Chimeras of brucella lumazine synthase and beta subunit of ab5 toxins
一种二氧四氢蝶啶、布鲁氏菌的技术，应用在寡聚蛋白复合体领域，能够解决Stx2B差免疫原等问题
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Embodiment 1
[0094] Molecular modeling of the chimeric protein BLS-Stx2
[0095] The chimera was designed by modeling its theoretical structure with the program PyMOL1.5 (www.pymol.org), combining the C-terminus of the Stx2B (PDB code: IR4P) structure with the BLS (PDB code: ID10) The N-terminus of the crystal structure was fused, a strategy similar to that previously described by Laplagne et al. (Laplagne et al., 2004. Proteins 57:820-828). The coding sequence of the first 8 residues at the end of BLSN was replaced with the coding sequence of Stx2B and the flexible G / S pentapeptide peptide linker, decapeptide peptide linker or 15 amino acid peptide linker connecting the two proteins. Both Stx2B and BLS form the same C5 symmetric pentameric structure, where each protomer interacts with the other two. The theoretical structure of the chimera obtained by molecular modeling ( figure 1 ) demonstrated that all linkers used were long enough to allow assembly of the Stx2B pentamer onto the pent...
Embodiment 2
[0097] The pET11a-BLS-Stx2B-L5 (joint with 5 amino acids) plasmid containing the gene with SEQ.ID.No.4 was constructed by the following scheme:
[0098] a) In order to clone the coding gene of 2,4-dioxotetrahydropteridine synthase (BLS) of Brucella (Brucellaspp.), utilize specific primer to amplify from Brucella abortus (B. abortus) genomic DNA to obtain the BLS sequence, and cloned into the pET11a vector (Novagen, USA). To develop the expression cassette, directed mutagenesis was performed on the sequence encoding the open reading frame of Brucella 2,4-dioxopteridine synthase (BLS) (Laplagne et al., 2004). Insert two new restriction sites in the 5' region of the BLS gene: an NsiI site in the first two codons at the 5' end; and a residue at position 8 and 9 containing the native amino acid sequence of BLS An AflII site in the two codons.
[0099] b) In order to insert the encoded sequence into the Stx2B protein (SEQ.ID.No.1), we designed an oligonucleotide to amplify the Stx...
Embodiment 3
[0109] The pCI-neo-BLS-Stx2B-L5 plasmid was constructed by the following protocol:
[0110] The gene sequence Seq. ID. No. 4 of BLS-Stx2B (L5) cloned in the pET11a vector was subcloned into the vector pCi-neo (Promega, USA). Therefore, the following oligonucleotides were prepared to be used as primers in PCR: ACCATG
[0111] BLS-DNA-chimera all (Seq.ID.No.12): 5'GTTTAAGAATTCGAAGGAGAT ACCACCATG CAT3′
[0112] BLS-Back (Seq.ID.No.13): 3'CGTAGCGCGAACAGACTCGATCGTACTGACCACCTGT5'
[0113] Primer BLS-DNA-chimera all added restriction site for EcoRI enzyme (bold) and Kozak consensus sequence (underlined); BLS-Back primer added restriction site for NheI enzyme (bold) .
[0114] Such sequences were amplified by PCR using the pET11a-BLS-Stx2B(L5) plasmid as a template. PCR reactions were performed as described in Example 2C. The cycling pattern was: 94°C for 5 minutes, 1 cycle; 94°C for 1 minute, 55°C for 1 minute and 74°C for 1 minute, 40 cycles; and 74°C for 8 minutes, 1 final c...
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