Colon, Identification And Analysis Of Efficient Promoters Which Can Transcript β-Galactosidase Gene Efficiently In Bacillus Licheniformis Bacteria

RNA and protein is the terminal expression produce of gene. Order to get mass of purified protein, expression vector which can express the exterior protein within high level should be constructed. Promoter is an important element of expression vector and one of factors influencing the mRNA synthesizing speed, so efficient promoter is necessary in expression vector expressing exterior protein with high level. In this research, we constructed the Bacillus Licheniformis partial genome library. With the method of gene-targeting, we separated a promoter using the shuttle as probe from the partial genome library. This promoter can transcript efficiently in bacillus expression system and can be used as an important element in constructing bacillus expression system. The main results of our research are bellow:(1) Screened and utilized a method with which clear supernatant sample after fermentation broth centrifuged in low temperature and protein procession buffer mixed with 1:1, then dealt with SDS-PAGE according to the relative regulation, we can obtain a satisfying electrophoretogram;(2) Screened a promoter having strong transcription ability. We constructed the Bacillus Licheniformis partial genome library and obtained a lots of efficient promoters using promoter probe with the gene-targeting method. Digested by restriction enzyme, we can know that these promoters have the correct vector fragment and insertion element. Named a promoter having the most efficient transcription activity in host Escherichia coli DH5αpB9, After the plasmid of promoter pB9 electrotransformation in bacillus, compared with pGlv promoter preserved by our laboratory induced by maltose, we can know that pB9 has strong transcription activity;(3) Strong promoter pB9 comes correctly from Bacillus Licheniformis. Sequenced the strong promoter pB9 nucleotide sequence and compared with Bacillus Licheniformis genome DNA sequence, we can know that strong promoter pB9 comes correctly from Bacillus Licheniformis LuxS gene;(4) Promoter pB9 has the classic structural feature of promoter in prokaryote and strong transcription activity. Determined enzyme activity curve of strong promoter pB9 within 48 hours, forecasted and analyzed the strong promoter nucleotide sequence with promoter forecasting software, we can know that pB9 has the classic structural feature of promoter in prokaryotel.