Burkholderia pyrrole endoglucanase and its recombinant expression method and application
A technology of Burkholderia pyrrole and endoglucanase, applied in the field of genetic engineering, can solve the problem of less research on bacterial sources and the like
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Embodiment 1
[0042] Example 1 Analysis of the BPEG01790 gene and 3D model of BPEG01790
[0043] A gene encoding the EG precursor was identified in the genomic sequence of B. Pyrrocinia B1213 by similarity analysis (BPEG01790). The open reading frame (ORF) of the BPEG01790 gene of BPEG01790 gene of BPEG01790 gene is 1218 bp (G + C content of 50%), encoding 406 amino acid (AA) residues, to assume the inner-cut glucanase, signal peptide is 40 amino acids The residue, wherein the full-length gene sequence encoding the signal peptide such as SEQ ID NO.1, the nucleotide sequence encoding the signal peptide coding sequence is shown in SEQ ID NO.3. The molecular weight and theoretical pi of BPEG01790 is 43.0kda and 9.50, 9 potential N-glycosylation sites, no potential O-glycosylation site ( figure 1 . Comparison with the amino acid sequence of the protein database indicates that the BPEG01790 has a similarity of 90% of the enzyme belonging to GH 8. BLAST analysis showed that BPEG01790 and the sequence...
Embodiment 2
[0045] Embodiment 2 of the cloning and recombinant carrier of the BPEG01790 gene
[0046] According to the manufacturer's instruction, the genomic DNA was separated from B1213 using a bacterial genome DNA purification kit from B1213. According to the EG gene sequence predicted in B.Pyrocinia B1213 (see SEG01790-PMD-18T-N and BPEG01790-PMD-18T-C and BPEG01790-PMD-18T-C and BPEG01790-PMD-18T-C primers, the entire BPEG01790 gene. use The high-grade DNA polymerase was carried out as the reaction conditions for PCR: 98 ° C for 2 min; 35 circulation, from 98 ° C 20 S, 67.5 ° C 20 S, 72 ° C 60 S, and extends at 72 ° C for 5 min, respectively. The amplified fragment was cloned into the plasmid PMD18-T and transformed into E. coli DH5α. The follow-up experiment was performed after the DNA sequencing was confirmed.
[0047] Table 1 primers used in experiments
[0048]
[0049] BPEG01790-PMD-18T-N and BPEG01790-PMD-18T-C were used to amplify the entire BPEG01790; BPEG01790-PET28A (+) - F ...
Embodiment 3
[0050] Example III, Expression of BPEG01790 and PET28A (+)
[0051] The PET plasmid is the preferred expression vector of the laboratory in the laboratory in the laboratory. Therefore, the gene is subcloned to PET28A (+) according to the description of the PLUS PCR kit. Complete the primers in Table 1, such as Figure 4 As shown, BPEG01790-PET28A (+) is generated, first express BPEG01790 using a PET28A (+) vector. Through the following primers:
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[0053]
[0054] The DNA fragment encoding the assigned BPEG01790 was amplified from B.Pyrociniausing PCR, and the PCR product was cloned into the expression vector PET28A (+) to give the expression vector BPEG01790-PET28a (+). The strain PCR and sequencing results show that BPEG01790 has been successfully attached to the PET28a (+) vector, and the constructed plasmid will be converted to E. coli BL21 (DE3) using conventional heating. The positive colonies were performed using 50 μg / ml kanamycin and the positive transform...
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