Method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces

Method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces

  • CN 105,132,450 A
  • Filed: 09/09/2015
  • Published: 12/09/2015
  • Est. Priority Date: 09/09/2015
  • Status: Active Application
First Claim
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1. a method for bacillus subtilis spore capsid protein Cot surface display trehalose synthase, is characterized in that, comprise the steps:

  • (1) genomic dna of subtilis is extracted, then be template PCR amplifications bacillus subtilis spore capsid protein gene with genomic dna, in pcr amplification process, BamHI and XbaI enzyme cutting site is introduced respectively, obtained bacillus subtilis spore capsid protein gene Cot in upstream and downstream primer;

    Described bacillus subtilis spore capsid protein gene is bacillus subtilis spore capsid protein gene CotG, bacillus subtilis spore capsid protein gene CotB, bacillus subtilis spore capsid protein gene CotC, bacillus subtilis spore capsid protein gene CotD, bacillus subtilis spore capsid protein gene CotH, bacillus subtilis spore capsid protein gene CotX, bacillus subtilis spore capsid protein gene CotY or bacillus subtilis spore capsid protein gene CotZ,(2) with Pseudomonas putidas genome for template, through pcr amplification, the nucleotide sequence of obtained TreP TreS;

    (3) terminator of the bacillus subtilis spore capsid protein gene Cot that removal step (1) is obtained, then the TreS gene adopting fusion DNA vaccine and step (2) to obtain merges, in fusion DNA vaccine process, the restriction enzyme site of restriction enzyme BamHI and AatII is introduced respectively, obtained fusion gene Cot-TreS in upstream and downstream;

    (4) fusion gene Cot-TreS step (3) obtained, after BamHI and AatII double digestion, connects with the same expression vector pHT01 through BamHI and AatII double digestion, obtained recombinant plasmid pHT01-Cot-TreS;

    (5) by recombinant plasmid pHT01-Cot-TreS Transforming B. subtilis (Bacillussubtilis) 168 obtained for step (4), screen through the LB flat board of coating containing paraxin after conversion, then carry out transformant qualification through iodine liquid colour developing mode, obtain positive strain;

    (6) positive strain step (5) obtained, through DSM substratum 35 ~ 37 DEG C of inducing culture 45 ~ 50h, obtains the recombinant spore of surface display trehalose synthase.

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