Nucleotide specific against 0-antigen of colibacillus 0012, 2 type Sh. Dysenterae and 15 type bao sh.bacillus
The technology of Shigella dysenteriae and Shigella baumannii is applied in the field of nucleotides specific to the O-antigen of Shigella baumannii type 15, Shigella dysenteriae type 2 and Escherichia coli O112 , which can solve problems such as indistinguishable
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Embodiment 1：：5mLLB37℃，。500ul 50mM Tris-HCl(pH8.0) and 10ul 0.4M EDTA，37℃20，10ul 10mg/ml20。3ul 20mg/mlK、15ul 10％SDS，50℃2，3ul 10mg/mlRNase，65℃30。，∶∶，。2DNA，DNA70％DNA，DNA30ul TE。DNA0.4％ test 。 Embodiment 2
[0044] Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (NewYork: Cold Spring Harbor Laboratory Press, 1989) are generally followed. Example 1: Genome extraction: Shigella was cultured overnight at 37° C. in 5 mL of LB medium, and the cells were collected by centrifugation. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / ml RNase, 65°C fo...
Embodiment 3
[0045] Embodiment 3: construct O-antigen gene cluster library:
[0046] The first is the acquisition of the ligation product: use the modified Novagen DNaseI shot gun method to construct the O-antigen gene cluster library. The reaction system is 300ng PCR purified product, 0.9ul 0.1M MnCl 2 , 1 ul of 1 mg / ml DNaseI diluted 1:2000, and the reaction was carried out at room temperature. Digest for 10 minutes to concentrate the DNA fragment size between 1kb-3kb, then add 2ul 0.1M EDTA to terminate the reaction. Combine 4 tubes of the same reaction system, extract once with an equal volume of phenol and phenol:chloroform:isoamyl alcohol, and then extract twice with an equal volume of ether, then precipitate DNA with 2.5 times the volume of absolute ethanol, and use The precipitate was washed with 70% ethanol, and finally resuspended in 18ul water. Then add 2.5ul dNTP (1mMdCTP, 1mMdGTP, 1mMdTTP, 10mMdATP), 1.25ul 100mM DTT and 5 units of T4 DNA polymerase to this mixture, 11°C fo...
Embodiment 4
[0049] Example 4: Sequencing the clones in the library:
[0050] From the library, 100 clones with inserts above 700 bp were selected, and the inserts in the clones were sequenced unidirectionally by Shanghai Bioengineering Co., Ltd. with an ABI377 DNA automatic sequencer, so that the sequence coverage reached 90%. For the remaining 10% of the sequence, primers were designed according to the obtained sequence, and then direct PCR was performed from the genomic DNA of Shigella baumannii type 15, and the PCR product was sequenced, so as to obtain all sequences of the O-antigen gene cluster. In Shigella baumannii type 15 we designed a pair of primers as follows: 5'-GGAGCGATCGTCCGGTCAC-3' and 5'-CAGCGCAGCTATGTGTCC-3'.
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