华根霉cDNA文库的构建及纤溶酶基因的筛选和功能鉴定
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摘要
血栓栓塞性疾病严重威胁人类生命和健康,溶栓治疗是血栓性疾病安全有效的治疗手段。目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵。因此研制高效、快速、副作用小,价廉的新型溶栓药物成为当前的迫切需求。
本试验构建了华根霉全长cDNA文库,并从中筛选出一种根霉纤溶酶基因；通过其在毕赤酵母中的表达,对该酶的酶学性质进行了初步的研究。
通过SMART (Switching Mechanism At 5'end of the RNA Transcript)技术构建了华根霉全长cDNA文库。大肠杆菌转化子平板检测和PCR鉴定表明,所构建的文库库容为1.6×109cfu／mL,重组率为91.6%,达到了用于目的基因的分离筛选及克隆表达的建库要求。
设计了四条简并引物并使用一种PCR方法从cDNA文库中分离出编码根霉纤溶酶的基因。该基因编码279个氨基酸,预测蛋白的分子量为30406 Da。根据基因序列比对得出此基因编码纤溶酶和其他纤溶酶类蛋白没有明显同源性。我们将此基因和pPIC9质粒相连接,构建了毕赤酵母表达载体,并通过在毕赤酵母中的表达对该纤溶酶功能进行了分析。实验结果表明重组工程菌分泌蛋白具有溶解血纤维蛋白的活性。
本文还对重组纤溶酶的酶学性质进行了研究。SDS-PAGE电泳测定纤溶酶分子量为30 KDa。预测该纤溶酶的等电点为10.23±0.2。重组纤溶酶比活力23.14U／mL,纤溶酶最适作用温度37℃,在4℃～42℃范围内酶活性受环境温度影响较小。讨论了酸碱对该酶活性的影响,得出该酶的最适反应pH 8.4,在pH 6.8～8.8该酶最稳定。该酶对酪蛋白具有水解作用,在和生色底物反应的实验中发现其能水解的底物为D-Ile-Phe-Lys-pNA和N-Sueeinyl-Ala-Ala-Pro-Phe-pNA。在金属离子对酶活的影响试验中得出Zn2+、Fe2+、Cu2+、Mn2+、Co2+离子对酶有不同程度的抑制作用,而且大多数表现为离子浓度越大抑制作用越强。Na+、K+、Ca2+、Mg2+等金属离子对该酶的激活作用并不显著。本实验研究与已报道的华根霉纤溶酶的性质有较大的区别,如等电点,最适温度,pH,底物专一性等方面。说明华根霉能够分泌多种不同性质的纤溶酶,本研究只克隆得到其中一种纤溶酶基因。
Thrombosis intimidates mankind's life and health seriously, fibrinolytic therapy was an effective and safety method to cure it. Currently, fibrinolytic enzymes available for clinical use are mostly plasminogen activators. Despite of their widespread applications, all these agents have undesired side effects and are very expensive. Therefore, searching for novel fibrinolytic enzymes from various sources.is emergency
In this research, a full-length cDNA library of Rhizopus chinensis has been constructed and a gene encoding fibrinolytic enzyme has been identified from Rhizopus chinensis cDNA library. Also, the characteristics of fibrinolytic enzyme expressed by recombinant Pichia pastor is were studied in this paper.
The full-length cDNA library of Rhizopus chinensis has been constructed by using SMART (Switching Mechanism At 5'end of RNA Transcript) technology. According to the results of phage plaques bright selection of host bacterial strain and PCR detection, the percentage of recombinant phages was 91.6% and the titer was 1.6×109 cfu/mL, which showed that the library was of high quality for cloning target genes and expressing target proteins.
Four degenerate primes were designated and a gene encoding hydrolysis fibrin activity protein from Rhizopus chinensis cDNA library was isolated by PCR. The coding region of 279 amino acids suggested a protein of 30 KDa. Sequences blasting, indicated that this protein did not shared homology to other proteins with hydrolysis fibrin activity secreted by animal and other microorganism. Expression vector pPIC9-rcfe was constructed and transformed into Pichia pastor is GS115. The function of rcfe was analyzed by the expression in Pichia pastoris. The result showed obviously that the protein secreted by transformed yeast cells had hydrolysis fibrin activity.
Further, the characteristics of the fibrinolytic enzyme from recombinant Pichia pastori were studied. The molecular mass was 30.4 KDa determined by SDS-PAGE. The isoelectric point was expected to be 10.23±0.2 as DNAMAN suggested. The enzyme had a specific activity of 23.14U/mL in the optimal temperature of 37℃, and its activity was stable in the temperature ranging from 4℃-42℃; and in the pH of 6.8-8.8; The optimal active temperature and pH was 37℃and 8.4 respectively. The enzyme showed hydrolysis activity against casein. Among the teste synthetic substrates, the enzyme can hydrolyze D-Ile-Phe-Lys-pNA and N-Sueeinyl-Ala-Ala-Pro-Phe-pNA. Zn2+, Fe2+, Fe3+, Cu2+, Mn2+ and Co2+ showed inhibition on the activity of the enzyme, and the inhibition activity was ever increasing as the concentration of these metal ion growing; on the other hand, K+, Na+, Ca2+, Mg2+had no obvious activation effect on the activity of the enzyme. Distinguish properties of fibrinolytic enzyme from Rhizopus chinensis were observed in our study compared with those in previously reported references. Rhizopus chinensis could secrete variety types of fibrinolytic enzyme; only one fibrinolytic enzyme gene has been cloned in this research.
本试验构建了华根霉全长cDNA文库,并从中筛选出一种根霉纤溶酶基因；通过其在毕赤酵母中的表达,对该酶的酶学性质进行了初步的研究。
通过SMART (Switching Mechanism At 5'end of the RNA Transcript)技术构建了华根霉全长cDNA文库。大肠杆菌转化子平板检测和PCR鉴定表明,所构建的文库库容为1.6×109cfu／mL,重组率为91.6%,达到了用于目的基因的分离筛选及克隆表达的建库要求。
设计了四条简并引物并使用一种PCR方法从cDNA文库中分离出编码根霉纤溶酶的基因。该基因编码279个氨基酸,预测蛋白的分子量为30406 Da。根据基因序列比对得出此基因编码纤溶酶和其他纤溶酶类蛋白没有明显同源性。我们将此基因和pPIC9质粒相连接,构建了毕赤酵母表达载体,并通过在毕赤酵母中的表达对该纤溶酶功能进行了分析。实验结果表明重组工程菌分泌蛋白具有溶解血纤维蛋白的活性。
本文还对重组纤溶酶的酶学性质进行了研究。SDS-PAGE电泳测定纤溶酶分子量为30 KDa。预测该纤溶酶的等电点为10.23±0.2。重组纤溶酶比活力23.14U／mL,纤溶酶最适作用温度37℃,在4℃～42℃范围内酶活性受环境温度影响较小。讨论了酸碱对该酶活性的影响,得出该酶的最适反应pH 8.4,在pH 6.8～8.8该酶最稳定。该酶对酪蛋白具有水解作用,在和生色底物反应的实验中发现其能水解的底物为D-Ile-Phe-Lys-pNA和N-Sueeinyl-Ala-Ala-Pro-Phe-pNA。在金属离子对酶活的影响试验中得出Zn2+、Fe2+、Cu2+、Mn2+、Co2+离子对酶有不同程度的抑制作用,而且大多数表现为离子浓度越大抑制作用越强。Na+、K+、Ca2+、Mg2+等金属离子对该酶的激活作用并不显著。本实验研究与已报道的华根霉纤溶酶的性质有较大的区别,如等电点,最适温度,pH,底物专一性等方面。说明华根霉能够分泌多种不同性质的纤溶酶,本研究只克隆得到其中一种纤溶酶基因。
Thrombosis intimidates mankind's life and health seriously, fibrinolytic therapy was an effective and safety method to cure it. Currently, fibrinolytic enzymes available for clinical use are mostly plasminogen activators. Despite of their widespread applications, all these agents have undesired side effects and are very expensive. Therefore, searching for novel fibrinolytic enzymes from various sources.is emergency
In this research, a full-length cDNA library of Rhizopus chinensis has been constructed and a gene encoding fibrinolytic enzyme has been identified from Rhizopus chinensis cDNA library. Also, the characteristics of fibrinolytic enzyme expressed by recombinant Pichia pastor is were studied in this paper.
The full-length cDNA library of Rhizopus chinensis has been constructed by using SMART (Switching Mechanism At 5'end of RNA Transcript) technology. According to the results of phage plaques bright selection of host bacterial strain and PCR detection, the percentage of recombinant phages was 91.6% and the titer was 1.6×109 cfu/mL, which showed that the library was of high quality for cloning target genes and expressing target proteins.
Four degenerate primes were designated and a gene encoding hydrolysis fibrin activity protein from Rhizopus chinensis cDNA library was isolated by PCR. The coding region of 279 amino acids suggested a protein of 30 KDa. Sequences blasting, indicated that this protein did not shared homology to other proteins with hydrolysis fibrin activity secreted by animal and other microorganism. Expression vector pPIC9-rcfe was constructed and transformed into Pichia pastor is GS115. The function of rcfe was analyzed by the expression in Pichia pastoris. The result showed obviously that the protein secreted by transformed yeast cells had hydrolysis fibrin activity.
Further, the characteristics of the fibrinolytic enzyme from recombinant Pichia pastori were studied. The molecular mass was 30.4 KDa determined by SDS-PAGE. The isoelectric point was expected to be 10.23±0.2 as DNAMAN suggested. The enzyme had a specific activity of 23.14U/mL in the optimal temperature of 37℃, and its activity was stable in the temperature ranging from 4℃-42℃; and in the pH of 6.8-8.8; The optimal active temperature and pH was 37℃and 8.4 respectively. The enzyme showed hydrolysis activity against casein. Among the teste synthetic substrates, the enzyme can hydrolyze D-Ile-Phe-Lys-pNA and N-Sueeinyl-Ala-Ala-Pro-Phe-pNA. Zn2+, Fe2+, Fe3+, Cu2+, Mn2+ and Co2+ showed inhibition on the activity of the enzyme, and the inhibition activity was ever increasing as the concentration of these metal ion growing; on the other hand, K+, Na+, Ca2+, Mg2+had no obvious activation effect on the activity of the enzyme. Distinguish properties of fibrinolytic enzyme from Rhizopus chinensis were observed in our study compared with those in previously reported references. Rhizopus chinensis could secrete variety types of fibrinolytic enzyme; only one fibrinolytic enzyme gene has been cloned in this research.
引文
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