纖溶酶基因在大腸桿菌和畢赤酵母中的表達
發(fā)布時間：2018-08-21 20:25
【摘要】：血栓性疾病已成為常見病和多發(fā)病,列致殘和致死病因的第一位,嚴重影響人類生存質(zhì)量和壽命,針對溶栓藥物的臨床應用要求,研發(fā)特異、高效、安全、價廉的纖溶酶類溶栓藥物具有廣闊應用前景。運用分子生物學技術(shù)和基因工程技術(shù),將現(xiàn)有溶栓藥的優(yōu)點集合起來,設計出完美的溶栓藥將是國內(nèi)外的研究熱點,同時,開發(fā)新型天然來源溶栓藥和探索新型結(jié)構(gòu)溶栓藥將是今后研究方向。 假蕈狀芽孢桿菌纖溶酶BpFE是土壤中分離得到的一種新型纖溶酶,基因全長1701 bp(GenBank FJ463037),其中954 bp為成熟肽基因,據(jù)報道基因全長編碼成熟肽之前的序列(基因長為747 bp)可能與調(diào)控有關,為了進一步探討之間的關系,即分析前端序列的作用及是否與活性有關,所以本工作進行了成熟肽基因的克隆與表達研究,并且為了將來的生產(chǎn)和研究摸索了其在畢赤酵母中的表達研究,主要研究內(nèi)容如下: 結(jié)合本實驗室對假蕈狀芽孢桿菌纖溶酶BpFE的報道,分析其基因序列,設計合成一對含EcoRI和XhoI酶切位點的引物,擴增得到纖溶酶成熟肽基因G,使用EcoRI和XhoI雙酶切表達載體pET-28a和纖溶酶成熟肽基因,通過連接構(gòu)建了重組質(zhì)粒pET-28a-G,利用熱激轉(zhuǎn)化法轉(zhuǎn)化宿主菌DH5α后測序。測序結(jié)果表明,插入序列長954bp,編碼317個氨基酸,與GenBank中BpFE成熟肽的954 bp序列一致。BpFE所包含的鋅蛋白酶家族結(jié)構(gòu)域(序列為VIGHELTHAV)沒有發(fā)生改變。 將重組質(zhì)粒pET-28a-G轉(zhuǎn)化大腸桿菌宿主菌BL21,成功獲得pET-28a-G/BL21工程菌。終濃度為1 mmol/L的IPTG誘導表達,經(jīng)SDS-PAGE檢測表達的融合蛋白表觀分子量約為40 kD,與理論值一致。誘導菌體超聲破壁后用纖維蛋白平板法檢測表達產(chǎn)物具有纖溶酶活性,并進一步鎳柱親和層析獲得純化,純化產(chǎn)物有纖溶酶活性。由此說明基因全長編碼成熟肽之前的序列在原基因組序列中起調(diào)控作用,對纖溶酶的活性無直接影響,因此成熟肽部分就表現(xiàn)有纖溶酶活性。 為利于外源基因的胞外表達便于工業(yè)生產(chǎn),將纖溶酶全長基因BpFE克隆到畢赤酵母中進行初步研究。根據(jù)所獲得的基因序列FJ463037和真核表達載體pPIC9K的多克隆位點重新設計含有EcoRI和Not I酶切位點的引物,構(gòu)建重組質(zhì)粒pPIC9K-G。將重組質(zhì)粒線性化后,電轉(zhuǎn)化畢赤酵母GS115中構(gòu)建重組菌株pPIC9K-G/GS115,經(jīng)MD、MM平板快慢型初篩和G418加壓篩選得到陽性轉(zhuǎn)化菌株,25℃甲醇誘導后,得到表觀分子量為64 kD的有纖溶活性的蛋白。鎳柱親和層析純化后用纖維蛋白平板法檢測到纖溶酶活性。 本文采用分子克隆及基因重組技術(shù),不但成功表達了纖溶酶BpFE成熟肽基因,探討了纖溶酶BpFE的全長基因和成熟肽基因之間的關系,而且纖溶酶BpFE全長基因在真核生物畢赤酵母中也得到了有效表達,為基因工程藥物的研究開發(fā)奠定必要的理論和實踐基礎。
[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 偽 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，
本文編號：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 偽 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，
本文編號：2196253
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