内生多粘芽孢杆菌纤溶酶的纯化、性质及其基因的克隆与表达
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摘要
血栓栓塞性疾病严重危害人类健康和生命,溶栓疗法是治疗血栓栓塞性疾病最为有效并且可靠的手段。目前临床应用的溶栓药物疗效肯定,明显降低了患者的死亡率和致残率,但还存在特异性不高、半衰期短、易出血及再栓塞等缺点。因此迫切需要研制开发高效、特异、安全、副作用小、价廉的新型溶栓药物。已有研究表明,许多中药具有抗血小板聚集、抗血栓形成的作用,包括生物碱类、黄酮类以及皂甙等化合物,尤其是活血化瘀中草药具有不同程度的抗凝血、血小板聚集及溶解血栓的作用。生活在植物组织内的内生菌,由于内生菌与宿主植物长期协同进化过程中,彼此构成了稳定的生态关系,使内生菌具有产生某些与植物相同或相似化合物的能力。鉴于此,从中药植物内生菌资源中可寻找和发现一些新型的溶栓药物。
本研究首次在内生细菌中筛选分离到一株具有纤溶活性较高和良好体外溶栓效果的EJS-3菌株,对其进行了形态特征、生理生化特征和分子生物学鉴定,并对内生多粘芽孢杆菌纤溶酶的分离纯化及其性质进行深入的研究。同时,采用基因工程技术将内生多粘芽孢杆菌纤溶酶基因克隆到表达载体上,实现内生多粘芽孢杆菌纤溶酶异源表达,并对表达产物的提纯和重组酶的酶学性质作了初步探讨,为研制和开发新型的溶栓药物提供重要理论依据。主要研究结果分述如下：
1.采用酪蛋白平板和纤维蛋白平板初筛、摇瓶复筛的筛选策略,从金银花、黄芩、蒲公英、黄连、连翘、爬山虎、鱼腥草、百部等植物的根、茎、叶分离筛选到7株具有纤溶活性的内生菌。其中从百部中分离到的内生菌株EJS-3酶活最高,达110 IU/mL。通过对其形态特征、生理生化特征进行鉴定,初步判断该菌株为多粘芽孢杆菌(Paenibacillus polymyxa)。对菌株EJS-3的16S rDNA进行PCR扩增,对得到的1500bpPCR产物纯化和序列测定,利用GenBank数据库进行同源搜索,并构建N-J系统发育树,结果表明：菌株EJS-3的16S rDNA序列(DQ120522)与多粘芽孢杆菌(Paenibacillus polymyxa)的16S rDNA序列的同源性均达98%以上,亲缘关系最近。结合常规的形态特征、生理生化特征鉴定,表明该菌株在细菌系统分类学上属于多粘芽孢杆菌(Paenibacillus polymyxa)。目前,国内外内生多粘芽孢杆菌产纤溶酶的研究尚未见报道。
2.内生菌株EJS-3发酵液经离心除菌,硫酸铵分级沉淀,Hiprep phenyl FF疏水层析,RESOURCEM Q离子交换层析和Sephacryl S-300HR凝胶过滤,获得电泳纯的多粘芽孢杆菌纤溶酶(PPFE-Ⅰ), HPLC为单一峰,纯度为94.1%。每升发酵液中可获得1.6 mg活性蛋白,每毫克蛋白活力达2096 IU,纯度提高了14.5,回收率为3.3%。
3. SDS-PAGE测定PPFE-Ⅰ的相对分子质量为63 KDa, MALDI-TOF质谱法准确测定其相对分子质量为63.3 KDa。该酶最适反应温度37℃,最适反应pH7.5,具有较好的热稳定性和pH稳定性。金属离子Cu2+和Ca2+能抑制其纤溶活性, Mg2+, Fe2+和Zn2+对该酶存在明显的激活作用。1 mmol/L PMSF完全抑制其纤溶活性,EDTA能部分抑制纤溶活性,这表明PPFE-Ⅰ属于丝氨酸蛋白酶,此酶还可能是一种金属蛋白酶。
4.利用几种人工合成的底物测定水解酰胺键活性的高低。结果表明,PPFE-Ⅰ的最适底物是枯草杆菌蛋白酶或胰凝乳蛋白酶的最适底物,即N-Succinyl-Ala-Ala-Pro-Phe -pNA;水解生色底物N-Succinyl-Ala-Ala-Pro-Phe-pNA的米氏常数Km为0.20 mM,表明PPFE-Ⅰ对该底物有较好的亲和性；PPFE-Ⅰ在普通纤维蛋白平板和加热平板上测定的纤溶活性没有显著的差别,这表明PPFE-Ⅰ对纤维蛋白具有直接的降解作用,不具有激活纤溶酶原形成纤溶酶的活性,因而PPFE-Ⅰ是一种纤溶酶,而不是纤溶酶原激活剂；通过PPFE-Ⅰ对人血纤维蛋白原的体外降解过程的研究,结果表明纤溶酶PPFE-Ⅰ最先降解α链,其次是β链,而对γ链降解最缓慢,1-4 h逐渐降解至完全；通过体外血凝块的溶解作用实验,结果表明当PPFE-Ⅰ酶溶液对血凝块作用24h时,其对体外血凝决的溶解率91.33%,表明PPFE-Ⅰ有明显的体外溶栓作用,直接溶解作用的结果,同时具有显著的抗凝活性,并且抗凝血效果优于尿激酶。
5.为了实现内生多粘芽孢杆菌纤溶酶的异源高效表达,以内生菌株EJS-3基因组DNA为模板,运用PCR扩增PPFE-Ⅰ基因,并克隆到pMD-19T载体上,构建克隆载体pMD-PPFE-Ⅰ,经测序正确后,将PPFE-Ⅰ克隆至表达载体pET-DsbA上,成功构建了pET-DsbA/PPFE-Ⅰ重组表达质粒,将其转化E. coli BL21(DE3)中,在IPTG诱导下实现了融合蛋白DsbA-PPFE-Ⅰ的表达,表达产物酶活性达228 IU/mL.表达产物用SDS-PAGE和Western blot进行鉴定。SDS-PAGE电泳检测融合蛋白主要以可溶形式表达,占菌体总蛋白的18.4%。Western blot结果表明在相应分子量处有一条特异性条带,证实该蛋白为DsbA-PPFE-Ⅰ融合蛋白。
6.表达产物通过Ni亲和柱、凝血酶酶切及Sephadex G-100等步骤进行分离纯化,并用MALDI-TOF质谱对重组酶进行了鉴定。纯化后的表达产物在纤维蛋白平板上表现出明显的纤溶活性。对纯化的重组纤溶酶进行最适温度及其稳定性、最适pH及其稳定性以及蛋白酶抑制剂、金属离子的影响等性质的研究,结果表明,重组酶与天然纤溶酶是一致的。
7.为了进一步探讨内生多粘芽孢杆菌纤溶酶基因异源表达特性,从内生菌株EJS-3总DNA中PCR扩增出PPFE-Ⅰ基因,将PPFE-Ⅰ基因克隆到本实验室构建大肠杆菌-枯草杆菌穿梭质粒pHPQ中,并转化枯草杆菌WB800中进行了分泌表达,在含氯霉素10μg/mL.红霉素10μg/mL的LB液体培养基中培养32h,纤溶酶活性为60 IU/mL.
Thromboenbolism disease is a serious threat to our health and life. Thrombolytic therapy, which can significantly decrease the patients mortality and mutilation rate, has been extensively investigated as a means of medical treatment. However, it is difficult to widely used in clinical application for the reason of low specificity, short half-life, hemorrhagic risk, and higher rate of thrombus reforming. So, the research and developments of the novel thrombolytic drugs with high efficiency, high specificity, high safety, little side effect, low cost are needed urgently to solve problems described above. Many traditional Chinese medicines, especially those herbs with activiting blood circulation, can inhibit blood platelet aggregation and thrombosis, including some alkaloids such as alkaloids, flavonoids and saponin. Endophytes are widely existing inside the tissues of healthy plants. Co-evolving with medicinal plants, some endophytes have developed the abilities to produce same or similar bioactive substances as what their host plants do. Therefore, it is a huge potential to screen novel, highly active fibrinolytic enzyme from endophytes.
In this study, strain EJS-3 with strongly fibrinolytic activity and thrombolytic effect test in vitro was screened from endophytes and later identified by morphological, physiological, biochemical tests and 16S rRNA gene sequence analysis. The fibrinolytic enzyme was purified and characterized. The gene encoding the fibrinolytic enzyme was cloned, sequenced, and overexpressed. Then, the recombinant enzyme was purified and characteried. All these results provided certain theoretical basis for developing and designing new thrombolytic drugs. Main study results as following:
1. Using improved method,7 strains with fibrinolytic activity were screened from the root, stem and leave of Chinese medicinal plants such as Japanese Honeysuckle, Skullcap, Mongol dandelion, Coptis chinensis Franch, Weeping Forsythia, Japan creeper, Houttuynia, and Japan Stemona. The screening strategies included the first selection with the casein plate and the further with shaking culture. Among them, the strain EJS-3 from the root of Japan Stemona had the highest fibrinolytic activity and later was preliminary identified as Paenibacillus polymyxa by morphological, physiological, and biochemical tests. The 16S rDNA gene of EJS-3 was amplified by polymerase chain reaction (PCR) from the genomic DNA. The PCR product (1.5 kb) was purified and sequenced. A similarity search was performed using Genbank databases and the neighbor-joining phylogenetic tree was constructed. The results showed that a typical characteristion of the Paenibacillus polymyxa 16S rDNA sequences (GenBank accession number DQ120522) comparison study supported a strong relationship between strain EJS-3 and members of genus Paenibacillus and particularly revealed the highest homology (98%) with Paenibacillus polymyxa. The result further indicated that strain EJS-3 belonged to Paenibacillus polymyxa. No work on Paenibacillus polymyxa producing fibrinolytic enzyme has been reported so far.
2. The purification of fibrinolytic enzyme from the endophytic strain EJS-3 was processed as the following procedure:centrifugal sterilization, fractional sedimentation with ammonium sulfate, Hiprep phenyl FF chromatography, RESOURCEM Q ion-exchange chromatography, Sephacryl S-300HR gel-filtration. The purified fibrinolytic enzyme PPFE-I was homogenous in SDS-PAGE electrophoresis, and a single peak in HPLC. Its purity was more than 94.1% estimated by HPLC. Compared with the crude enzyme extraction, the specific activity of the purified enzyme increased by 14.5 folds with a recovery of 3.3%, and 1.6mg of active proteins with an activity of 2096 IU·mg-1 could be obtained from 1L fermentation broth.
3. Estimation of the molecular weight of PPFE-Ⅰby SDS-PAGE and MALDI-TOF gave the values of 63 kDa and 63.3KDa respectively. The optimum pH and temperature of PPFE-Ⅰwere pH7.5 and37℃, respectively. The PPFE-Ⅰexcreted by isolate EJS-3 showed a greater degree of pH and thermal stability. The fibrinolytic enzyme was inhibited by Cu2+ and Ca2+, while enhanced by Mg2+, Fe2+, and Zn2+. The PPFE-Ⅰwas inhibited completely by PMSF, partly inhibited by EDTA, indicating that the PPFE-Ⅰwas a serine metalloprotease.
4. The proteolytic activity of PPFE-Ⅰwas identified and measured using synthetic substrates with arginyl bonds. The rusult showed that the optimum substrate of PPFE-Ⅰwas Suc-Ala-Ala-Pro-Phe-pNA, which was the same as that of subtilisin or chymotrypsin. The Michaelis constant Km value of PPFE-Ⅰusing N-Succinyl-Ala-Ala-Pro-Phe-pNA as substrate was 0.20mM, which showed that the enzyme had a great affinity with the substrate. Fibrin clots could be effectively degraded by PPFE-Ⅰvia direct fibrinolysis. The PPFE-Ⅰwas a kind of fibrinolytic enzyme but not plasminogen activator. Theα-subunits of fibrinogen were firstly cleaved, followed by theβ-chains, while theγ-chains were resistant to the enzyme digestion. Throniholysis was evaluated by constant temperature natural lysis in vitro. The dissolving rate of blood clots was 91.33% after incubation 24h showed that the PPFE-Ⅰhad significantly thrombolysis effect in vitro. The anticoagulant activity of the PPFE-Ⅰwas more significant than that of urokinase.
5. The mature PPFE-Ⅰgene was amplified from purified strain EJ-3 genomic DNA by PCR. The PCR product was isolated and ligated into the pMD-19T vector and sequenced. The vector constructed above was pMD-PPFE-Ⅰ. The PPFE-Ⅰgene was cloned into pET-DsbA and integrated into E. coli BL21(DE3). Active recombinant fibrinolytic enzyme had been successfully expressed in Escherichia coli as protein fusions with DsbA by addition of IPTG. The fibrinolytic activity of recombinant enzyme was 228 IU·mL-1. The expression of the recombinant enzyme was cinfirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed the recombinant enzyme was soluble and was about 18.4% of total cell protein. The recombinant enzyme exhibited a special band on Western blotting, which showed that the recombinant protein was DsbA-PPFE-Ⅰ.
6. The recombinant enzyme was purified from the supernatant by Ni affinity column, thrombin digestion, and sephadex G-100 gel-filtration. Its fibrinolysis activity was detected by fibrin plate method. The characterization of the purified recombinant enzyme such as optimal temperature, thermal stability, optimal pH, pH stability, substrate specificity, protease inhibitors, and metal ions were determined. There were no different characterization between the natural enzyme and recombinant enzyme.
7. The PPFE-Ⅰgene was amplified by PCR and cloned to the Escherichia coli-Bacillus subtilis shuttle expression vector pHPQ constructed in our laboratory, and then integrated into Bacillus subtilis WB800. The fibrinolytic enzyme of recombinant enzyme whose activity in LB media containing 10μg·mL-1 chloramphenicol and 10μg·mL-1 erythromycin was 60 IU·mL-1 at 32h incubation time.
本研究首次在内生细菌中筛选分离到一株具有纤溶活性较高和良好体外溶栓效果的EJS-3菌株,对其进行了形态特征、生理生化特征和分子生物学鉴定,并对内生多粘芽孢杆菌纤溶酶的分离纯化及其性质进行深入的研究。同时,采用基因工程技术将内生多粘芽孢杆菌纤溶酶基因克隆到表达载体上,实现内生多粘芽孢杆菌纤溶酶异源表达,并对表达产物的提纯和重组酶的酶学性质作了初步探讨,为研制和开发新型的溶栓药物提供重要理论依据。主要研究结果分述如下：
1.采用酪蛋白平板和纤维蛋白平板初筛、摇瓶复筛的筛选策略,从金银花、黄芩、蒲公英、黄连、连翘、爬山虎、鱼腥草、百部等植物的根、茎、叶分离筛选到7株具有纤溶活性的内生菌。其中从百部中分离到的内生菌株EJS-3酶活最高,达110 IU/mL。通过对其形态特征、生理生化特征进行鉴定,初步判断该菌株为多粘芽孢杆菌(Paenibacillus polymyxa)。对菌株EJS-3的16S rDNA进行PCR扩增,对得到的1500bpPCR产物纯化和序列测定,利用GenBank数据库进行同源搜索,并构建N-J系统发育树,结果表明：菌株EJS-3的16S rDNA序列(DQ120522)与多粘芽孢杆菌(Paenibacillus polymyxa)的16S rDNA序列的同源性均达98%以上,亲缘关系最近。结合常规的形态特征、生理生化特征鉴定,表明该菌株在细菌系统分类学上属于多粘芽孢杆菌(Paenibacillus polymyxa)。目前,国内外内生多粘芽孢杆菌产纤溶酶的研究尚未见报道。
2.内生菌株EJS-3发酵液经离心除菌,硫酸铵分级沉淀,Hiprep phenyl FF疏水层析,RESOURCEM Q离子交换层析和Sephacryl S-300HR凝胶过滤,获得电泳纯的多粘芽孢杆菌纤溶酶(PPFE-Ⅰ), HPLC为单一峰,纯度为94.1%。每升发酵液中可获得1.6 mg活性蛋白,每毫克蛋白活力达2096 IU,纯度提高了14.5,回收率为3.3%。
3. SDS-PAGE测定PPFE-Ⅰ的相对分子质量为63 KDa, MALDI-TOF质谱法准确测定其相对分子质量为63.3 KDa。该酶最适反应温度37℃,最适反应pH7.5,具有较好的热稳定性和pH稳定性。金属离子Cu2+和Ca2+能抑制其纤溶活性, Mg2+, Fe2+和Zn2+对该酶存在明显的激活作用。1 mmol/L PMSF完全抑制其纤溶活性,EDTA能部分抑制纤溶活性,这表明PPFE-Ⅰ属于丝氨酸蛋白酶,此酶还可能是一种金属蛋白酶。
4.利用几种人工合成的底物测定水解酰胺键活性的高低。结果表明,PPFE-Ⅰ的最适底物是枯草杆菌蛋白酶或胰凝乳蛋白酶的最适底物,即N-Succinyl-Ala-Ala-Pro-Phe -pNA;水解生色底物N-Succinyl-Ala-Ala-Pro-Phe-pNA的米氏常数Km为0.20 mM,表明PPFE-Ⅰ对该底物有较好的亲和性；PPFE-Ⅰ在普通纤维蛋白平板和加热平板上测定的纤溶活性没有显著的差别,这表明PPFE-Ⅰ对纤维蛋白具有直接的降解作用,不具有激活纤溶酶原形成纤溶酶的活性,因而PPFE-Ⅰ是一种纤溶酶,而不是纤溶酶原激活剂；通过PPFE-Ⅰ对人血纤维蛋白原的体外降解过程的研究,结果表明纤溶酶PPFE-Ⅰ最先降解α链,其次是β链,而对γ链降解最缓慢,1-4 h逐渐降解至完全；通过体外血凝块的溶解作用实验,结果表明当PPFE-Ⅰ酶溶液对血凝块作用24h时,其对体外血凝决的溶解率91.33%,表明PPFE-Ⅰ有明显的体外溶栓作用,直接溶解作用的结果,同时具有显著的抗凝活性,并且抗凝血效果优于尿激酶。
5.为了实现内生多粘芽孢杆菌纤溶酶的异源高效表达,以内生菌株EJS-3基因组DNA为模板,运用PCR扩增PPFE-Ⅰ基因,并克隆到pMD-19T载体上,构建克隆载体pMD-PPFE-Ⅰ,经测序正确后,将PPFE-Ⅰ克隆至表达载体pET-DsbA上,成功构建了pET-DsbA/PPFE-Ⅰ重组表达质粒,将其转化E. coli BL21(DE3)中,在IPTG诱导下实现了融合蛋白DsbA-PPFE-Ⅰ的表达,表达产物酶活性达228 IU/mL.表达产物用SDS-PAGE和Western blot进行鉴定。SDS-PAGE电泳检测融合蛋白主要以可溶形式表达,占菌体总蛋白的18.4%。Western blot结果表明在相应分子量处有一条特异性条带,证实该蛋白为DsbA-PPFE-Ⅰ融合蛋白。
6.表达产物通过Ni亲和柱、凝血酶酶切及Sephadex G-100等步骤进行分离纯化,并用MALDI-TOF质谱对重组酶进行了鉴定。纯化后的表达产物在纤维蛋白平板上表现出明显的纤溶活性。对纯化的重组纤溶酶进行最适温度及其稳定性、最适pH及其稳定性以及蛋白酶抑制剂、金属离子的影响等性质的研究,结果表明,重组酶与天然纤溶酶是一致的。
7.为了进一步探讨内生多粘芽孢杆菌纤溶酶基因异源表达特性,从内生菌株EJS-3总DNA中PCR扩增出PPFE-Ⅰ基因,将PPFE-Ⅰ基因克隆到本实验室构建大肠杆菌-枯草杆菌穿梭质粒pHPQ中,并转化枯草杆菌WB800中进行了分泌表达,在含氯霉素10μg/mL.红霉素10μg/mL的LB液体培养基中培养32h,纤溶酶活性为60 IU/mL.
Thromboenbolism disease is a serious threat to our health and life. Thrombolytic therapy, which can significantly decrease the patients mortality and mutilation rate, has been extensively investigated as a means of medical treatment. However, it is difficult to widely used in clinical application for the reason of low specificity, short half-life, hemorrhagic risk, and higher rate of thrombus reforming. So, the research and developments of the novel thrombolytic drugs with high efficiency, high specificity, high safety, little side effect, low cost are needed urgently to solve problems described above. Many traditional Chinese medicines, especially those herbs with activiting blood circulation, can inhibit blood platelet aggregation and thrombosis, including some alkaloids such as alkaloids, flavonoids and saponin. Endophytes are widely existing inside the tissues of healthy plants. Co-evolving with medicinal plants, some endophytes have developed the abilities to produce same or similar bioactive substances as what their host plants do. Therefore, it is a huge potential to screen novel, highly active fibrinolytic enzyme from endophytes.
In this study, strain EJS-3 with strongly fibrinolytic activity and thrombolytic effect test in vitro was screened from endophytes and later identified by morphological, physiological, biochemical tests and 16S rRNA gene sequence analysis. The fibrinolytic enzyme was purified and characterized. The gene encoding the fibrinolytic enzyme was cloned, sequenced, and overexpressed. Then, the recombinant enzyme was purified and characteried. All these results provided certain theoretical basis for developing and designing new thrombolytic drugs. Main study results as following:
1. Using improved method,7 strains with fibrinolytic activity were screened from the root, stem and leave of Chinese medicinal plants such as Japanese Honeysuckle, Skullcap, Mongol dandelion, Coptis chinensis Franch, Weeping Forsythia, Japan creeper, Houttuynia, and Japan Stemona. The screening strategies included the first selection with the casein plate and the further with shaking culture. Among them, the strain EJS-3 from the root of Japan Stemona had the highest fibrinolytic activity and later was preliminary identified as Paenibacillus polymyxa by morphological, physiological, and biochemical tests. The 16S rDNA gene of EJS-3 was amplified by polymerase chain reaction (PCR) from the genomic DNA. The PCR product (1.5 kb) was purified and sequenced. A similarity search was performed using Genbank databases and the neighbor-joining phylogenetic tree was constructed. The results showed that a typical characteristion of the Paenibacillus polymyxa 16S rDNA sequences (GenBank accession number DQ120522) comparison study supported a strong relationship between strain EJS-3 and members of genus Paenibacillus and particularly revealed the highest homology (98%) with Paenibacillus polymyxa. The result further indicated that strain EJS-3 belonged to Paenibacillus polymyxa. No work on Paenibacillus polymyxa producing fibrinolytic enzyme has been reported so far.
2. The purification of fibrinolytic enzyme from the endophytic strain EJS-3 was processed as the following procedure:centrifugal sterilization, fractional sedimentation with ammonium sulfate, Hiprep phenyl FF chromatography, RESOURCEM Q ion-exchange chromatography, Sephacryl S-300HR gel-filtration. The purified fibrinolytic enzyme PPFE-I was homogenous in SDS-PAGE electrophoresis, and a single peak in HPLC. Its purity was more than 94.1% estimated by HPLC. Compared with the crude enzyme extraction, the specific activity of the purified enzyme increased by 14.5 folds with a recovery of 3.3%, and 1.6mg of active proteins with an activity of 2096 IU·mg-1 could be obtained from 1L fermentation broth.
3. Estimation of the molecular weight of PPFE-Ⅰby SDS-PAGE and MALDI-TOF gave the values of 63 kDa and 63.3KDa respectively. The optimum pH and temperature of PPFE-Ⅰwere pH7.5 and37℃, respectively. The PPFE-Ⅰexcreted by isolate EJS-3 showed a greater degree of pH and thermal stability. The fibrinolytic enzyme was inhibited by Cu2+ and Ca2+, while enhanced by Mg2+, Fe2+, and Zn2+. The PPFE-Ⅰwas inhibited completely by PMSF, partly inhibited by EDTA, indicating that the PPFE-Ⅰwas a serine metalloprotease.
4. The proteolytic activity of PPFE-Ⅰwas identified and measured using synthetic substrates with arginyl bonds. The rusult showed that the optimum substrate of PPFE-Ⅰwas Suc-Ala-Ala-Pro-Phe-pNA, which was the same as that of subtilisin or chymotrypsin. The Michaelis constant Km value of PPFE-Ⅰusing N-Succinyl-Ala-Ala-Pro-Phe-pNA as substrate was 0.20mM, which showed that the enzyme had a great affinity with the substrate. Fibrin clots could be effectively degraded by PPFE-Ⅰvia direct fibrinolysis. The PPFE-Ⅰwas a kind of fibrinolytic enzyme but not plasminogen activator. Theα-subunits of fibrinogen were firstly cleaved, followed by theβ-chains, while theγ-chains were resistant to the enzyme digestion. Throniholysis was evaluated by constant temperature natural lysis in vitro. The dissolving rate of blood clots was 91.33% after incubation 24h showed that the PPFE-Ⅰhad significantly thrombolysis effect in vitro. The anticoagulant activity of the PPFE-Ⅰwas more significant than that of urokinase.
5. The mature PPFE-Ⅰgene was amplified from purified strain EJ-3 genomic DNA by PCR. The PCR product was isolated and ligated into the pMD-19T vector and sequenced. The vector constructed above was pMD-PPFE-Ⅰ. The PPFE-Ⅰgene was cloned into pET-DsbA and integrated into E. coli BL21(DE3). Active recombinant fibrinolytic enzyme had been successfully expressed in Escherichia coli as protein fusions with DsbA by addition of IPTG. The fibrinolytic activity of recombinant enzyme was 228 IU·mL-1. The expression of the recombinant enzyme was cinfirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed the recombinant enzyme was soluble and was about 18.4% of total cell protein. The recombinant enzyme exhibited a special band on Western blotting, which showed that the recombinant protein was DsbA-PPFE-Ⅰ.
6. The recombinant enzyme was purified from the supernatant by Ni affinity column, thrombin digestion, and sephadex G-100 gel-filtration. Its fibrinolysis activity was detected by fibrin plate method. The characterization of the purified recombinant enzyme such as optimal temperature, thermal stability, optimal pH, pH stability, substrate specificity, protease inhibitors, and metal ions were determined. There were no different characterization between the natural enzyme and recombinant enzyme.
7. The PPFE-Ⅰgene was amplified by PCR and cloned to the Escherichia coli-Bacillus subtilis shuttle expression vector pHPQ constructed in our laboratory, and then integrated into Bacillus subtilis WB800. The fibrinolytic enzyme of recombinant enzyme whose activity in LB media containing 10μg·mL-1 chloramphenicol and 10μg·mL-1 erythromycin was 60 IU·mL-1 at 32h incubation time.
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