Claim document
1, a nucleotide with specificity for O- antigens of Shigella dysenteriae 12 and Escherichia coli O 152, characterized in that
Therefore, it is an isolated nucleotide as shown in SEQ ID NO: 1, with a total length of 12235 bases.
2. O- antigens of Shigella dysenteriae 1 2 and Escherichia coli O 152 according to claim1.
The characteristic of nucleotide is that it consists of 10 genes, which are located between galF gene and gnd gene.
3. A nucleus specific for O- antigens of Shigella dysenteriae 12 and Escherichia coli O 152 according to claim 2.
The glycoside acid is characterized in that the gene is:
Genes related to transportation and processing include wzx gene or genes with similar functions to wzx, wzy gene or genes related to WZX.
Wzy has genes with similar functions;
Glycosyltransferase genes, including orf5, orf7, orf9 and orf 10 genes; Wherein the gene:
Orf5 is a nucleotide with 4626 to 5663 bases in SEQ ID NO: 1;
Wzx is a nucleotide with 5663 to 6874 bases in SEQ ID NO: 1;
Orf7 is the nucleotide of bases 6877th to 7869th in SEQ ID NO: 1;
Wzy is a nucleotide with 7882 to 8955 bases in SEQ ID NO: 1;
Orf9 is a nucleotide with 8960 to 972 1 bases in SEQ ID NO: 1;
Orf 10 is the nucleotide of 97 18 to 10788 in SEQ ID NO: 1.
4. The O- antigen specific for Shigella dysenteriae 12 and Escherichia coli O 152 according to claim 1-2.
Nucleotide is characterized in that it is derived from wzx gene, wzy gene or glycosyltransferase gene orf5, orf7,
Orf9 and orf 10 genes; Or oligonucleotides in genes of sugar synthesis pathway; And mixtures thereof or combinations thereof.
5. The core specific for O- antigens of Shigella dysenteriae 12 and Escherichia coli O 152 according to claim 4.
The nucleotide is characterized in that the oligonucleotide is:
Oligonucleotide pairs derived from orf5:
4866-4886 nucleotides and 5573-559 1 nucleotide in seq id no: 1,
Nucleotide from 5 156 to 5 176 and nucleotide from 5587 to 5595 in SEQ ID NO: 1,
Seq id no: 464 1 to 4660 nucleotides and 5302 to 5320 nucleotides in 1;
Oligonucleotide pairs derived from wzx:
Seq id no: 189 to 6207 nucleotides and 1 to 6750 nucleotides,
Seq id no: 57 13 to 5733 nucleotides and 6724 to 674 1 nucleotides in1,
Seq id no: 6463 to 648 1 nucleotides and 682 1 to 6839 nucleotides in1;
Oligonucleotide pairs derived from orf7:
7024 to 704 1 nucleotides and 7779 to 7800 nucleotides in SEQ ID NO: 1,
Nucleotides at bases 6972-6992 and nucleotides at bases 7578-7596 in SEQ ID NO: 1,
SEQ ID number: nucleotides with bases from 7 13 1 to 7 148 and nucleotides with bases from 7693 to 7674 in 1;
Oligonucleotide pair from wzy:
7883-7903 nucleotides and 8903-892 1 nucleotides in seq id no: 1,
Nucleotides at bases 8049-8069 and nucleotides at bases 8876-8894 in SEQ ID NO: 1,
Seq id no: 197 to 82 14 and 8 197 to 82 14;
Oligonucleotide pairs derived from orf9:
9020 to 9040 base nucleotides and 9630 to 9647 base nucleotides in SEQ ID NO: 1,
Nucleotide at bases 8982-9000th and nucleotide at bases 9443rd-9462nd in SEQ ID NO: 1,
Seq id no: 9 165 to 9 183 and 9525 to 9543 in 1;
Oligonucleotide pairs derived from orf 10:
Seq id no: 9752-9770 nucleotide and 10567- 10586 nucleotide are in 1,
The nucleotides from 9 165 to 9 183 and the nucleotides from 9525 to 9543 in SEQ ID NO: 1,
Seq id no: 9934-9952 and 1 0513-10530.
6. The nucleotide specific for the O- antigen of Shigella dysenteriae 1 2 and Escherichia coli O 152 according to claim1.
Application of detecting bacteria expressing O- antigen in vitro.
7. Nuclei specific for O- antigens of Shigella dysenteriae 1 2 and Escherichia coli O 152 according to claim1.
The application of glycosidic acid is characterized by being used as a primer for PCR and as a probe for hybridization reaction, fluorescence detection and manufacturing.
Gene chips or microarrays are used to detect Shigella dysenteriae 12 or Escherichia coli O 152 in vitro.
8. The nucleotide specific for the O- antigen of Shigella dysenteriae 1 2 and Escherichia coli O 152 according to claim1.
The separation method is characterized by comprising the following steps:
1) genome extraction.
Shigella was cultured overnight in LB medium at 37℃, and the cells were collected by centrifugation. Tris-HCl with a pH of 8.0 was used.
Then incubate at 37℃ for 20min minutes, add lysozyme to decompose bacteria, and add protease K and SDS to degrade eggs.
White matter, then adding ribonuclease to remove RNA；; Adding equal volume of phenol to extract the mixture, collecting supernatant, and eluting with equal volume of phenol: chloroform:
Isoamyl alcohol was extracted twice to remove enzyme and protein, and then residual phenol was extracted with equal volume of ether. Multiply diploid by b
The DNA was precipitated with alcohol, rolled out with glass fiber, washed with 70% ethanol, and finally resuspended with 30μlTE.
Group A DNA was detected by 0.4% agarose gel electrophoresis.
2) Long PCR amplification of Shigella dysenteriae 12 O- antigen gene cluster.
Firstly, the upstream primer ATT was designed according to the common jump initiation sequence in the promoter region of O- antigen gene cluster.
GTG GCT GCA GGG ATC AAA GAA AT, and then design the downstream primers according to the gnd gene downstream of the O- antigen gene cluster.
-TAG TCG CGT GNG CCT GGA TTA AGT TCG C； Using the extended length of Boehringer Mannheim
O- antigen gene cluster was amplified by template PCR, and the PCR reaction procedure was: pre-denaturing at 94℃ for 2 minutes; then
Denaturation at 94℃ 10 second, annealing at 60℃ for 30 seconds, extension at 68℃ 15 minutes, and so on for 30 times; Finally, at 68
Continue extension for 7 minutes at℃ to obtain PCR products; 6-tube PCR products were combined and Wizard of Promega company was used.
The PCR products were purified by PCR Preps purification kit.
3) Construction of O-antigen gene cluster library
O- antigen gene cluster library was constructed by improved Novagen DNaseI shotgun method, and the reaction system was 300ng.
The purified product by PCR was diluted with 0.9μl 0. 1M MnCl2, 1 μ l 1: 2000 and reacted at room temperature.
Execution; Enzymatic digestion 10 minute makes the size of digested DNA fragment concentrated between 1kb-3kb, and then 2μl 0. 1m EDTA is added.
Terminating the reaction, combining four identical reaction systems, extracting once with equal volume of phenol, and extracting once with equal volume of phenol: chloroform;
Extract that mixed solution of isopropanol once, wherein the mix volume ratio of phenol: chloroform: isoamyl alcohol is 25: 24:1; Then use the same volume.
After extracting with ether once, DNA was precipitated with 2.5 times of anhydrous ethanol, washed with 70% ethanol, and finally resuspended in
18μl water; Subsequently, 2.5 μ l of DNTP was added to the mixture, which contained 1mMdCTP, 1mMdGTP and 1mMdTTP.
And 10mMdATP, 1.25 μ l 100 MMD DTT and 5 units of T4DNA polymerase, and the product was digested at 0℃ for 30 minutes.
After the reaction was terminated at 75℃, 5 units of Tth DNA polymerase and its buffer were added.
The system was amplified to 80μl, and the dA tail was added to the 3' end of DNA. Mix the mixture with chloroform
Isoamyl alcohol mixed solution extraction and ether extraction, and then connected with pGEM-T-Easy carrier at 65438 06℃ for 24 hours, with a total volume of
90μl, including 25 units of T4DNA ligase and 9μl of buffer containing 10 times T4DNA ligase, and finally110.
The ligation mixture was precipitated with 3M NaAc with a volume of pH = 5.2 and twice the volume of anhydrous ethanol, washed with 70% ethanol, and then dissolved in 30μl l.
The ligation product was obtained in water, and the competent large intestine rod was prepared by the method of preparing competent cells by electro-transformation of Bio-Rad company.
Strain DH5α cells, take 2-3μl of ligation product and 50μl of competent Escherichia coli DH5α, and then transfer to Bio-Rad.
The company's 0.2cm electric shock cup shocked, with a voltage of 2.5 kV and a time of 5.0 milliseconds to 6.0 milliseconds. After the electric shock,
Add 1ml SOC culture medium to the cup to revive the bacteria, and then coat the bacteria with ampicillin, X-Gal and IPTG.
Cultured overnight on LB solid medium at 37℃, blue and white colonies were obtained the next day. The white colony obtained by transformation is a white clone.
Cultured on LB solid medium containing ampicillin, at the same time, plasmids were extracted from each clone and digested with EcoRI.
The size of the inserted fragment was identified, and the obtained white clone group constituted the O- antigen gene of Shigella dysenteriae 12 type.
Cluster library;
4) obtaining the complete sequence of O- antigen gene cluster.
120 clones with insertion fragments above 700bp were screened from O- antigen gene bank produced by Shanghai Bioengineering Company.
The company used ABI377 DNA automatic sequencer to unidirectionally sequence the inserted fragments in the clone, making the sequencing reach 80%.
The remaining 20% sequence was designed according to the obtained sequence, and then primers were designed from the bases of Shigella dysenteriae 12 type.
All the sequences of O- antigen gene cluster were obtained by direct PCR in DNA and sequencing of PCR products, and all the sequences were obtained by using British sword.
Pregap4, a software package of Staden package published by Molecular Biology Laboratory of Bridge Medical Research Council.
All the sequences were spliced and edited by Gap4 software, and the core of Shigella dysenteriae 12 O- antigen gene cluster was obtained.
The quality of the full-length nucleotide sequence is mainly ensured by two aspects: (1) six long PCR for each genome.
Reacting, and then mixing the products to generate a library; (2) For each base, ensure the high-quality coverage of more than three bases;
5) obtaining the structure of the O- antigen gene cluster.
Discovery of Shigella dysenteriae by orffinder of national biotechnology information center
The gene was found in the nucleotide sequence of O- antigen gene cluster of 12 strain blast, and 10 open reading frames were found.
The software compares the genes in GenBank, finds the functions of these open reading frames and determines what genes they are, and then
Gene annotation was completed by Artemis software of sanger Center in the UK, and DNA and protein sequencing was completed by ClustTralw software.
Through accurate comparison between columns, the structure of Shigella dysenteriae 12 O- antigen gene cluster was finally obtained.