A kind of recombinant bacillus subtilis and its construction method and application
A technology of Bacillus subtilis and mature peptides, which is applied in the field of recombinant Bacillus subtilis and its construction, can solve problems such as cumbersome methods, and achieve the effects of simplifying steps, saving costs, and having a large inhibition zone
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Embodiment 1
[0025] Embodiment 1: Construction of recombinant shuttle plasmid pHT43-papA1
[0026] 1. Plant lactobacillus mature peptide gene fragment papA1 Synthesis.
[0027] In order to facilitate purification, a His tag and a thrombin cleavage site were added before the gene encoding the mature peptide fragment of plant lactobacillus papA, and the DNA sequence is shown in SEQ ID NO. 1. In order to enhance expression in Bacillus subtilis, the sequence is base-optimized according to the codon usage frequency of Bacillus subtilis, and the optimized sequence is SEQ ID NO. 3 ( papA1 ), and at the 5´ and 3´ ends respectively add Bam HI and Xba Ⅰ Restriction site. The sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
[0028] 2, papA1 Synthesis of fragments and digestion of plasmid pHT43.
[0029] restriction endonuclease Bam HI and Xba Ⅰ (Dalian TaKaRa Company) for Escherichia coli-Bacillus subtilis shuttle expression vector pHT43 and synthetic papA1 The ...
Embodiment 2
[0032] Example 2: Recombinant Bacillus subtilis containing recombinant shuttle plasmid pHT43-papA1 Bacillus subtilis Construction of DBN-SKL-PA1
[0033] Use the plasmid extraction kit (Dalian TaKaRa) to extract the plasmid from the Escherichia coli Tans10 positive transformant containing the recombinant shuttle expression plasmid pHT43-papA1 constructed in Example 1; use the Spizizen transformation method to transform pHT43-papA1 into Bacillus subtilis WB800N strain , screened on a 2YT plate containing 5 μg / mL chloramphenicol, and obtained the Bacillus subtilis containing the plasmid pHT43-papA1 after colony PCR verification, named as Bacillus subtilis DBN-SKL-PA1, whose taxonomic designation is Bacillus subtilis Bacillus subtilis . The strain was deposited in China General Microorganism Culture Collection Center (CGMCC) on August 2, 2021. The preservation date is August 2, 2021. The preservation number is: CGMCCNO: 23101, and the preservation address is Beichen West R...
Embodiment 3
[0034] Example 3: Induced expression and protein purification of Bacillus subtilis DBN-SKL-PA1
[0035] 1. Preparation of seed suspension:
[0036] 2YT liquid medium: yeast powder 10.0 g / L, tryptone 16.0 g / L, sodium chloride 5.0 g / L, pH7.0-7.2, filled with 50 mL / 250 mL Erlenmeyer flask, sterilized at 121 ℃ for 20 min.
[0037] 2YT solid medium: yeast powder 5.0g / L, tryptone 10.0g / L, sodium chloride 10.0g / L, agar 20.0g / L, solvent is deionized water, pH 7.0~7.2 , filled with 100mL / 250mL Erlenmeyer flask, sterilized at 121°C for 20min.
[0038] Streak the Bacillus subtilis DBN-SKL-PA1 on the 2YT solid medium containing 5 μg / mL chloramphenicol for activation, and culture it upside down at 37°C for 24 h; pick a single colony and inoculate it on the 2YT solid medium containing 5 μg / mL chloramphenicol Cultivate overnight at 37°C, 200rpm in liquid medium;
[0039] 2. Induced expression of recombinant PA1 protein
[0040] The DBN-SKL-PA1 seed solution was inoculated into 2YT liquid...
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