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Cloning And Heterologous Expression Of Lumbrokinase Gene

Posted on:2018-12-02Degree:MasterType:Thesis
Country:ChinaCandidate:L F LiFull Text:PDF
GTID:2310330512989696Subject:Microbiology
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Lumbrukinase?LK?is a kind of protein with fibrinolytic activity isolated from earthworm crude extract.It not only activates plasminogen into plasmin,stimulates the release of tissue plasminogen activator?t-PA?,then indirectly promotes the dissolution of fibrin,and can directly hydrolyze fibrin.In addition,lumbrokinase has a good thrombolytic effect,which can improve microcirculation,increase vascular elasticity,reduce blood viscosity,inhibit the formation of thrombosis,thus is widely used in clinical containg cardiovascular,cerebrovascular,endocrine,respiratory systems and other diseases in the prevention and treatment.In this paper,we mainly studied the expression of EFLK in Escherichia coli and Pichia pastoris,and optimized the fermentation conditions of P.pastoris recombinant.Besides,we purified and studied the enzymatic properties of lumbrokinase.The main works are as follows:?1?Genome DNA was extracted from wild earthworm.Through molecular biology methods,we identified the species of samples and constructed the phylogenetic trees.The results showed that this wild earthworm belonged to Eisenia foetida.?2?The total RNA was extracted from the wild-type Eisenia foetida,and then the single-stranded cDNA was reversed from mRNA by the kit,which was utilized as a template to clone the lumbrokinase gene Eflk?GenBank:KY452025?.Bioinformatics software analysis results showed that the full-length of Eflk was 738 bp,which encoded 245 amino acids that contain a signal peptide with 7 amino acids.The similarity of lumbrokinase EFLK combined with F-III-2?GenBank:AB045719?was 96.21%.The results of DS2.5 software indicated that the active center of EFLK is Arg15-Phe19-Pro20-Glu66-Asn113.?3?The recombinant plasmid pET-28a?+?-Eflk was constructed and conversed into E.coli BL21?DE3?.EFLK was successfully expressed in E.coli.The sorbitol,sucrose,glycine and ethanol were added to the medium to increase the activity of the enzyme by up to 15.6 U/mL.?4?Recombinant plasmid pPIC9K-Eflk was constructed and linearized by Sac I.Through conversion by electric,the EFLK was successfully expressed in P.pastoris GS115.The fibrinolytic activity was detected in the fermentation supernatant,of which the highest activity was 224.8 U/mL peaked at 72 h.?5?The fermentation conditions of P.pastoris was optimized by single factor and orthogonal experiment methods.Four key factors were determined by the single factor experiment:initial pH,induction temperature,inoculum amount and additional methanol concentration.Orthogonal experiment was designed with 4-factors and 3-levels in this paper to obtain the best fermentation conditions.The results showed that the optimum fermentation conditions were as follows:the initial pH was 5.5,the induction temperature was 30 ?,the inoculation amount was OD600= 2.0 and the concentration of methanol was 1%.The optimized fermentation activity was 259.4 U/mL peaked at 72 h.?6?EFLK was purified by Ni+ column with a specific activity on fibrin?4545.84 U/mg?.Studies of enzymatic properties show that the optimum pH of EFLK was 8.0.The EFLK activity was losed off 20%by incubating at 37 ? for 12 h?pH 7.4?.The residual vitality was 67.33%after 48 hours.In addition,the EFLK was stable at low temperatures?below 40 ??.On the other hand,the EFLK still maintained a high vitality while its activity was losed off 41.63%by incubating for 48 h at pH 8.0.
Keywords/Search Tags:Eisenia foetida, Lumbrokinase, Escherichia coli, Pichia pastoris, Enzymatic properties
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