纤溶酶基因在大肠杆菌和毕赤酵母中的表达
发布时间：2018-08-21 20:25
【摘要】：血栓性疾病已成为常见病和多发病,列致残和致死病因的第一位,严重影响人类生存质量和寿命,针对溶栓药物的临床应用要求,研发特异、高效、安全、价廉的纤溶酶类溶栓药物具有广阔应用前景。运用分子生物学技术和基因工程技术,将现有溶栓药的优点集合起来,设计出完美的溶栓药将是国内外的研究热点,同时,开发新型天然来源溶栓药和探索新型结构溶栓药将是今后研究方向。 假蕈状芽孢杆菌纤溶酶BpFE是土壤中分离得到的一种新型纤溶酶,基因全长1701 bp(GenBank FJ463037),其中954 bp为成熟肽基因,据报道基因全长编码成熟肽之前的序列(基因长为747 bp)可能与调控有关,为了进一步探讨之间的关系,即分析前端序列的作用及是否与活性有关,所以本工作进行了成熟肽基因的克隆与表达研究,并且为了将来的生产和研究摸索了其在毕赤酵母中的表达研究,主要研究内容如下: 结合本实验室对假蕈状芽孢杆菌纤溶酶BpFE的报道,分析其基因序列,设计合成一对含EcoRI和XhoI酶切位点的引物,扩增得到纤溶酶成熟肽基因G,使用EcoRI和XhoI双酶切表达载体pET-28a和纤溶酶成熟肽基因,通过连接构建了重组质粒pET-28a-G,利用热激转化法转化宿主菌DH5α后测序。测序结果表明,插入序列长954bp,编码317个氨基酸,与GenBank中BpFE成熟肽的954 bp序列一致。BpFE所包含的锌蛋白酶家族结构域(序列为VIGHELTHAV)没有发生改变。 将重组质粒pET-28a-G转化大肠杆菌宿主菌BL21,成功获得pET-28a-G/BL21工程菌。终浓度为1 mmol/L的IPTG诱导表达,经SDS-PAGE检测表达的融合蛋白表观分子量约为40 kD,与理论值一致。诱导菌体超声破壁后用纤维蛋白平板法检测表达产物具有纤溶酶活性,并进一步镍柱亲和层析获得纯化,纯化产物有纤溶酶活性。由此说明基因全长编码成熟肽之前的序列在原基因组序列中起调控作用,对纤溶酶的活性无直接影响,因此成熟肽部分就表现有纤溶酶活性。 为利于外源基因的胞外表达便于工业生产,将纤溶酶全长基因BpFE克隆到毕赤酵母中进行初步研究。根据所获得的基因序列FJ463037和真核表达载体pPIC9K的多克隆位点重新设计含有EcoRI和Not I酶切位点的引物,构建重组质粒pPIC9K-G。将重组质粒线性化后,电转化毕赤酵母GS115中构建重组菌株pPIC9K-G/GS115,经MD、MM平板快慢型初筛和G418加压筛选得到阳性转化菌株,25℃甲醇诱导后,得到表观分子量为64 kD的有纤溶活性的蛋白。镍柱亲和层析纯化后用纤维蛋白平板法检测到纤溶酶活性。 本文采用分子克隆及基因重组技术,不但成功表达了纤溶酶BpFE成熟肽基因,探讨了纤溶酶BpFE的全长基因和成熟肽基因之间的关系,而且纤溶酶BpFE全长基因在真核生物毕赤酵母中也得到了有效表达,为基因工程药物的研究开发奠定必要的理论和实践基础。
[Abstract]:Thrombotic disease has become a common disease and frequent disease, the first cause of disability and death, seriously affecting the quality of life and life span of human beings, to the clinical application of thrombolytic drugs, the development of specific, efficient, safe, Cheap fibrinolytic enzyme thrombolytic drugs have broad application prospects. Combining the advantages of the existing thrombolytic agents with molecular biological and genetic engineering techniques, it will be a hot research topic at home and abroad to design perfect thrombolytic agents. Developing new natural thrombolytic agents and exploring new structural thrombolytic agents will be the research direction in the future. The plasminogen BpFE of Bacillus fungoides is a new type of plasminase isolated from soil. The gene length is 1701 bp (GenBank FJ463037), of which 954bp is a mature peptide gene. It has been reported that the sequence prior to the full-length encoding of the mature peptide (747 BP in length) may be related to regulation. In order to further explore the relationship between the sequence, that is, to analyze the role of the front-end sequence and whether it is related to activity, Therefore, the cloning and expression of mature peptide gene were studied, and the expression of mature peptide gene in Pichia pastoris was explored for the future production and research. The main contents are as follows: according to the report of our laboratory on the plasminogen BpFE of Bacillus fungoides, the gene sequence was analyzed and a pair of primers containing EcoRI and XhoI restriction sites were designed and synthesized. The plasminase mature peptide gene was amplified. The expression vector pET-28a and plasminogen mature peptide gene were digested by EcoRI and XhoI. The recombinant plasmid pET-28a-Gwas constructed by ligation. The recombinant plasmid pET-28a-Gwas transformed into host strain DH5 伪 by heat shock transformation and sequenced. The sequencing results showed that the inserted sequence was 954 BP, encoding 317 amino acids, which was consistent with the 954 BP sequence of BpFE mature peptide in GenBank. The zinc protease family domain (sequence of VIGHELTHAV) contained by BpFE did not change. The recombinant plasmid pET-28a-G was transformed into Escherichia coli host strain BL21, and pET-28a-G/BL21 engineering strain was successfully obtained. When the final concentration of IPTG was 1 mmol/L, the apparent molecular weight of the fusion protein detected by SDS-PAGE was about 40 KD, which was consistent with the theoretical value. Fibrinolytic enzyme activity was detected by fibrin plate method and purified by nickel column affinity chromatography. The purified product had plasminogen activity. It is concluded that the sequence before the full-length encoding mature peptide plays a regulatory role in the primordial genome sequence, and has no direct effect on the activity of plasminogen, so the mature peptide has plasminolytic activity. In order to facilitate the extracellular expression of exogenous genes for industrial production, the full-length plasminase gene BpFE was cloned into Pichia pastoris for preliminary study. According to the obtained gene sequence FJ463037 and the polyclonal site of eukaryotic expression vector pPIC9K, the primers containing EcoRI and Not I restriction sites were redesigned to construct the recombinant plasmid pPIC9K-G. The recombinant plasmid was linearized and electrotransformed into Pichia pastoris GS115 to construct the recombinant strain pPIC9K-G / GS115.After screening with MD-MM plate and G418, the positive transformed strain was induced by methanol at 25 鈩，
本文编号：2196253
[Abstract]:Thrombotic disease has become a common disease and frequent disease, the first cause of disability and death, seriously affecting the quality of life and life span of human beings, to the clinical application of thrombolytic drugs, the development of specific, efficient, safe, Cheap fibrinolytic enzyme thrombolytic drugs have broad application prospects. Combining the advantages of the existing thrombolytic agents with molecular biological and genetic engineering techniques, it will be a hot research topic at home and abroad to design perfect thrombolytic agents. Developing new natural thrombolytic agents and exploring new structural thrombolytic agents will be the research direction in the future. The plasminogen BpFE of Bacillus fungoides is a new type of plasminase isolated from soil. The gene length is 1701 bp (GenBank FJ463037), of which 954bp is a mature peptide gene. It has been reported that the sequence prior to the full-length encoding of the mature peptide (747 BP in length) may be related to regulation. In order to further explore the relationship between the sequence, that is, to analyze the role of the front-end sequence and whether it is related to activity, Therefore, the cloning and expression of mature peptide gene were studied, and the expression of mature peptide gene in Pichia pastoris was explored for the future production and research. The main contents are as follows: according to the report of our laboratory on the plasminogen BpFE of Bacillus fungoides, the gene sequence was analyzed and a pair of primers containing EcoRI and XhoI restriction sites were designed and synthesized. The plasminase mature peptide gene was amplified. The expression vector pET-28a and plasminogen mature peptide gene were digested by EcoRI and XhoI. The recombinant plasmid pET-28a-Gwas constructed by ligation. The recombinant plasmid pET-28a-Gwas transformed into host strain DH5 伪 by heat shock transformation and sequenced. The sequencing results showed that the inserted sequence was 954 BP, encoding 317 amino acids, which was consistent with the 954 BP sequence of BpFE mature peptide in GenBank. The zinc protease family domain (sequence of VIGHELTHAV) contained by BpFE did not change. The recombinant plasmid pET-28a-G was transformed into Escherichia coli host strain BL21, and pET-28a-G/BL21 engineering strain was successfully obtained. When the final concentration of IPTG was 1 mmol/L, the apparent molecular weight of the fusion protein detected by SDS-PAGE was about 40 KD, which was consistent with the theoretical value. Fibrinolytic enzyme activity was detected by fibrin plate method and purified by nickel column affinity chromatography. The purified product had plasminogen activity. It is concluded that the sequence before the full-length encoding mature peptide plays a regulatory role in the primordial genome sequence, and has no direct effect on the activity of plasminogen, so the mature peptide has plasminolytic activity. In order to facilitate the extracellular expression of exogenous genes for industrial production, the full-length plasminase gene BpFE was cloned into Pichia pastoris for preliminary study. According to the obtained gene sequence FJ463037 and the polyclonal site of eukaryotic expression vector pPIC9K, the primers containing EcoRI and Not I restriction sites were redesigned to construct the recombinant plasmid pPIC9K-G. The recombinant plasmid was linearized and electrotransformed into Pichia pastoris GS115 to construct the recombinant strain pPIC9K-G / GS115.After screening with MD-MM plate and G418, the positive transformed strain was induced by methanol at 25 鈩，
本文编号：2196253
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