence of p-Cl-phe and the strain not expressing the PheS mutant survives. This reverse screening principle determines that PheS reverse screening markers established in other bacteria cannot be used in Streptococcus suis, but rather require: 1) Identifying its own pheS gene for streptococcus suis; 2) Identifying the amino acids corresponding to T251 and A294, and carrying out substitution mutation; 3) Introducing a large number of synonymous mutations, and on the premise of keeping the protein sequence consistent with the wild PheS protein, reducing the similarity of the nucleic acid sequences as much as possible, and avoiding recombination of the two; 4) Screening strong promoter drive against Streptococcus suis, and increasing the expression level of mutant PheS protein, thereby competing with wild PheS protein.
Based on the above, the invention starts from the identification of PheS protein and mutant amino acid of streptococcus suis, and develops a high-efficiency streptococcus suis reverse screening marker by introducing double mutation, synonymous mutation and screening promoter, and determines the action condition and efficiency of the streptococcus suis reverse screening marker.
Disclosure of Invention
One of the purposes of the present invention is to provide a reverse screening marker for Streptococcus suis.
The second object of the invention is to provide streptococcus suis containing the reverse screening marker and application thereof in streptococcus suis gene editing.
The invention further aims at providing a streptococcus suis traceless gene editing method.
In order to achieve the above purpose, the invention adopts the following technical means:
firstly, the invention provides a reverse screening marker of streptococcus suis, wherein the reverse screening marker is a mutated pheS gene encoding phenylalanine tRNA synthetase alpha subunit of streptococcus suis, the mutated pheS gene leads to T261S and A315G double substitution mutation of the encoded phenylalanine tRNA synthetase alpha subunit of streptococcus suis, the PheS mutant competes with wild PheS protein of the strain, phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe) is erroneously doped into the synthesized protein in the translation process, so that the strain expressing the PheS mutant dies in the presence of p-Cl-Phe, and the strain not expressing the PheS mutant survives.
Wherein, preferably, the nucleotide sequence of the pheS gene of the alpha subunit of the streptococcus suis phenylalanine tRNA synthetase is shown as SEQ ID NO. 1.
Preferably, the amino acid sequence of the PheS mutant encoded by the pheS gene encoding the alpha subunit of the phenylalanine tRNA synthetase of streptococcus suis after mutation is shown as SEQ ID NO. 2.
The invention further provides streptococcus suis containing the reverse screening marker.
Wherein, preferably, the reverse screening marker consists of a promoter P 0177 、P 0530 、P 1503 、P 1815 Or P 1868 Driving the expression.
Preferably, the streptococcus suis is prepared by the following steps:
(1) Synthesis of the mPES mutant Gene
Synthesizing an mPES mutant gene sequence shown in SEQ ID NO. 1;
(2) Construction of fusion fragment P-mPES of mPES and strong promoter
1) Cloning of the mphs with 5 strong promoter fragments:
PCR primers were designed and the nucleotide sequences were as follows:
P 0177 -F:5’-CCGGCGGAAGAAGGAGTAATTGGTAAGAGAAATGTGAGTG-3’
P 0177 -R:5’-GTTGTTGCTCGATGTTAGACATATCTTTATAAGACATGATATCCTC-3’
P 0530 -F:5’-CCGGCGGAAGAAGGAGTAAGTAGGATAACTGAATGGAGAA-3’
P 0530 -R:5’-GTTGTTGCTCGATGTTAGACATTTTGGTAAAAGCCTCCAATAA-3’
P 1503 -F:5’-CCGGCGGAAGAAGGAGTAATGTTTCGCCAGAGGCTT-3’
P 1503 -R:5’-GTTGTTGCTCGATGTTAGACATTATATTACTCTCCTTTGAGTTT-3’
P 1815 -F:5’-CCGGCGGAAGAAGGAGTAACAGCGCCTCAAAAACTA-3’
P 1815 -R:5’-GTTGTTGCTCGATGTTAGACATAAGTCCTCCATATAAGTACTTC-3’
P 1868 -F:5’-CCGGCGGAAGAAGGAGTAAAAAAACAGCAAGGATTGTAG-3’
P 1868 -R:5’-GTTGTTGCTCGATGTTAGACATAAAACACCTCTGTTTTCTTT-3’
mPheS-F:5’-ATGTCTAACATCGAGCAAC-3’
mPheS-R:5’-TTAGAATTGTTCTGAGAAACGAAC-3’
taking the mPIS gene synthesized in the step (1) as a template and taking the mPIS-F/mPIS-R as a primer to obtain an amplification product of the mPIS gene; the genome DNA of streptococcus suis 05ZYH33 strain is used as a template, and P is respectively used as a template 0177 -F/P 0177 -R、P 0530 -F/P 0530 -R、P 1503 -F/P 1503 -R、P 1815 -F/P 1815 -R、P 1868 -F/P 1868 R is primer to obtain 5 promoters P 0177 、P 0530 、P 1503 、P 1815 、P 1868 Is a product of amplification of (a);
3) Construction of fusion fragment P-mPHS
The amplified products of the promoters and the amplified products of the mPES are respectively mixed as templates, and fusion is carried out by overlapping extension PCR, so as to obtain products, namely fusion fragments P-mPES of the mPES and the strong promoters, which are respectively named P 0177 -mPheS、P 0530 -mPheS、P 1503 -mPheS、P 1815 -mPES and P 1868 -mPheS；
(3) Integration of P-mPES fusion fragments into Streptococcus suis genome by erythromycin resistance Gene (erm)
1) Streptococcus suis ssu05_0630 gene upstream sequence and erm gene containing downstream sequence amplification
PCR primers were designed and the nucleotide sequences were as follows:
UP 0630 -F:5’-TGCTAACGATGCTACAAATGC-3’
UP 0630 -R:5’-TTACTCCTTCTTCCGCCGG-3’
Erm-DN 0630 -F:5’-CGTTCGTTTCTCAGAACAATTCTAAAGAAGGAGGGATTCGTCATG-3’
Erm-DN 0630 -R:5’-CAAAGATAGCGGTGGTCGT-3’
respectively with UP 0630 -F/UP 0630 -R and Erm-DN 0630 -F/Erm-DN 0630 R is a primer, and PCR amplification is carried out by taking the erm gene to replace the genomic DNA of a streptococcus suis ssu05_0630 gene deletion strain (Chen Ping, liu Ran, huang Mengmeng, zhu Jinlu, xie Fang, ni Hongbo, liu Saiguo, zhang Yueling) of the ssu05_0630 gene, wherein the strain 05ZYH33 of the streptococcus suis has the identification and functional research of N-acetylglucosaminidase activity protein, and the genome DNA of the national institute of preventive veterinary science, 2019, 41 (05): 462-467) is a template, so as to obtain an upstream sequence amplification product UP of the ssu05_0630 gene 0630 And the downstream sequence amplification product Erm-DN of the ssm-05_0630 gene 0630 ；
2) Fusion fragment UP 0630 -P-mPheS-Erm-DN 0630 Construction of (3)
UP is taken into account 0630 And Erm-DN 0630 The fragment and the 5 fusion fragments P-mPES are respectively combined to be used as templates, and UP is used 0630 -F/UP 0630 R is a primer, overlap extension PCR is carried out by adopting the PCR reaction conditions, 5 corresponding fusion fragments are obtained by amplification, and the fusion fragments are respectively named as UP-P 0177 -mPheS-Erm-DN、UP-P 0530 -mPheS-Erm-DN、UP-P 1503 -mPheS-Erm-DN、UP-P 1815 -mPES-Erm-DN and UP-P 1868 -mPheS-Erm-DN；
3) Peptide-induced transformation and integration identification
Synthesizing polypeptide shown in SEQ ID NO.3 with purity of 95%, dissolving in deionized water to a final concentration of 5mM, subpackaging, and storing at-20deg.C for use; after overnight incubation of Streptococcus suis 05ZYH33 strain, it was inoculated at 1:100 into fresh TSBS medium, 37℃and 5% CO 2 Under the condition, standing and culturing for 1.5h, 2h, 2.5h and 3h; 50 μl of bacterial liquid is taken at each time point, and is respectively mixed with 2.5 μl of polypeptide and 1 μg of fusion fragments of 5 UP-P-mPhs-Erm-DN obtained in the step 2), and after the mixture is cultured for 4 hours, a TSAS plate (TSAS-Erm) added with erythromycin is coated;
PCR primers were designed and the nucleotide sequences were as follows:
SeqF:5’-GCGGAGCCCTTACCAG-3’
SeqR:5’-AATACAGAAGTTAAACGATTTGT-3’
selecting single colony on a TSAS-Erm plate, culturing, carrying out PCR identification by adopting SeqF/SeqR primers, detecting the sizes of 2004bp, 1820bp, 1717bp, 1872bp and 1777bp respectively by 1% agarose electrophoresis, and confirming that each UP-P-mPES-Erm-DN fragment is integrated into a target site, which means that a fragment with P-mPES-Erm integrated in a genome, namely PPE for short is obtained; these strains were designated as gP respectively 0177 PE、gP 0530 PE、gP 1503 PE、gP 1815 PE and gP 1868 PE。
The invention further provides application of the streptococcus suis in streptococcus suis traceless gene editing.
Finally, the invention also provides a streptococcus suis traceless gene editing method, which comprises the following steps: amplifying upstream UP1 and downstream DN1 sequences of mutant genes from a Streptococcus suis WT strain; amplifying from the genome DNA of the streptococcus suis strain to obtain a fragment PPE containing a promoter, a reverse screening marker and an erythromycin resistance gene; amplifying to obtain a fusion DNA fragment UP1-PPE-DN1 by overlap extension PCR; fragment transformation of streptococcus suis WT strain and screening on TSAS-Erm plate, colony obtaining positive colony by PCR identification, obtaining intermediate strain containing reverse screening mark P-mPHS;
amplifying upstream UP2 and downstream DN2 sequences of mutant genes from a Streptococcus suis WT strain; the upstream and downstream sequences of the mutant gene are either fused directly together, fused to a specific gene or fragment, or fused to the mutant gene to obtain a second fragment for a second transformation; fragments were transformed with the above p-Cl-phe-sensitive intermediate strain and reverse screened on TSAS plates containing 0.05% p-Cl-phe; randomly picking 100 colonies, culturing, and carrying out PCR identification on the colonies to obtain the target mutation.
Compared with the prior art, the invention has the beneficial effects that:
streptococcus suis is an important zoonosis pathogen, and seriously threatens the pig industry and human public health. Intensive studies on Streptococcus suis and vaccine preparation are in urgent need for an effective traceless gene manipulation system, and the efficiency of reverse screening markers directly determines the efficiency of traceless gene manipulation. Two reverse screening markers based on SacB and YeoB have been developed in streptococcus suis, but they are not efficient enough and relatively complex to operate. In the invention, under the genetic background of streptococcus suis, the method comprises the steps of identifying the pheS gene of the streptococcus suis, introducing mutation, screening a promoter, and searching for proper concentration of the chloro-phenylalanine (p-Cl-phe) in the streptococcus suis through a genome integration way, and identifying screening efficiency, wherein the high-efficiency reverse screening marker of the streptococcus suis based on the pheS mutant gene is developed, and the screening efficiency reaches 100 percent and is the highest-efficiency reverse screening marker in the current streptococcus suis. By utilizing the efficient reverse screening marker, a high-efficiency streptococcus suis traceless gene operation strategy is established, and the efficient streptococcus suis traceless gene screening marker is used for realizing efficient traceless gene deletion, gene fusion and gene mutation, and other traceless gene operations, and has good application prospects in the aspects of researching physiological and pathological mechanisms of streptococcus suis and preparing vaccine strains.
Drawings
FIG. 1 is a comparison of wtPheS and mPHS related sequences;
FIG. 2 is a schematic representation of the construction of different P-mPHS genome integration strains and detection of P-Cl-phe sensitivity;
FIG. 3 is P 1503 -phes as a reverse screening marker to construct ireB gene deleted strains;
FIG. 4 is a diagram based on P 1503 -schematic representation of a high-efficiency streptococcus suis traceless gene manipulation strategy for the inverse selection marker of mhes.
Detailed Description
The invention is further illustrated by the following experiments in conjunction with examples, which are to be understood as being for illustrative purposes only and in no way limiting the scope of the invention.
Example 1 design and Synthesis of a mPIS mutant Gene
BLAST was performed using the pheS of Streptococcus mutans, and the wild-type pheS gene (wtPheS) was identified from the genome of Streptococcus suis 05ZYH33 (GenBank: CP 000407). The amino acids T261 and A315 corresponding to E.coli PheS proteins T251 and A294 were identified by ClustalW comparing the PheS proteins of E.coli, streptococcus mutans, listeria monocytogenes, acidophilic bacteria and Streptococcus suis (FIG. 1A). The codons ACT and GCC were changed to TCA and GGT, respectively, to achieve T261S and A315G double substitution mutation (FIG. 1B, dotted rectangle). Then 275 synonymous mutations were finally introduced by means of the codon adaptation software Jcat and manual mutation, the similarity of the obtained mhes gene (shown in SEQ ID No. 1) to the wtPheS gene was 73.7%, the longest continuous identical sequence between them not exceeding 8bp (fig. 1B). Their corresponding mPES (shown in SEQ ID NO. 2) and wtPheS proteins were identical in amino acid sequence except for the T261S/A315G double substitution (FIG. 1C). The mphs gene was synthesized by the hua major gene company.
EXAMPLE 2 construction of a fusion fragment of mPHS and a strong promoter P-mPHS
(1) The mhes was cloned with 5 potential strong promoter fragments:
PCR primers were designed and the nucleotide sequences were as follows:
P 0177 -F:5’-CCGGCGGAAGAAGGAGTAATTGGTAAGAGAAATGTGAGTG-3’
P 0177 -R:5’-GTTGTTGCTCGATGTTAGACATATCTTTATAAGACATGATATCCTC-3’
P 0530 -F:5’-CCGGCGGAAGAAGGAGTAAGTAGGATAACTGAATGGAGAA-3’
P 0530 -R:5’-GTTGTTGCTCGATGTTAGACATTTTGGTAAAAGCCTCCAATAA-3’
P 1503 -F:5’-CCGGCGGAAGAAGGAGTAATGTTTCGCCAGAGGCTT-3’
P 1503 -R:5’-GTTGTTGCTCGATGTTAGACATTATATTACTCTCCTTTGAGTTT-3’
P 1815 -F:5’-CCGGCGGAAGAAGGAGTAACAGCGCCTCAAAAACTA-3’
P 1815 -R:5’-GTTGTTGCTCGATGTTAGACATAAGTCCTCCATATAAGTACTTC-3’
P 1868 -F:5’-CCGGCGGAAGAAGGAGTAAAAAAACAGCAAGGATTGTAG-3’
P 1868 -R:5’-GTTGTTGCTCGATGTTAGACATAAAACACCTCTGTTTTCTTT-3’
mPheS-F:5’-ATGTCTAACATCGAGCAAC-3’
mPheS-R:5’-TTAGAATTGTTCTGAGAAACGAAC-3’
the PCR reaction system is as follows:
the PCR reaction conditions were: denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 5-10s/kb,32 cycles. Except that the mPIS gene synthesized in example 1 was used as a template for the mPIS amplification, the genomic DNA of Streptococcus suis 05ZYH33 strain was used as a template.
P 0177 -F/P 0177 -R、P 0530 -F/P 0530 -R、P 1503 -F/P 1503 -R、P 1815 -F/P 1815 -R、P 1868 -F/P 1868 PCR products amplified by six pairs of primers of-R and mPES-F/mPES-R are 5 promoters P respectively 0177 、P 0530 、P 1503 、P 1815 、P 1868 And the mPES gene, sizes of 484bp, 300bp, 197bp, 352bp, 257bp and 1044bp, respectively, were detected by 1% agarose electrophoresis (FIG. 2A).
(2) Construction of fusion fragment P-mPHS: fusion was performed by overlap extension PCR using a mixture of the promoter fragments and the mPES fragments as templates according to the template and primer combinations shown in Table 1.
The PCR reaction conditions are the same, and the obtained product is the fusion fragment P-mPES of mPES and a strong promoter, which are respectively named as P 0177 -mPheS、P 0530 -mPheS、P 1503 -mPheS、P 1815 -mPES and P 1868 The sizes of-mPES, detected by 1% agarose electrophoresis, were 1528bp, 1344bp, 1241bp, 1396bp and 1301bp, respectively (FIG. 2B).
Example 3 integration of the P-mPHS fusion fragment into Streptococcus suis genome by erythromycin resistance Gene (erm)
(1) Streptococcus suis ssu05_0630 gene upstream sequence and erm gene-containing ssu05_0630 gene downstream sequence amplification
PCR primers were designed and the nucleotide sequences were as follows:
UP 0630 -F:5’-TGCTAACGATGCTACAAATGC-3’
UP 0630 -R:5’-TTACTCCTTCTTCCGCCGG-3’
Erm-DN 0630 -F:5’-CGTTCGTTTCTCAGAACAATTCTAAAGAAGGAGGGATTCGTCATG-3’
Erm-DN 0630 -R:5’-CAAAGATAGCGGTGGTCGT-3’
respectively with UP 0630 -F/UP 0630 -R and Erm-DN 0630 -F/Erm-DN 0630 R is a primer, and PCR amplification is carried out by taking the erm gene to replace the genomic DNA of a streptococcus suis ssu05_0630 gene deletion strain (Chen Ping, liu Ran, huang Mengmeng, zhu Jinlu, xie Fang, ni Hongbo, liu Saiguo, zhang Yueling) of the ssu05_0630 gene, wherein the strain 05ZYH33 of the streptococcus suis has the identification and functional research of N-acetylglucosaminidase activity protein, and the genome DNA of the national institute of preventive veterinary science, 2019, 41 (05): 462-467) is a template, so as to obtain an upstream sequence amplification product UP of the ssu05_0630 gene 0630 And the downstream sequence amplification product Erm-DN of the ssm-05_0630 gene 0630 . Product UP 0630 And Erm-DN 0630 The detected sizes were 1024bp and 2413bp, respectively (FIG. 2B).
(2) Fusion fragment UP 0630 -P-mPheS-Erm-DN 0630 Construction of (3)
As shown in Table 2, UP 0630 And Erm-DN 0630 Fragments were combined with the 5P-mPES fragments, and the mixture was used as a template for UP 0630 -F/UP 0630 R is a primer, overlap extension PCR is carried out by adopting the PCR reaction conditions, and 5 corresponding fusion fragments UP-P are obtained by amplification 0177 -mPheS-Erm-DN、UP-P 0530 -mPheS-Erm-DN、UP-P 1503 -mPheS-Erm-DN、UP-P 1815 -mPES-Erm-DN and UP-P 1868 -mPHS-Erm-DN. Sizes were 4983bp, 4799bp, 4696bp, 4851bp and 4756bp, respectively, as detected by 1% agarose electrophoresis (FIG. 2C).
TABLE 2 overlap extension PCR templates and primer combinations
(3) Peptide-induced transformation and integration identification
Peptide-induced transformation references were made (Zdacaria et al, 2014). The polypeptide (SEQ ID NO.3, GNWGTWVEE) was synthesized by GenScript (China) at a purity of 95%. Dissolving in deionized water to a final concentration of 5mM, packaging, and storing at-20deg.C. After overnight incubation of Streptococcus suis 05ZYH33 strain, it was inoculated at 1:100 into fresh TSBS medium, 37℃and 5% CO 2 Under the condition, the culture is carried out for 1.5h, 2h, 2.5h and 3h. 50 μl of the bacterial liquid was taken at each time point and mixed with 2.5 μl of the polypeptide and 1 μg of the fusion fragment of 5 UP-P-mPHS-Erm-DN obtained in step (2), respectively. After 4 hours of incubation of the mixture, the TSAS plate (TSAS-Erm) was coated with erythromycin.
PCR primers were designed and the nucleotide sequences were as follows:
SeqF:5’-GCGGAGCCCTTACCAG-3’
SeqR:5’-AATACAGAAGTTAAACGATTTGT-3’
single colonies on TSAS-Erm plates are selected, after culturing, PCR identification is carried out by adopting SeqF/SeqR primers, and the sizes of 2004bp, 1820bp, 1717bp, 1872bp and 1777bp (figure 2D) are detected by 1% agarose electrophoresis, and the integration of the UP-P-mPES-Erm-DN fragments into target sites is confirmed, which means that the fragments with the integrated P-mPES-Erm in the genome, namely PPE for short, are obtained. These strains were designated as gP respectively 0177 PE、gP 0530 PE、gP 1503 PE、gP 1815 PE and gP 1868 PE (g stands for genomic integration) (FIG. 2E).
Example 4 sensitivity detection of different gPPE strains to p-Cl-phe
After overnight culture of Streptococcus suis strains, they were transferred to fresh medium and grown to OD 600nm After 0.6, 5. Mu.l were dropped onto TSAS plates containing p-Cl-phe at the indicated concentrations. At 37℃with 5% CO 2 After 24 hours of stationary culture under the condition, the growth of the strain was observed and photographed. The Minimum Inhibitory Concentration (MIC) of p-Cl-phe for each strain was defined as the minimum concentration that inhibited the growth of that strain. p-Cl-phe vs gP 0177 PE、gP 0530 PE、gP 1503 PE、gP 1815 PE and gP 1868 The MIC of the PE strain was 0.02%, 0.01%, 0.08% and 0.06%, respectively (FIG. 2F), whereas the wild strain (WT) was insensitive to p-Cl-phe at concentrations below 0.15%. It was demonstrated that 5P-mPHS each confer sensitivity to P-Cl-phe on the strain, where gP 0530 PE and gP 1503 MIC of PE is lowest, description P 0530 -mPES and P 1503 The sensitivity conferred by mPES is the strongest and is most suitable as a reverse screening marker for Streptococcus suis.
Example 5P 1503 Screening efficiency of-mPES as a reverse screening marker
To detect P 1503 Efficiency of mhes as a reverse screening marker for streptococcus suis, for use in traceless deletion of the ireB gene. PCR primers were designed and the nucleotide sequences were as follows:
UP1-F:5’-GAAGAAGCTCCTGTTGTTGC-3’
UP1-R:5’-CTTCGGTAAATCCCATACTTAC-3’
PPE-F:5’-GTAAGTATGGGATTTACCGAAGTGTTTCGCCAGAGGCTT-3’
PPE-R:5’-GTCAATCCCATTCCCTTTCCCAAATTCCCCGTAGGC-3’
DN1-F:5’-GAAAGGGAATGGGATTGAC-3’
DN1-R:5’-GCGTCTTCTGGGATAGGTT-3’
UP2-F:5’-ACAACGCCTGGTGGACG-3’
UP2-R:5’-ACTTACACCTTCTTTCCCT-3’
DN2-F:5’-AGGGAAAGAAGGTGTAAGTTGAGAATAATGGGATTAGACGT-3’
DN2-R:5’-TGATAGGCTGGATAGTTTTGATA-3’
ireB-F:5’-GAAACGACTTCAAGTGGGC-3’
ireB-R:5’-GTTCGGTCAAACGCTCCA-3’
amplifying upstream UP1 and downstream DN1 of the ireB gene from the strain of Streptococcus suis WT by using UP1-F/UP1-R and DN1-F/DN1-R primers, respectively; from gP using PPE-F/PPE-R primers 1503 Amplification of P in PE Strain genomic DNA 1503 PE sequences (PPE for short) (fig. 3A). The fusion DNA fragment UP1-PPE-DN1 was amplified by overlap extension PCR (FIG. 3B). Fragments were transformed into streptococcus suis WT strains and screened on TSAS-Erm plates and colonies were PCR identified to obtain positive colonies (fig. 3C). Positive colony inoculation TSAS plates with 0.05% P-Cl-phe added confirm their sensitivity to P-Cl-phe to obtain a negative selection marker P 1503 Intermediate strains of mPIS (FIG. 3D).
The upstream sequence UP2 and downstream sequence DN2 of the ireB gene were amplified from the Streptococcus suis WT strain using UP2-F/UP2-R and DN2-F/DN2-R primers, respectively (FIG. 3E). The fusion DNA fragment UP2-DN2 was amplified by overlap extension PCR (FIG. 3F). Fragments were transformed with the above-described P-Cl-phe-sensitive intermediate strain and reverse screened on TSAS plates containing 0.05% P-Cl-phe. Randomly picking 100 colonies, culturing, and performing PCR identification with primer ireB-F/ireB-R, wherein all colonies with tolerance of 0.05% of P-Cl-phe contain target ireB gene traceless deletion (FIG. 3G), indicating P 1503 As a reverse screening marker, mPES was used with a screening efficiency of 100%. Three times of repetitionExperiments have further confirmed this.
Example 6 at P 1503 Streptococcus suis traceless gene manipulation strategy with-mPES as reverse screening marker
P 1503 100% screening efficiency of mPIS makes it possible to perform efficient traceless genetic manipulation of Streptococcus suis. By inserting and removing P 1503 The two steps of the-mPES and the p-Cl-phe are taken as inhibitors, thus forming an efficient traceless gene operation strategy of streptococcus suis which only needs two steps. As shown in FIG. 4, this strategy only requires two fusion fragments and two transformations. First, the upstream and downstream sequences of the mutation site are compared with a mutant sequence containing P 1503 -mPES tag P 1503 PE fragment fusion to obtain UP-P for first transformation 1503 PE-DN fragment selected by positive selection marker erm carried by the fragment to obtain a fragment containing P 1503 Intermediate strains of PE. Then, the upstream and downstream sequences of the mutation site are either fused directly together (for gene deletion), fused with a specific gene or fragment (for example, a fluorescent protein gene or FLAG tag for gene fusion), or fused with a mutant gene (for gene mutation), to obtain a second fragment for the second transformation, and after transformation of the intermediate strain, the reverse selection is performed by p-Cl-phe to obtain the desired mutation.
Sequence listing
<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin division center of Chinese animal health and epidemiology center)
<120> a reverse screening marker for Streptococcus suis, streptococcus suis containing the same and use thereof
<141> 2022-06-23
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 1044
<212> DNA
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atgtctaaca tcgagcaaca acttgcagaa ctttcacaaa ctactttgga aaaacttaag 60
gaaattcaac accaaggtga aaaggaattg caagatcttc gtgtagctgt acttggcaaa 120
aaaggttcac ttactgattt gcttaaaggt cttaaggatt tgtcaaacga tatgaagcct 180
attgtaggca aacaagtaaa cgaagttcgt gacgttttga ctacagcttt cgaagagaca 240
gcacaaaaag ttgctgctgc taaaatccaa caacaattgg catcagaaac tatcgacgtt 300
actcttcctg gacgtcaagt aaaagttggt aaacgtcacg ttcttactca aacatcagaa 360
gaaatcgaag acatcttcct tggtatgggt ttccaaattg tagatggttt cgaagttgaa 420
aaagattatt ataacttcga acgtatgaac cttccaaaag accaccctgc tcgtgacatg 480
caagacactt tctacattac tgaagagatc ttgatgcgta ctcacacttc accagtacaa 540
gctcgtacta tggatcaaca cgacttctct aagggcgcac ttaaaatgat ctcaccaggc 600
cgtgtattcc gccgcgacac tgacgacgct actcactcac accaattcca ccaaatcgaa 660
ggtcttgtag ttggcgaaaa cgtttcaatg ggtgacttga aaggcacttt ggaaatgatc 720
attaaaaaaa tgttcggtga agaacgtcaa atccgtcttc gcccttctta tttccctttc 780
tcagaaccat cagtagaagt tgacgtatca tgtttcaaat gtggaggtga tggctgtaac 840
gtttgtaaaa aaactggttg gatcgaaatc ttgggtgctg gtatggttca cccacaagtt 900
cttgaaatgt caggtatcga ttctactaaa tactcaggtt tcggtttcgg cttgggacaa 960
gagcgtatcg ctatgttgcg ctacggaatt aacgatattc gtggcttcta ccaaggcgac 1020
gttcgtttct cagaacaatt ctaa 1044
<210> 2
<211> 347
<212> PRT
<213> artificial sequence
<400> 2
Met Ser Asn Ile Glu Gln Gln Leu Ala Glu Leu Ser Gln Thr Thr Leu
1 5 10 15
Glu Lys Leu Lys Glu Ile Gln His Gln Gly Glu Lys Glu Leu Gln Asp
20 25 30
Leu Arg Val Ala Val Leu Gly Lys Lys Gly Ser Leu Thr Asp Leu Leu
35 40 45
Lys Gly Leu Lys Asp Leu Ser Asn Asp Met Lys Pro Ile Val Gly Lys
50 55 60
Gln Val Asn Glu Val Arg Asp Val Leu Thr Thr Ala Phe Glu Glu Thr
65 70 75 80
Ala Gln Lys Val Ala Ala Ala Lys Ile Gln Gln Gln Leu Ala Ser Glu
85 90 95
Thr Ile Asp Val Thr Leu Pro Gly Arg Gln Val Lys Val Gly Lys Arg
100 105 110
His Val Leu Thr Gln Thr Ser Glu Glu Ile Glu Asp Ile Phe Leu Gly
115 120 125
Met Gly Phe Gln Ile Val Asp Gly Phe Glu Val Glu Lys Asp Tyr Tyr
130 135 140
Asn Phe Glu Arg Met Asn Leu Pro Lys Asp His Pro Ala Arg Asp Met
145 150 155 160
Gln Asp Thr Phe Tyr Ile Thr Glu Glu Ile Leu Met Arg Thr His Thr
165 170 175
Ser Pro Val Gln Ala Arg Thr Met Asp Gln His Asp Phe Ser Lys Gly
180 185 190
Ala Leu Lys Met Ile Ser Pro Gly Arg Val Phe Arg Arg Asp Thr Asp
195 200 205
Asp Ala Thr His Ser His Gln Phe His Gln Ile Glu Gly Leu Val Val
210 215 220
Gly Glu Asn Val Ser Met Gly Asp Leu Lys Gly Thr Leu Glu Met Ile
225 230 235 240
Ile Lys Lys Met Phe Gly Glu Glu Arg Gln Ile Arg Leu Arg Pro Ser
245 250 255
Tyr Phe Pro Phe Ser Glu Pro Ser Val Glu Val Asp Val Ser Cys Phe
260 265 270
Lys Cys Gly Gly Asp Gly Cys Asn Val Cys Lys Lys Thr Gly Trp Ile
275 280 285
Glu Ile Leu Gly Ala Gly Met Val His Pro Gln Val Leu Glu Met Ser
290 295 300
Gly Ile Asp Ser Thr Lys Tyr Ser Gly Phe Gly Phe Gly Leu Gly Gln
305 310 315 320
Glu Arg Ile Ala Met Leu Arg Tyr Gly Ile Asn Asp Ile Arg Gly Phe
325 330 335
Tyr Gln Gly Asp Val Arg Phe Ser Glu Gln Phe
340 345
<210> 3
<211> 9
<212> PRT
<213> artificial sequence
<400> 3
Gly Asn Trp Gly Thr Trp Val Glu Glu
1 5
Claims (7)
1. A reverse selectable marker for streptococcus suis, wherein the reverse selectable marker is a mutated pheS gene encoding a phenylalanine tRNA synthetase α subunit of streptococcus suis, wherein the mutated pheS gene results in T261S and a315G double substitution mutations of the encoded phenylalanine tRNA synthetase α subunit of streptococcus suis, wherein the pheS mutant comprises a phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe) incorporated erroneously into a synthetic protein during translation by competing with a wild pheS protein of the strain, such that in the presence of p-Cl-Phe, the strain expressing the pheS mutant dies and the strain not expressing the pheS mutant survives, and wherein the mutated pheS gene encoding the phenylalanine tRNA synthetase α subunit of streptococcus suis has a nucleotide sequence as set forth in SEQ ID No. 1.
2. The inverted selection marker of streptococcus suis according to claim 1, wherein the amino acid sequence of the pheS mutant encoded by the pheS gene encoding the alpha subunit of phenylalanine tRNA synthetase of streptococcus suis after mutation is shown as SEQ ID No. 2.
3. A streptococcus suis comprising the reverse screening marker of claim 1 or 2.
4. A streptococcus suis as claimed in claim 3, wherein the reverse selectable marker consists of promoter P 0177 、P 0530 、P 1503 、P 1815 Or P 1868 Driving the expression.
5. The streptococcus suis of claim 3, wherein said streptococcus suis is prepared by the process of:
(1) Synthesis of the mPES mutant Gene
Synthesizing an mPES mutant gene sequence shown in SEQ ID NO. 1;
(2) Construction of fusion fragment P-mPES of mPES and strong promoter
1) Cloning of the mphs with 5 strong promoter fragments:
PCR primers were designed and the nucleotide sequences were as follows:
P 0177 -F:
5’-CCGGCGGAAGAAGGAGTAATTGGTAAGAGAAATGTGAGTG-3’
P 0177 -R:
5’-GTTGTTGCTCGATGTTAGACATATCTTTATAAGACATGATATCCTC-3’
P 0530 -F:
5’-CCGGCGGAAGAAGGAGTAAGTAGGATAACTGAATGGAGAA-3’
P 0530 -R:
5’-GTTGTTGCTCGATGTTAGACATTTTGGTAAAAGCCTCCAATAA-3’
P 1503 -F:
5’-CCGGCGGAAGAAGGAGTAATGTTTCGCCAGAGGCTT-3’
P 1503 -R:
5’-GTTGTTGCTCGATGTTAGACATTATATTACTCTCCTTTGAGTTT-3’
P 1815 -F:
5’-CCGGCGGAAGAAGGAGTAACAGCGCCTCAAAAACTA-3’
P 1815 -R:
5’-GTTGTTGCTCGATGTTAGACATAAGTCCTCCATATAAGTACTTC-3’
P 1868 -F:
5’-CCGGCGGAAGAAGGAGTAAAAAAACAGCAAGGATTGTAG-3’
P 1868 -R:
5’-GTTGTTGCTCGATGTTAGACATAAAACACCTCTGTTTTCTTT-3’
mPheS-F:5’-ATGTCTAACATCGAGCAAC-3’
mPheS-R:5’-TTAGAATTGTTCTGAGAAACGAAC-3’
taking the mPIS gene synthesized in the step (1) as a template and taking the mPIS-F/mPIS-R as a primer to obtain an amplification product of the mPIS gene; the genome DNA of streptococcus suis 05ZYH33 strain is used as a template, and P is respectively used as a template 0177 -F/P 0177 -R、P 0530 -F/P 0530 -R、P 1503 -F/P 1503 -R、P 1815 -F/P 1815 -R、P 1868 -F/P 1868 R is primer to obtain 5 promoters P 0177 、P 0530 、P 1503 、P 1815 、P 1868 Is a product of amplification of (a);
2) Construction of fusion fragment P-mPHS
The amplified products of the promoters and the amplified products of the mPES are respectively mixed as templates, and fusion is carried out by overlapping extension PCR, so as to obtain products, namely fusion fragments P-mPES of the mPES and the strong promoters, which are respectively named P 0177 -mPheS、P 0530 -mPheS、P 1503 -mPheS、P 1815 -mPES and P 1868 -mPheS；
(3) Integration of P-mPES fusion fragments into Streptococcus suis genome by erythromycin resistance Gene (erm)
1) Streptococcus suis ssu05_0630 gene upstream sequence and erm gene-containing ssu05_0630 gene downstream sequence amplification
PCR primers were designed and the nucleotide sequences were as follows:
UP 0630 -F:5’-TGCTAACGATGCTACAAATGC-3’
UP 0630 -R:5’-TTACTCCTTCTTCCGCCGG-3’
Erm-DN 0630 -F:
5’-CGTTCGTTTCTCAGAACAATTCTAAAGAAGGAGGGATTCGTCATG-3’
Erm-DN 0630 -R:5’-CAAAGATAGCGGTGGTCGT-3’
respectively with UP 0630 -F/UP 0630 -R and Erm-DN 0630 -F/Erm-DN 0630 R is a primer, and the erm gene is used for replacing the genome DNA of a streptococcus suis ssu05_0630 gene deletion strain of the ssu05_0630 gene as a template, and PCR amplification is carried out to obtain an amplified product UP of an upstream sequence of the ssu05_0630 gene 0630 And the downstream sequence amplification product Erm-DN of the ssm-05_0630 gene 0630 ；
2) Fusion fragment UP 0630 -P-mPheS-Erm-DN 0630 Construction of (3)
UP is taken into account 0630 And Erm-DN 0630 Fragments and upperThe 5 fusion fragments P-mPES are respectively combined to be used as templates, and UP is used 0630 -F/UP 0630 R is a primer, overlap extension PCR is carried out by adopting the PCR reaction conditions, 5 corresponding fusion fragments are obtained by amplification, and the fusion fragments are respectively named as UP-P 0177 -mPheS-Erm-DN、UP-P 0530 -mPheS-Erm-DN、UP-P 1503 -mPheS-Erm-DN、UP-P 1815 -mPES-Erm-DN and UP-P 1868 -mPheS-Erm-DN；
3) Peptide-induced transformation and integration identification
Synthesizing polypeptide shown in SEQ ID NO.3 with purity of 95%, dissolving in deionized water to a final concentration of 5mM, subpackaging, and storing at-20deg.C for use; after overnight incubation of Streptococcus suis 05ZYH33 strain, it was inoculated at 1:100 into fresh TSBS medium, 37℃and 5% CO 2 Under the condition, standing and culturing for 1.5h, 2h, 2.5h and 3h; 50 μl of bacterial liquid is taken at each time point, and is respectively mixed with 2.5 μl of polypeptide and 1 μg of fusion fragments of 5 UP-P-mPhs-Erm-DN obtained in the step 2), and after the mixture is cultured for 4 hours, a TSAS plate added with erythromycin is coated;
PCR primers were designed and the nucleotide sequences were as follows:
SeqF:5’-GCGGAGCCCTTACCAG-3’
SeqR:5’-AATACAGAAGTTAAACGATTTGT-3’
selecting single colony on a TSAS-Erm plate, culturing, carrying out PCR identification by adopting SeqF/SeqR primers, detecting the sizes of 2004bp, 1820bp, 1717bp, 1872bp and 1777bp respectively by 1% agarose electrophoresis, and confirming that each UP-P-mPES-Erm-DN fragment is integrated into a target site, which means that a fragment with P-mPES-Erm integrated in a genome, namely PPE for short is obtained; these strains were designated as gP respectively 0177 PE、gP 0530 PE、gP 1503 PE、gP 1815 PE and gP 1868 PE。
6. Use of streptococcus suis according to any one of claims 3-5 for the traceless gene editing of streptococcus suis.
7. A method for editing a streptococcus suis traceless gene, which is characterized by comprising the following steps: amplifying upstream UP1 and downstream DN1 sequences of mutant genes from a Streptococcus suis WT strain; amplifying from the genomic DNA of a Streptococcus suis strain of any one of claims 3-5 a fragment PPE comprising a promoter, a reverse selectable marker and an erythromycin resistance gene; amplifying to obtain a fusion DNA fragment UP1-PPE-DN1 by overlap extension PCR; fragment transformation of streptococcus suis WT strain and screening on TSAS-Erm plate, colony obtaining positive colony by PCR identification, obtaining intermediate strain containing reverse screening mark P-mPHS;
amplifying upstream UP2 and downstream DN2 sequences of mutant genes from a Streptococcus suis WT strain; the upstream and downstream sequences of the mutant gene are either fused directly together, fused to a specific gene or fragment, or fused to the mutant gene to obtain a second fragment for a second transformation; fragments were transformed with the above p-Cl-phe-sensitive intermediate strain and reverse screened on TSAS plates containing 0.05% p-Cl-phe; randomly picking colonies, culturing, and carrying out PCR identification on the colonies to obtain the target mutation.
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| CN105820252A (en) * | 2010-05-03 | 2016-08-03 | Atyr 医药公司 | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-trna synthetases |
| CN112300973A (en) * | 2019-08-02 | 2021-02-02 | 南京理工大学 | Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse screening marker |
| CN112980982A (en) * | 2021-05-18 | 2021-06-18 | 至善时代智能科技(北京)有限公司 | Primers and detection kit for identifying multiple staphylococci based on pheS gene and application thereof |
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| CN105820252A (en) * | 2010-05-03 | 2016-08-03 | Atyr 医药公司 | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-trna synthetases |
| CN104780935A (en) * | 2012-06-27 | 2015-07-15 | 梅里亚有限公司 | Attenuated streptococcus suis vaccines and methods of making and use thereof |
| JP2016032440A (en) * | 2014-07-31 | 2016-03-10 | 国立研究開発法人産業技術総合研究所 | Novel counter selection marker |
| CN112300973A (en) * | 2019-08-02 | 2021-02-02 | 南京理工大学 | Rhodococcus gene editing method using phenylalanyl-tRNA synthetase gene mutant as reverse screening marker |
| CN112980982A (en) * | 2021-05-18 | 2021-06-18 | 至善时代智能科技(北京)有限公司 | Primers and detection kit for identifying multiple staphylococci based on pheS gene and application thereof |
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