Cloning And Expression Of Bacillus Plasmin Gene

With the accelerated pace of human life,thrombosis was a common disease that en-dangers human life and health.The demand for thrombolytic drugs is gradually increasing.researchers have studied various aspects of microbial source plasmin.Such as optimizing various enzyme-producing conditions,genetic engineering method to transform the plasmin gene,and fibrinolytic strains were induced by some physical and chemical ways,etc.This study is mainly to screen and identify fibrinolytic enzyme-producing strains and to enable the expression of plasmin gene in E.coli expression system through genetic engineering.The research has achieved the following results:1 The 13 strains isolated from the samples of daqu,fermented grains and pit mud were used as starting strains by the Microbiology Laboratory of Guizhou University.These13 strains were able to produce Maotai-flavor and form spores.13 strains were cultivated by skim milk powder plates to obtain 6 strains that with strong protease-producing ability;The fibrinolytic enzyme strain GZHZ_V-8-8 was screened from 6 strains by fibrin double-layer plate,this strain has a plasmin activity of 2344.23 IU/mL.2 According to the third edition of"Microbiology Experiment"（Shen Ping et al.,1999）,the"BERGEY’S MANUAL^?OF Systematic Bacteriology"（David R.Boone et al.,2001）,the ninth edition and the"Handbook of Common Bacterial Identification"（Dong Xiu-zhu,2001）.The GZHZ_V-8-8 was identified by morphological characteristics and physiological and biochemical identification,and molecular biological identification method,GZHZV-8was identified as Bacillus amyloliquefaciens;3 In this research,genome DNA of Bacillus amyloliquefaciens GZHZ_V-8-8 was extracted,usd as the PCR template,the DNA segments of 1205bp were cloned using PCR,the ORF region contains 1089 bp,encoding 363 amino acid residues;the online cloning sequence analysis indicated that the cloned plasmin gene and the AprE gene from B.amyloliquefa-ciens（NCBI accession number GU825966.1）had a similarity of 99.65%at the nucleic a-cid level and a similarity at the amino acid level of 100%.After the relevant bioinforma-tics online website predicts and analyzes,the molecular weight of the protein was 36.99kDa.The isoelectric point（PI）was 8.73,a stable and hydrophilic protein with no signifi-cant transmembrane region,a signal peptide,predicted to be a secreted protein,containing two conserved domains.The prediction of protein structure showed that the random coil ratio was 36.64%,theα-helix ratio was 28.37%,the extended chain ratio was 23.69%,and theβ-turn angle ratio was 11.29%.The three-dimensional structure model has a simi-larity to the template 3 whi.1.A of 86.36%,and has a binding region of DNA.4 The restriction endonuclease was used to digest the target DNA and the expression empty plasmid,and ligated by T4 DNA Ligase enzyme,and transferred into E.coli BL21（DE3）competent cells.The recombinant strain was induced to grow with IPTG at a con-centration of 1 mmol/L for 48 hours,The plasmin activity of the supernatant of the fer-mentation supernatant,the cell supernatant and the cell pellet was 613.26 IU/mL.407.38IU/mL,993.12 IU/mL.The expression strain was expressed at a higher level after 7 hours of IPTG induction at 1 mmol/L.and fermentation supernatant had a thrombolytic effect.