CN116042664A - Preparation method and application of acyl-CoA oxidase - Google Patents

Preparation method and application of acyl-CoA oxidase Download PDF

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CN116042664A
CN116042664A CN202211704989.5A CN202211704989A CN116042664A CN 116042664 A CN116042664 A CN 116042664A CN 202211704989 A CN202211704989 A CN 202211704989A CN 116042664 A CN116042664 A CN 116042664A
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coa oxidase
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王佳
张伟
蒋析文
吴宇鹏
刘文丽
罗小丽
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Abstract

The application discloses a preparation method and application of acyl-CoA oxidase. The invention screens out bacterial sources capable of expressing high-enzyme activity soluble acyl-CoA oxidase from a large number of bacterial sources containing acyl-CoA oxidase by genetic engineering technology, and provides a corresponding method for industrially producing the acyl-CoA oxidase.

Description

酰基辅酶A氧化酶的制备方法和应用Preparation method and application of acyl-CoA oxidase

技术领域technical field

本发明涉及生物医药领域,特别涉及酰基辅酶A氧化酶的制备方法和应用。The invention relates to the field of biomedicine, in particular to a preparation method and application of acyl-CoA oxidase.

背景技术Background technique

酰基辅酶A氧化酶(Acyl-CoA oxidase,ACX/ACO)广泛存在于多种生物体中,位于过氧化物酶体,参与过氧化物酶体中各类脂肪酸β氧化的第一步反应,该酶也是脂肪酸活化代谢的限速酶。ACX基因家族共有6个同工酶,根据催化反应中识别碳链长短不同可分为三类。分别是:识别长链脂肪酸的Long-chain ACX(LACX)、识别中链脂肪酸的Medium-chainACX(MACX)和识别短链脂肪酸的Short-chain ACX(SACX)。在体外诊断领域,酰基辅酶A氧化酶催化酰基辅酶A的氧化反应,可用于血液中游离脂肪酸浓度的检测。Acyl-CoA oxidase (Acyl-CoA oxidase, ACX/ACO) widely exists in a variety of organisms, is located in peroxisomes, and participates in the first step of the β-oxidation of various fatty acids in peroxisomes. The enzyme is also the rate-limiting enzyme in fatty acid activation metabolism. There are 6 isoenzymes in the ACX gene family, which can be divided into three types according to the length of the carbon chain recognized in the catalytic reaction. They are: Long-chain ACX (LACX) that recognizes long-chain fatty acids, Medium-chain ACX (MACX) that recognizes medium-chain fatty acids, and Short-chain ACX (SACX) that recognizes short-chain fatty acids. In the field of in vitro diagnosis, acyl-CoA oxidase catalyzes the oxidation reaction of acyl-CoA and can be used to detect the concentration of free fatty acids in blood.

酰基辅酶A氧化酶可以从天然产物中制备,比如植物、微生物,这类制备方法多采用Tris-HCl缓冲液、磷酸钾缓冲液萃取法,为了提高纯度,经常多种纯化工艺联用,比如:丙酮提取,热处理,硫酸铵沉淀和苯基琼脂糖层析,羟基磷灰石分子筛柱。这些步骤复杂,需要多种物质材料,产率较低,很难应用于工业上大规模生产。如文献《热带假丝酵母酰基辅酶A氧化酶的纯化及性质研究》中公开了从热带假丝酵母菌中提取目的蛋白并进行纯化的方法,该方法产率低,且产物比活仅有21.8U/mg。基于基因工程制备重组酰基辅酶A氧化酶适宜用于工业制备,但受限于产物大多是包涵体表达且酶活差等因素,仍然难以实现大规模工业制备。因此,本领域尚需开发一种适宜大规模工业生产的酰基辅酶A氧化酶制备方法。Acyl-CoA oxidase can be prepared from natural products, such as plants and microorganisms. Such preparation methods mostly use Tris-HCl buffer and potassium phosphate buffer extraction methods. In order to improve the purity, multiple purification processes are often combined, such as: Acetone extraction, heat treatment, ammonium sulfate precipitation and phenyl sepharose chromatography, hydroxyapatite molecular sieve column. These steps are complex, require a variety of materials, and the yield is low, so it is difficult to apply to large-scale industrial production. For example, the document "Purification and Properties of Candida tropicalis Acyl-CoA Oxidase" discloses a method for extracting and purifying a target protein from Candida tropicalis. The yield of this method is low, and the specific activity of the product is only 21.8 U/mg. The preparation of recombinant acyl-CoA oxidase based on genetic engineering is suitable for industrial production, but it is still difficult to achieve large-scale industrial production due to factors such as the expression of inclusion bodies and poor enzyme activity. Therefore, there is still a need in the art to develop a method for preparing acyl-CoA oxidase suitable for large-scale industrial production.

发明内容Contents of the invention

本发明的目的在于提供一种酰基辅酶A氧化酶的制备方法。The purpose of the present invention is to provide a preparation method of acyl-CoA oxidase.

本发明的另一目的在于提供编码酰基辅酶A氧化酶的多核苷酸序列。Another object of the present invention is to provide a polynucleotide sequence encoding acyl-CoA oxidase.

本发明的另一目的在于提供与编码酰基辅酶A氧化酶的多核苷酸序列适配的载体。Another object of the present invention is to provide a vector compatible with the polynucleotide sequence encoding acyl-CoA oxidase.

本发明的另一目的在于提供含有编码酰基辅酶A氧化酶的多核苷酸序列的试剂盒。Another object of the present invention is to provide a kit comprising a polynucleotide sequence encoding acyl-CoA oxidase.

为解决上述技术问题,本发明第一方面,提供了一种编码酰基辅酶A氧化酶的多核苷酸,所述多核苷酸经密码子优化,且所述多核苷酸选自以下任一种:In order to solve the above technical problems, the first aspect of the present invention provides a polynucleotide encoding acyl-CoA oxidase, the polynucleotide is codon-optimized, and the polynucleotide is selected from any of the following:

(i)如SEQ ID NO.1所示序列的多核苷酸;(i) the polynucleotide of sequence as shown in SEQ ID NO.1;

(ii)与如SEQ ID NO.1所示序列的同源性大于95%的多核苷酸;和(ii) polynucleotides with greater than 95% homology to the sequence shown in SEQ ID NO.1; and

(iii)与(i)或(ii)中所述的多核苷酸序列互补的多核苷酸。(iii) A polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

本发明第二方面,提供了一种表达载体,所述表达载体包括本发明第一方面提供的多核苷酸。The second aspect of the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

在一些优选的方案中,所述表达载体为大肠杆菌表达载体,更优选为pET-28a(+)。In some preferred schemes, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

本发明第三方面,提供了一种宿主细胞,所述宿主细胞包括本发明第二方面提供的表达载体;或者In the third aspect of the present invention, a host cell is provided, and the host cell includes the expression vector provided in the second aspect of the present invention; or

所述宿主细胞的基因组中整合有如本发明第一方面提供的多核苷酸。The polynucleotide provided in the first aspect of the present invention is integrated in the genome of the host cell.

在一些优选的方案中,所述宿主细胞为大肠杆菌(Escherichia coli)。In some preferred embodiments, the host cell is Escherichia coli (Escherichia coli).

在一些优选的方案中,所述宿主细胞为大肠杆菌BL21(DE3)菌株。In some preferred schemes, the host cell is Escherichia coli BL21 (DE3) strain.

本发明第四方面提供了一种制备酰基辅酶A氧化酶的方法,所述方法包括步骤:培养本发明第三方面所述的宿主细胞,以表达出目的蛋白;和The fourth aspect of the present invention provides a method for preparing acyl-CoA oxidase, the method comprising the steps of: cultivating the host cell described in the third aspect of the present invention to express the target protein; and

分离所述目的蛋白,即得所述酰基辅酶A氧化酶;isolating the target protein to obtain the acyl-CoA oxidase;

在一些优选的方案中,所述宿主细胞通过含有本发明第一方面所述多核苷酸的质粒转化大肠杆菌获得。In some preferred schemes, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide according to the first aspect of the present invention.

在一些优选的方案中,使用SB、TB、LB、SOC培养基培养所述的宿主细胞,更优选为使用TB培养基培养所述的宿主细胞。In some preferred schemes, the host cells are cultured using SB, TB, LB, SOC medium, more preferably TB medium.

在一些优选的方案中,在振荡的环境中培养所述的宿主细胞。In some preferred embodiments, the host cells are cultured in a shaking environment.

在一些优选的方案中,在温度为16至19℃下或35至39℃下培养所述宿主细胞,更优选为在温度为16至19℃下培养所述宿主细胞。In some preferred schemes, the host cells are cultured at a temperature of 16 to 19°C or 35 to 39°C, more preferably the host cells are cultured at a temperature of 16 to 19°C.

在一些优选的方案中,培养所述的宿主细胞时,所用培养基中含有卡那霉素抗性基因。In some preferred schemes, when culturing the host cells, the culture medium used contains a kanamycin resistance gene.

在一些优选的方案中,培养所述的宿主细胞时,使用IPTG进行诱导,以表达出目的蛋白。In some preferred schemes, when the host cells are cultured, IPTG is used to induce the expression of the target protein.

在一些优选的方案中,培养所述的宿主细胞时,培养至OD600在0.6至0.8,后使用IPTG进行诱导,以表达出目的蛋白。In some preferred schemes, when the host cells are cultured, they are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

在一些优选的方案中,所述分离所述目的蛋白的步骤包括:In some preferred schemes, the step of isolating the protein of interest comprises:

将经破碎的目的蛋白上清液与流动相同时通过层析柱进行洗脱,收集洗脱液。The crushed target protein supernatant is eluted through the chromatographic column when the flow is the same, and the eluate is collected.

在一些优选的方案中,所述层析柱为Ni-柱亲和层析柱(Ni-NTA)。In some preferred schemes, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

本发明第五方面提供了一种试剂盒,所述试剂盒包括:如本发明第一方面提供的的多核苷酸;或者The fifth aspect of the present invention provides a kit, said kit comprising: the polynucleotide as provided in the first aspect of the present invention; or

如本发明第二方面提供的表达载体;或者The expression vector as provided in the second aspect of the present invention; or

如本发明第三方面所述的宿主细胞;或者The host cell as described in the third aspect of the present invention; or

或者根据本发明第四方面所述的方法制备的酰基辅酶A氧化酶。Or the acyl-CoA oxidase prepared according to the method described in the fourth aspect of the present invention.

本发明相对于现有技术而言,至少具有下述优点:Compared with the prior art, the present invention has at least the following advantages:

(1)本发明通过基因工程技术从大量含酰基辅酶A氧化酶的菌种源中筛选出可表达高酶活可溶性酰基辅酶A氧化酶的菌源,并提供了相应的工业化生产酰基辅酶A氧化酶的方法,该方法纯化步骤简单、产出的酶活性高、蛋白表达量高且生产成本低;(1) The present invention selects a bacterial source capable of expressing high-enzyme activity soluble acyl-CoA oxidase from a large number of strain sources containing acyl-CoA oxidase through genetic engineering technology, and provides a corresponding industrial production of acyl-CoA oxidase Enzyme method, which has simple purification steps, high enzyme activity, high protein expression and low production cost;

(2)本发明优选的实施方式中,通过同义密码子偏好性优化筛选获得了可溶性表达量高的优化密码子,进一步提高了产量和生产效率。(2) In a preferred embodiment of the present invention, optimized codons with high soluble expression levels are obtained through synonymous codon preference optimization screening, further improving yield and production efficiency.

应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

附图说明Description of drawings

一个或多个实施例通过与之对应的附图中的图片进行示例性说明,这些示例性说明并不构成对实施例的限定。One or more embodiments are exemplified by pictures in the accompanying drawings, and these exemplifications are not intended to limit the embodiments.

图1是根据本发明实施例中巴纳氏杆菌来源的酰基辅酶A氧化酶在LB培养基中表达产物SDS-PAGE鉴定图(重组质粒中含有优化密码子Ⅰ);1 is an SDS-PAGE identification diagram of the expression product of acyl-CoA oxidase derived from Barnerella in LB medium according to an embodiment of the present invention (the recombinant plasmid contains optimized codon I);

图2是根据本发明实施例中巴纳氏杆菌来源的酰基辅酶A氧化酶在TB培养基中表达产物SDS-PAGE鉴定图(重组质粒中含有优化密码子Ⅰ);Figure 2 is an SDS-PAGE identification diagram of the expression product of acyl-CoA oxidase derived from Barnerella in TB medium according to an embodiment of the present invention (the recombinant plasmid contains optimized codon I);

图3根据本发明实施例中变形虫来源的酰基辅酶A氧化酶在TB和LB培养基中18℃诱导表达产物SDS-PAGE鉴定图;Figure 3 is an SDS-PAGE identification diagram of the induced expression product of the amoeba-derived acyl-CoA oxidase in TB and LB medium at 18°C according to the embodiment of the present invention;

图4是根据本发明实施例中假单胞菌来源的酰基辅酶A氧化酶在TB和LB培养基中18℃诱导表达产物SDS-PAGE鉴定图;Figure 4 is an SDS-PAGE identification diagram of the induced expression product of acyl-CoA oxidase derived from Pseudomonas in TB and LB medium at 18°C according to an embodiment of the present invention;

图5是根据本发明实施例中纯化后的巴纳氏杆菌来源的酰基辅酶A氧化酶的SDS-PAGE图(重组质粒中含有优化密码子Ⅰ);Fig. 5 is the SDS-PAGE figure of the purified acyl-CoA oxidase derived from Barnerella according to the embodiment of the present invention (optimized codon I is contained in the recombinant plasmid);

图6是根据本发明实施例中酰基辅酶A氧化酶测活标准曲线图。Fig. 6 is a standard curve diagram of measuring activity of acyl-CoA oxidase according to the embodiment of the present invention.

具体实施方式Detailed ways

本发明人经过广泛而深入的研究,开发了基于基因工程的制备物酶活高的酰基辅酶A氧化酶的方法,提高了可溶性蛋白表达量,并适宜工业大规模生产。本发明中基于基因工程的制备物酶活高的酰基辅酶A氧化酶的方法的开发基于如下程序:S1:首先通过基因工程技术进行菌种源筛选,摒弃那些在大肠杆菌中表达产物为包涵体的菌种源;S2:收集上清可溶蛋白粗产品进行酶活检测,进一步摒弃那些在大肠杆菌中表达产物无酶活的菌种源,选定在大肠杆菌中存在可溶表达且酶活高的菌种源;S3:对最终选定的菌种源中编码酰基辅酶A氧化酶的基因序列进行同义密码子偏好性优化,从若干优化密码子中选择可溶蛋白产量高的优化密码子,基于该优化密码子开发配套的酰基辅酶A氧化酶表达方法,培养获得大量可溶的高酶活酰基辅酶A氧化酶。After extensive and in-depth research, the present inventors have developed a method based on genetic engineering to prepare acyl-CoA oxidase with high enzyme activity, which improves the expression of soluble protein and is suitable for large-scale industrial production. In the present invention, the development of the method for acyl-CoA oxidase with high enzyme activity based on the preparation of genetic engineering is based on the following procedure: S1: firstly, strain source screening is carried out by genetic engineering technology, and those expressed in Escherichia coli are discarded as inclusion bodies S2: Collect the supernatant soluble protein crude product for enzyme activity detection, further discard those bacterial strains that express the product in Escherichia coli without enzyme activity, and select soluble expression and enzyme activity in Escherichia coli High strain source; S3: optimize the synonymous codon preference for the gene sequence encoding acyl-CoA oxidase in the finally selected strain source, and select the optimized codon with high soluble protein yield from several optimized codons Based on the optimized codon, a matching acyl-CoA oxidase expression method was developed, and a large amount of soluble acyl-CoA oxidase with high enzymatic activity was cultivated.

本发明中适宜工业大规模生产的制备酰基辅酶A氧化酶的方法,涉及1)获取目的基因/获取目的蛋白有关核酸序列的步骤;2)对目的基因进行同义密码子优化获得优化密码子的步骤;3)将优化密码子导入载体的步骤;4)将载体导入宿主细胞的步骤;和5)培养宿主细胞并获得目的蛋白的步骤。优选还涉及6)纯化目的蛋白的步骤。The method for preparing acyl-CoA oxidase suitable for large-scale industrial production in the present invention involves 1) the steps of obtaining the target gene/obtaining the relevant nucleic acid sequence of the target protein; 2) performing synonymous codon optimization on the target gene to obtain optimized codons 3) the step of introducing the optimized codon into the vector; 4) the step of introducing the vector into the host cell; and 5) the step of culturing the host cell and obtaining the target protein. Preferably, 6) the step of purifying the protein of interest is also involved.

获取目的基因/获取目的蛋白有关的核酸序列Obtain the target gene/acquire the nucleic acid sequence related to the target protein

本发明中目的基因或目的蛋白相关的核酸序列通过分析不同菌源中含酰基辅酶A氧化酶的氨基酸序列获得其核酸序列信息。不同生物来源的同种蛋白具有不同的氨基酸序列,以不同来源的目的蛋白为基础获得的基因序列,其重组表达产物通常具有不可预知的功能活性。在本发明的一个实施方式中,通过NCBI数据库对大量不同来源的目的蛋白的氨基酸序列进行分析,获得不同来源的目的基因序列信息。一些实施例中,通过NCBI数据库对巴纳氏杆菌源、变形虫源和假单胞菌源的酰基辅酶A氧化酶进行分析,获取对应的酰基辅酶A氧化酶基因序列信息。The nucleic acid sequence information related to the target gene or target protein in the present invention is obtained by analyzing the amino acid sequence of the acyl-CoA oxidase in different bacterial sources. The same protein from different biological sources has different amino acid sequences, and the gene sequence obtained based on the target protein from different sources usually has unpredictable functional activity. In one embodiment of the present invention, the amino acid sequences of a large number of target proteins from different sources are analyzed through the NCBI database to obtain target gene sequence information from different sources. In some embodiments, the acyl-CoA oxidases derived from Barnerella, Amoeba and Pseudomonas are analyzed through the NCBI database to obtain the corresponding acyl-CoA oxidase gene sequence information.

获取目的蛋白序列信息后,可溶通过本领域技术人员熟知的方法制备得到多核苷酸序列。本发明中目的蛋白或其元件的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据已公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。After obtaining the sequence information of the target protein, the soluble polynucleotide sequence is prepared by methods well known to those skilled in the art. The full-length nucleotide sequence or its fragments of the target protein or its elements in the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择,并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

同义密码子偏好性优化Synonymous codon preference optimization

本发明涉及经同义密码子偏好性优化的多核苷酸序列。对获取的目的基因序列进行同义密码子偏好性优化,经同义密码子偏好性优化的目的基因序列可表达与目的蛋白相同的氨基酸序列。在本发明的一些实施方式中,通过巴纳氏杆菌源的基因序列进行大肠杆菌同义密码子偏好性优化,获得了若干优化密码子,示例性地如SEQ ID NO:1所示的优化密码子Ⅰ、SEQ ID NO:4-7所示的优化密码子Ⅳ、优化密码子Ⅴ、优化密码子Ⅵ和优化密码子Ⅶ。在另一实施方式中,通过对变形虫源的基因序列进行大肠杆菌同义密码子偏好性优化,获得若干优化密码子,示例性地如SEQ ID NO:2所示的优化密码子Ⅱ。在另一实施方式中,通过对假单胞菌源的基因序列进行大肠杆菌同义密码子偏好性优化,获得若干优化密码子,示例性地如SEQ ID NO:3所示的优化密码子Ⅲ。The present invention relates to polynucleotide sequences optimized for synonymous codon bias. The obtained target gene sequence is optimized for synonymous codon bias, and the target gene sequence optimized for synonymous codon bias can express the same amino acid sequence as the target protein. In some embodiments of the present invention, E. coli synonymous codon preference optimization is carried out through the gene sequence derived from Barnerella, and several optimized codons are obtained, exemplarily the optimized codon shown in SEQ ID NO:1 Optimized codon IV, optimized codon V, optimized codon VI and optimized codon VII shown in codon I, SEQ ID NO:4-7. In another embodiment, several optimized codons are obtained by optimizing the preference of Escherichia coli synonymous codons to the amoeba-derived gene sequence, for example, the optimized codon II shown in SEQ ID NO:2. In another embodiment, several optimized codons are obtained by optimizing the preference of Escherichia coli synonymous codons to the gene sequence derived from Pseudomonas, for example, the optimized codon III shown in SEQ ID NO:3 .

同种来源的目的蛋白基因,经不同方式同义密码子偏好性优化获得的若干优化密码子,通常地,这些优化密码子虽可在宿主细胞中表达活性相当的目的蛋白,但表达量并不相同。在本发明的一些实施方式中,通过巴纳氏杆菌源的酰基辅酶A氧化酶基因序列进行大肠杆菌同义密码子偏好性优化得到的优化密码子中,大量包涵体表达,部分存在可溶性表达,示例性地如优化密码子Ⅰ和优化密码子Ⅴ、优化密码子Ⅵ和优化密码子Ⅶ,只有优化密码子Ⅰ存在可溶性表达。相同条件下,存在可溶性表达的优化密码子,可溶蛋白表达量也存在明显差异,示例性地如优化密码子Ⅰ和优化密码子Ⅳ,优化密码子Ⅰ导入大肠杆菌可溶蛋白表达量明显高于优化密码子Ⅳ。The target protein gene from the same source, several optimized codons obtained through optimization of synonymous codon preference in different ways, usually, although these optimized codons can express the target protein with equivalent activity in the host cell, the expression level is not same. In some embodiments of the present invention, among the optimized codons obtained by optimizing the synonymous codon preference of Escherichia coli through the acyl-CoA oxidase gene sequence derived from Barnerella, a large number of inclusion bodies are expressed, and some of them are soluble. Exemplarily such as optimized codon I and optimized codon V, optimized codon VI and optimized codon VII, only optimized codon I has soluble expression. Under the same conditions, there are optimized codons for soluble expression, and there are obvious differences in the expression of soluble proteins. For example, optimized codon I and optimized codon IV, the expression of optimized codon I into E. coli is significantly higher in optimizing codon IV.

不同种来源的目的蛋白基因,经同义密码子偏好性优化后,导入宿主细胞得到的表达产物存在较大的活性差异。在一个实施方式中,经大肠杆菌同义密码子偏好性优化后的巴纳氏杆菌源优化密码子Ⅰ和变形虫源的优化密码子Ⅱ,在大肠杆菌中的表达产物活性差异显著,仅巴纳氏杆菌源的优化密码子Ⅰ具有酶活。The target protein genes from different sources, after optimization of synonymous codon preference, have large activity differences in the expression products obtained by introducing into host cells. In one embodiment, the optimized codon I derived from Barnerella and the optimized codon II derived from amoebae after optimizing the preference of synonymous codons in E. The optimized codon Ⅰ derived from Nessella has enzymatic activity.

本发明还涉及与SEQ ID NO:1-7所示序列的同源性大于80%,优选大于85%,更优选大于90%,更优选大于91%,更优选大于95%的多核苷酸;和与SEQ ID NO:1-7所示序列互补的多核苷酸。The present invention also relates to a polynucleotide having a homology greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, more preferably greater than 95% with the sequence shown in SEQ ID NO: 1-7; and polynucleotides complementary to the sequences shown in SEQ ID NO: 1-7.

目的基因的载体Carrier of target gene

本发明中还涉及包含本发明的多核苷酸(优化密码子)的载体。本发明中“载体”表示线性或环状DNA分子,其包含编码目的蛋白的片段,所述目的蛋白可操作地连接到提供其转录的其它片段。这样的附加片段可以包括启动子和终止子序列,并且可以任选地包括一个或多个复制起点,一个或多个可选择标记,增强子,多腺苷酸化信号,载体等。载体片段可以衍生自宿主生物体,另一生物体,质粒或病毒DNA,或可以是合成的。载体可以是合成的或方便地进行重组DNA操作的任何表达载体,载体的选择通常取决于载体要导入的宿主细胞。因此,载体可以是自主复制载体,即载体,其作为染色体外实体存在,染色体外实体的复制与染色体复制无关,例如质粒。或者,载体可以是当引入宿主细胞时整合到宿主细胞基因组中并与其整合的染色体一起复制的载体。在一个实施方案中,本发明的载体是表达载体。本发明的一个实施例中选择pET-28a(+)作为载体,以获取更高效的表达效率。Also contemplated in the present invention are vectors comprising the polynucleotides (codon optimized) of the present invention. "Vector" in the present invention means a linear or circular DNA molecule comprising a segment encoding a protein of interest operably linked to other segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. A vector segment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector can be any expression vector that is synthetic or convenient for recombinant DNA manipulation, and the choice of the vector usually depends on the host cell into which the vector is to be introduced. Thus, a vector may be an autonomously replicating vector, ie a vector, which exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome of the host cell and replicates with the chromosome into which it has integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pET-28a(+) is selected as the vector to obtain higher expression efficiency.

本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。示例性地,使用DNA内切酶将载体DNA分子切割成可与外源基因连接的线性分子,然后将经密码子优化的目的基因片段连接于载体,可选用单酶切位点的黏端连接、双酶切片段的定向克隆、不同限制酶切位点的黏端连接、平端连接、人工接头连接或同寡核苷酸末端连接实现外源DNA片段的插入。Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the DNA endonuclease is used to cut the carrier DNA molecule into a linear molecule that can be connected with the foreign gene, and then the codon-optimized target gene fragment is connected to the carrier, and the sticky end connection with a single enzyme cutting site can be selected , Directional cloning of double-digested fragments, sticky-end ligation of different restriction enzyme sites, blunt-end ligation, artificial adapter ligation or ligation with oligonucleotide ends to achieve the insertion of exogenous DNA fragments.

含有目的基因的载体转化宿主细胞Transform host cells with the vector containing the gene of interest

本发明还涉及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞。含有经密码子优化的目的基因的载体可以通过已知的方法插入、转染或乙其他方式转化到宿主细胞中,从而获得含有本发明经密码子优化的目的基因并能够表达目的蛋白的转化体。本发明中“宿主细胞”是引入了外源多核苷酸和/或载体的细胞。宿主细胞可以是真核宿主细胞或原核宿主细胞,宿主细胞优选是细菌,并且优选是大肠杆菌,更优选是大肠杆菌ROSETTA(DE3)菌种(Escherichia coli Rosetta(DE3)strain)。The present invention also relates to host cells produced by genetic engineering using the vector or fusion protein coding sequence of the present invention. The vector containing the codon-optimized gene of interest can be inserted, transfected or otherwise transformed into the host cell by known methods, thereby obtaining a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest . A "host cell" in the present invention is a cell into which exogenous polynucleotides and/or vectors have been introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell, and the host cell is preferably a bacterium, and preferably Escherichia coli, more preferably Escherichia coli Rosetta (DE3) strain.

制备目的蛋白的方法Method for preparing target protein

本发明还涉及制备目的蛋白的方法,可利用本发明的多核苷酸序列表达或生产重组蛋白。一般来说有以下步骤:The present invention also relates to a method for preparing the target protein, and the polynucleotide sequence of the present invention can be used to express or produce the recombinant protein. Generally speaking, there are the following steps:

(1)用本发明的编码本发明蛋白的多核苷酸(或变异体),或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞;(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2)在合适的培养基中培养的宿主细胞;(2) host cells cultured in a suitable medium;

(3)从培养基或细胞中分离、纯化蛋白质。(3) Separation and purification of protein from culture medium or cells.

其中,步骤(1)含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞可通过本领域技术人员熟知的常规技术进行,当宿主是大肠杆菌时,可选用热击法和电转化法等。Wherein, step (1) transforming or transducing suitable host cells with the recombinant expression vector containing the polynucleotide can be carried out by conventional techniques well known to those skilled in the art. When the host is Escherichia coli, heat shock and electroporation can be selected. law etc.

获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基,优选为SB、TB、LB或SOC培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。为促进目的蛋白的表达并提升可溶蛋白的表达量,本发明的一个优选的实施方式,使用TB或LB培养基培养的宿主细胞,且所用培养基中含有卡那霉素抗性基因。The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. According to the host cells used, the medium used in the culture can be selected from various conventional mediums, preferably SB, TB, LB or SOC medium. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time. In order to promote the expression of the target protein and increase the expression level of the soluble protein, in a preferred embodiment of the present invention, host cells cultured in TB or LB medium are used, and the medium used contains a kanamycin resistance gene.

为进一步促进目的蛋白的可溶性表达,在本发明的一个优选的实施方式中,培养宿主细胞至OD600在0.6-0.8之间后,采用IPTG进行诱导,并在17至19℃下或35至39℃下继续培养约8至12小时。In order to further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, after culturing the host cells until the OD 600 is between 0.6-0.8, IPTG is used to induce, and at 17 to 19 ° C or 35 to 39 Cultivation was continued for about 8 to 12 hours at °C.

在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。因此本发明中,在成功培养得到目的蛋白后,还涉及对其进行分离和纯化的步骤,例如步骤(3)中,从培养基中分离和纯化蛋白质以获得高纯度的目的蛋白。虽纯化目的蛋白的方法可是本领域技术人员熟知的常规手段,包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。本发明的优选的实施方式中提供了分离目的蛋白的方法,步骤包括:将经破碎的目的蛋白上清液与流动相同时通过层析柱进行洗脱,收集洗脱液;流动相包括BufferA、BufferB和/或BufferC;其中,BufferA包括三羟甲基氨基甲烷(Tris,浓度50mM)和NaCl溶液(浓度50mM);BufferB包括三羟甲基氨基甲烷(Tris,浓度50mM)、NaCl溶液(浓度50mM)和咪唑(Imidazole,浓度500mM);BufferC包括三羟甲基氨基甲烷(Tris,浓度50mM)和NaCl溶液(浓度1M)。The protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. Therefore, in the present invention, after the target protein is successfully cultured, it also involves the steps of isolating and purifying it, for example, in step (3), separating and purifying the protein from the culture medium to obtain a high-purity target protein. Although the method of purifying the target protein can be a conventional means well known to those skilled in the art, including but not limited to: conventional refolding treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic destruction, supertreatment, ultracentrifugation , Molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods. A preferred embodiment of the present invention provides a method for separating the target protein, the steps include: eluting the broken target protein supernatant through a chromatographic column when the flow is the same, and collecting the eluate; the mobile phase includes BufferA, BufferB and/or BufferC; Wherein, BufferA comprises tris (Tris, concentration 50mM) and NaCl solution (concentration 50mM); BufferB comprises tris (Tris, concentration 50mM), NaCl solution (concentration 50mM ) and imidazole (Imidazole, concentration 500mM); BufferC includes tris (Tris, concentration 50mM) and NaCl solution (concentration 1M).

优选地,洗脱步骤中,所述洗脱的程序包括第一阶段、第二阶段;所述第一阶段中,使用的流动相为BufferA;所述第二阶段中,使用的流动相为BufferA和BufferB的混合物,其中,BufferA的体积百分含量从100%逐渐降低至40%,BufferB的体积百分含量从0%逐渐增加至60%。Preferably, in the elution step, the elution procedure includes a first stage and a second stage; in the first stage, the mobile phase used is BufferA; in the second stage, the mobile phase used is BufferA and BufferB, wherein the volume percentage of BufferA gradually decreases from 100% to 40%, and the volume percentage of BufferB gradually increases from 0% to 60%.

更优选地,所述洗脱的程序还包括第三阶段,所述第三阶段中,使用的流动相为BufferB。More preferably, the elution procedure further includes a third stage, and in the third stage, the mobile phase used is BufferB.

将洗脱纯化后的目的蛋白产品进行透析,并收集透析样品。透析样品可通过BCA法测量浓度,计算产量。The eluted and purified target protein product is dialyzed, and the dialyzed sample is collected. Dialyzed samples can be measured by BCA method concentration, to calculate the yield.

在本发明中,使用针对本文中的某些实施例提供的任何示例性或示例性措辞(例如,“”)只是为了更好地呈现本发明,而不限制以其它方式要求权利的本发明的范围。本文中的任何措辞都不应被解释为表示本发明实施中不可缺少的权利要求中未描述的要素。In this disclosure, use of any exemplary or exemplary language (eg, "") provided with respect to certain embodiments herein is for the purpose of better presenting the invention only and does not limit the scope of the invention otherwise claimed. scope. Nothing in the text should be construed as indicating any non-recited element in the claims indispensable to the practice of the invention.

如果引用文献中的术语的定义或使用与本文中描述的术语的定义不一致或不一致,则使用本文中描述的术语的定义,而不使用引用文献中的术语的定义。If the definition or use of a term in a cited document is inconsistent or inconsistent with the definition of the term described herein, the definition of the term described herein applies instead of the definition of the term in the cited document.

本文中使用的各种术语如下所示。如果权利要求中使用的术语未在下文中定义,则应给出本领域技术人员给出的该术语的最广泛定义,以反映在申请时印刷的出版物或所发布的专利中。Various terms used in this article are as follows. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the art have given that term as reflected in printed publications or issued patents at the time of filing.

如本文中所用的,术语“分离的”是指与核酸或多肽在其天然来源中存在的至少一种其它组分(例如核酸或多肽)分离的核酸或多肽。在一个实施方案中,发现核酸或多肽仅存在(如果有的话)通常存在于其溶液中的溶剂,缓冲液,离子或其它组分。术语“分离的”和“纯化的”不包括存在于其天然来源中的核酸或多肽。As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (eg, nucleic acid or polypeptide) of the nucleic acid or polypeptide in its natural source. In one embodiment, a nucleic acid or polypeptide is found only in the presence, if any, of solvents, buffers, ions or other components normally present in solution thereof. The terms "isolated" and "purified" do not include nucleic acids or polypeptides as they exist in their natural source.

如本文中所用的,术语“多核苷酸”和“多核苷酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码多肽的功能。The present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.

如本文中所用的,术语“密码子优化”是指根据实际做蛋白表达或生产的生物(包括大肠杆菌、酵母、哺乳动物血细胞、植物细胞、昆虫细胞等)表现出的密码子利用差异,避免使用低利用率或稀有的密码子,来提高基因合成效率的方式。As used herein, the term "codon optimization" refers to avoiding codon usage differences based on the actual expression or production of proteins (including Escherichia coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) A way to improve the efficiency of gene synthesis by using low-utilization or rare codons.

如本文中所用的,术语“同源性”和“同一性”可互换使用,是指两个或更多个多核苷酸或多肽之间相同(即相同)核苷酸或氨基酸的百分比。可以通过以下方法测量两个或多个多核苷酸或多肽之间的序列同一性。排列多核苷酸或多肽的核苷酸或氨基酸序列,对排列的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置的数量进行评分,并将其与排列的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置的数量进行比较。多核苷酸可以在一个位置上不同,例如,根据包含不同的核苷酸(即,替换或变异)或核苷酸的缺失(即,在多核苷酸中插入或缺失一个或两个核苷酸)。多肽可以例如通过含有氨基酸(即,取代或变异)或氨基酸的缺失(即,插入一个或两个多肽中的氨基酸或氨基酸的缺失)在一个位置上不同。可以通过将含有相同核苷酸或氨基酸残基的位置的数量除以多核苷酸或多肽中氨基酸残基的总数来计算序列同一性。例如,百分比同一性可通过将含有相同核苷酸或氨基酸残基的位置的数量除以多核苷酸或多肽中核苷酸或氨基酸残基的总数,然后乘以100来计算。As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (ie identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. Align the nucleotide or amino acid sequences of polynucleotides or polypeptides, score the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue, and compare this with the aligned polynucleotides or polypeptides Compare the number of positions containing different nucleotide or amino acid residues in . Polynucleotides may differ at a position, for example, by the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide ). Polypeptides may differ at one position, for example, by the inclusion of amino acids (ie, substitutions or variations) or deletions of amino acids (ie, insertions or deletions of amino acids in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

如本文中所用的,术语“序列互补”和“反向序列互补”可互换使用,指的是与原多核苷酸序列的方向相反,且与原多核苷酸序列互补的序列。例如,如果原始多核苷酸序列是ACTGAAC,则其反向互补序列是GTTCAT。As used herein, the terms "sequence complementarity" and "reverse sequence complementarity" are used interchangeably to refer to a sequence that is oriented in the opposite direction to an original polynucleotide sequence and that is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

如本文中所用的,术语“表达”包括涉及宿主细胞中多肽产生的任何步骤,包括但不限于转录,翻译,翻译后修饰和分泌。表达后可收获,即回收宿主细胞或表达产物。As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Following expression, it can be harvested, ie, the host cells or the expression product are recovered.

为使本发明实施例的目的、技术方案和优点更加清楚,下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

除非另有指明,本文所用的技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义,需要注意的是,本文所用的术语仅为了描述具体实施方式,而非意图限制本申请的示例性实施方式。Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the application belongs. Exemplary implementation of the application.

实施例1Example 1

本实施例中,合成了含有巴纳氏杆菌来源的、变形虫来源的和假单胞菌来源的编码酰基辅酶A氧化酶的优化密码子的质粒,并将其导入大肠杆菌培养获得单克隆。In this example, plasmids containing optimized codons encoding acyl-CoA oxidase derived from Barnerella, Amoeba and Pseudomonas were synthesized and introduced into Escherichia coli to obtain single clones.

(1)构建酰基辅酶A氧化酶质粒(1) Construction of an acyl-CoA oxidase plasmid

获取巴纳氏杆菌来源的酰基辅酶A氧化酶的基因序列,并对其进行大肠杆菌同义密码子偏好性优化,得到优化密码子Ⅰ(SEQ ID NO.1),连接到pET-28a(+)载体,并委托苏州金维智生物科技有限公司合成。The gene sequence of the acyl-CoA oxidase derived from Barnerella was obtained, and the synonymous codon preference of Escherichia coli was optimized to obtain the optimized codon Ⅰ (SEQ ID NO.1), which was connected to pET-28a (+ ) carrier, and entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize it.

获取变形虫来源的酰基辅酶A氧化酶的基因序列,并对其进行大肠杆菌同义密码子偏好性优化,得到优化密码子Ⅱ(SEQ ID NO.2)。连接到pET-28a(+)载体,并委托苏州金维智生物科技有限公司合成。The gene sequence of acyl-CoA oxidase derived from amoeba was obtained, and the synonymous codon preference optimization of Escherichia coli was carried out to obtain the optimized codon II (SEQ ID NO.2). Connected to the pET-28a(+) vector, and entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize it.

获取假单胞菌来源的酰基辅酶A氧化酶的基因序列,并对其进行大肠杆菌同义密码子偏好性优化,得到优化密码子Ⅲ(SEQ ID NO.3)。连接到pET-28a(+)载体,并委托苏州金维智生物科技有限公司合成。The gene sequence of acyl-CoA oxidase derived from Pseudomonas was obtained, and the synonymous codon preference of Escherichia coli was optimized to obtain the optimized codon III (SEQ ID NO.3). Connected to the pET-28a(+) vector, and entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize it.

(2)重组质粒导入宿主大肠杆菌(2) Recombinant plasmid introduced into host Escherichia coli

取上述步骤(1)中制备的表达质粒1μL,在冰浴条件下,加入到30μL大肠杆菌感受态BL21(DE3)中,冰浴放置30min,42℃水浴45s,立刻冰上放置2min,加入400μL不含抗生素的SOC培养基,37℃、230rpm振荡培养45min。取100μL菌液均匀涂布到含100μg/mL卡那抗性的LB平板上,37℃培养箱培养过夜。Take 1 μL of the expression plasmid prepared in the above step (1), add it to 30 μL of Escherichia coli competent BL21(DE3) in an ice bath, place it in an ice bath for 30 minutes, and place it in a water bath at 42°C for 45 seconds, immediately place it on ice for 2 minutes, and add 400 μL In the SOC medium without antibiotics, shake culture at 37°C and 230rpm for 45min. Take 100 μL of the bacterial solution and evenly spread it on the LB plate containing 100 μg/mL kana-resistant, and cultivate overnight in a 37°C incubator.

(3)目的基因的表达(3) Expression of the target gene

挑取步骤(2)中的制备的单克隆,无菌操作分别接种于含100μg/mL卡那抗性的LB和TB培养基中,做两个重复,37℃220rpm振荡培养至OD600在0.6-0.8之间,IPTG进行诱导,分别放置于37℃和18℃振荡培养过夜。取样超声破碎进行SDS-PAGE鉴定。鉴定结果见图1至4。Pick the single clones prepared in step (2), and inoculate them in LB and TB medium containing 100 μg/mL kana resistance respectively by aseptic operation, do two repetitions, and culture at 37°C with shaking at 220rpm until the OD600 is between 0.6- Between 0.8, IPTG was induced, respectively placed at 37 ° C and 18 ° C shaking culture overnight. Samples were sonicated for SDS-PAGE identification. The identification results are shown in Figures 1 to 4.

根据图1至4,巴纳氏杆菌和假单胞菌来源的酰基辅酶A氧化酶在大肠杆菌中可以上清表达,变形虫来源的酰基辅酶A氧化酶为包涵体表达。According to Figures 1 to 4, the acyl-CoA oxidase derived from Barnerella and Pseudomonas can be expressed supernatantly in Escherichia coli, and the acyl-CoA oxidase derived from amoebae can be expressed in inclusion bodies.

(4)目的蛋白的纯化(4) Purification of target protein

称取约4g步骤(3)制备的巴纳氏杆菌和假单胞菌来源重组菌体,加入20mL LysisBuffer在冰上使用分散机分散。超声破碎细胞:Ф10探头,功率10%,工作5.5s,停止9.9s,超声破碎30min。20000rpm,4℃离心30min,取上清,0.22μm膜过滤。用1mL Ni-NTA纯化,流动相各组分的配制参考表1,流动相流速为0.5mL/min,上完样使用20mL Lysis Buffer冲洗UV和电导至基线。洗脱程序包括:Step 1:0%B,10CV,1.5ml/min;Step 2:0—60%B,20CV,1.5ml/min;Step 3:100%B,15CV,1.5ml/min。Weigh about 4 g of the Barnerella and Pseudomonas-derived recombinant cells prepared in step (3), add 20 mL of LysisBuffer and disperse on ice using a disperser. Ultrasonic disruption of cells: Ф10 probe, power 10%, working for 5.5s, stopping for 9.9s, ultrasonic disruption for 30min. Centrifuge at 20,000 rpm at 4°C for 30 min, take the supernatant, and filter through a 0.22 μm membrane. Use 1mL Ni-NTA to purify, refer to Table 1 for the preparation of mobile phase components, the mobile phase flow rate is 0.5mL/min, use 20mL Lysis Buffer to wash the UV and conductance to the baseline after loading the sample. The elution program includes: Step 1: 0% B, 10CV, 1.5ml/min; Step 2: 0—60% B, 20CV, 1.5ml/min; Step 3: 100% B, 15CV, 1.5ml/min.

表1Table 1

Figure BDA0004026028410000101
Figure BDA0004026028410000101

Figure BDA0004026028410000111
Figure BDA0004026028410000111

收集纯化产物进行后续酶活测试。部分来源纯化产物电泳结果见图5(图5为巴纳氏杆菌来源的酰基辅酶A氧化酶收样后电泳结果,由图5可知,目的蛋白可以挂柱,并在130mM咪唑浓度下开始洗脱根据SDS结果,选择洗脱液B1-B7进行透析,透析后得到的样品13mL。采用BCA测浓度,结果为:R2=0.996,其浓度为1.917mg/mL,产量为24.921mg,得率为6.23mg/g菌)。The purified product was collected for subsequent enzyme activity testing. The electrophoresis results of the purified products from some sources are shown in Figure 5 (Figure 5 is the electrophoresis results of the acyl-CoA oxidase derived from Barnerella bacillus after collection. It can be seen from Figure 5 that the target protein can be hung on the column and begins to elute at a concentration of 130mM imidazole According to SDS result, select eluent B1-B7 to carry out dialysis, the sample 13mL that obtains after dialysis.Use BCA to measure concentration, the result is: R2=0.996, and its concentration is 1.917mg/mL, and output is 24.921mg, and yield rate is 6.23 mg/g bacteria).

SEQ ID NO.1SEQ ID NO.1

ATGACCGAAGTAGTTGACCGTGCTTCTTCTCCGGCTTCTCCTGGTTCCACTACCGCTGCGGCAGATGGTGCGAAAGTAGCATGACCGAAGTAGTTGACCGTGCTTTCTTCTCCGGCTTCTCCTGGTTCCACTACCGCTGCGGCAGATGGTGCGAAAGTAGC

GGTTGAACCGCGTGTTGACGTTGCAGCTCTGGGTGAACAACTGCTGGGCCGTTGGGCGGATATCCGTCTGCACGCACGTGGGTTGAACCGCGTGTTGACGTTGCAGCTCTGGGTGAACAACTGCTGGGCCGTTGGGCGGATATCCGTCTGCACGCACGTG

ACCTGGCTGGTCGTGAGGTGGTTCAGAAAGTTGAGGGCCTGACTCACACTGAACACCGTTCTCGTGTTTTTGGTCAACTGACCTGGCTGGTCGTGAGGTGGTTCAGAAAGTTGAGGGCCTGACTCACACTGAACACCGTTCTCGTGTTTTTGGTCAACTG

AAATACCTGGTTGACAACAACGCCGTACACCGTGCATTCCCATCTCGTCTGGGCGGTTCTGACGATCATGGTGGCAATATAAATACCTGGTTGACAACAACGCCGTACACCGTGCATTCCCATCTCGTCTGGGCGGTTCTGACGATCATGGTGGCAATAT

CGCAGGCTTCGAAGAACTGGTTACTGCTGATCCGAGCCTGCAGATCAAAGCGGGCGTACAGTGGGGTCTGTTTGGTAGCGCGCAGGCTTCGAAGAACTGGTTACTGCTGATCCGAGCCTGCAGATCAAAGCGGGCGTACAGTGGGGTCTGTTTGGTAGCG

CGGTTATGCACCTGGGCACCCGTGAACATCACGATAAATGGCTGCCGGGTATCATGTCCCTGGAGATCCCGGGCTGCTTCCGGTTATGCACCTGGGCACCCGTGAACATCACGATAAATGGCTGCCGGGTATCATGTCCCTGGAGATCCCGGGCTGCTTC

GCAATGACCGAGACTGGTCACGGCTCCGACGTCGCTTCTATTGCAACCACCGCAACCTACGATGAAGAAACTCAGGAATTGCAATGACCGAGACTGGTCACGGCTCCGACGTCGCTTCTATTGCAACCACCGCAACCTACGATGAAGAAACTCAGGAATT

TGTGATCGACACCCCATTCCGTGCAGCATGGAAAGACTACATCGGCAACGCAGCAAACGATGGTCTGGCGGCGGTTGTTTTGTGATCGACACCCCATTCCGTGCAGCATGGAAAGACTACATCGGCAACGCAGCAAACGATGGTCTGGCGGCGGTTGTTT

TCGCCCAGCTGATCACGCGTAAAGTAAACCATGGTGTGCATGCTTTCTACGTTGATCTGCGTGACCCGGCGACTGGTGACTCGCCCAGCTGATCACGCGTAAAGTAAACCATGGTGTGCATGCTTTCTACGTTGATCTGCGTGACCCGGCGACTGGTGAC

TTTCTGCCGGGTATTGGTGGTGAGGACGACGGTATCAAAGGTGGCCTGAACGGTATTGACAATGGCCGTCTGCATTTCACTTTCTGCCGGGTATTGGTGGTGAGGACGACGGTATCAAAGGTGGCCTGAACGGTATTGACAATGGCCGTCTGCATTTCAC

CAACGTTCGTATTCCGCGTACCAATCTGCTGAACCGTTACGGCGACGTAGCTGTTGACGGTACCTACAGCAGCACTATCGCAACGTTCGTATTCCGCGTACCAATCTGCTGAACCGTTACGGCGACGTAGCTGTTGACGGTACCTACAGCAGCACTATCG

AATCCCCGGGTCGTCGCTTTTTCACCATGCTGGGCACTCTGGTTCAAGGTCGCGTAAGCCTGGATGGCGCAGCCGTTGCAAATCCCCGGGTCGTCGCTTTTTTCACCATGCTGGGCACTCTGGTTCAAGGTCGCGTAAGCCTGGATGGCGCAGCCGTTGCA

GCATCTAAAGTGGCTCTGCAGAGCGCTATTCATTATGCAGCGGAACGTCGCCAGTTCAACGCTACTAGCCCTACCGAAGAGCATCTAAAGTGGCTCTGCAGAGCGCTATTCATTATGCAGCGGAACGTCGCCAGTTCAACGCTACTAGCCCTACCGAAGA

AGAAGTCCTGCTGGACTACCAGCGTCATCAGCGCCGTCTGTTCACGCGTCTGGCAACCACCTACGCTGCATCCTTCGCTCAGAAGTCCTGCTGGACTACCAGCGTCATCAGCGCCGTCTGTTCACGCGTCTGGCAACCACCTACGCTGCATCCTTCGCTC

ACGAGCAGCTGCTGCAGAAATTCGATGATGTTTTCAGCGGCGCACACGATACCGATGCAGATCGTCAGGATCTGGAAACCACGAGCAGCTGCTGCAGAAATTCGATGATGTTTTCAGCGGCGCACACGATACCGATGCAGATCGTCAGGATCTGGAAACC

CTGGCGGCTGCGCTGAAACCGCTGTCTACTTGGCACGCACTGGATACGCTGCAGGAATGCCGTGAAGCTTGTGGTGGCGCCTGGCGGCTGCGCTGAAACCGCTGTCTACTTGGCACGCACTGGATACGCTGCAGGAATGCCGTGAAGCTTGTGGTGGCGC

GGGTTTCCTGATTGAAAACCGTTTCGCTTCCCTGCGCGCTGACCTGGATGTGTACGTCACCTTTGAAGGTGATAACACCGGGGTTTCCTGATTGAAAACCGTTTCGCTTCCCTGCGCGCTGACCTGGATGTGTACGTCACCTTTGAAGGTGATAACACCG

TTCTGCTGCAGCTGGTTGCGAAACGTCTGCTGGCTGACTACGCTAAAGAATTCCGTGGCGCTAACTTTGGTGTTCTGGCGTTCTGCTGCAGCTGGTTGCGAAACGTCTGCTGGCTGACTACGCTAAAGAATTCCGTGGCGCTAACTTTGGTGTTCTGGCG

CGCTATGTGGTTGATCAAGCTGCGGGTGTAGCACTGCACCGTACTGGTCTGCGTCAGGTGGCTCAGTTCGTCGCTGATTCCGCTATGTGGTTGATCAAGCTGCGGGTGTAGCACTGCACCGTACTGGTCTGCGTCAGGTGGCTCAGTTCGTCGCTGATTC

TGGTTCTGTTCAGAAAAGCGCTCTGGCACTGCGCGACGAAGAGGGTCAGCGTACTCTGCTGACCGACCGTGTGCAATCTATGGTTCTGTTCAGAAAAGCGCTCTGGCACTGCGCGACGAAGAGGGTCAGCGTACTCTGCTGACCGACCGTGTGCAATCTA

TGGTGGCTGAAGTTGGTGCTGCGCTGAAAGGTGCTGGTAAACTGCCGCAGCATCAGGCAGCAGCGCTGTTTAACCAGCACTGGTGGCTGAAGTTGGTGCTGCGCTGAAAGGTGCTGGTAAACTGCCGCAGCATCAGGCAGCAGCGCTGTTTAACCAGCAC

CAGAACGAACTGATCGAAGCGGCTCAAGCGCATGCTGAACTGCTGCAATGGGAAGCGTTCACCGAAGCACTGGCGAAAGTCAGAACGAACTGATCGAAGCGGCTCAAGCGCATGCTGAACTGCTGCAATGGGAAGCGTTCACCGAAGCACTGGCGAAAGT

AGATGACGCGGGCACGAAAGAAGTTCTGACCCGCCTGCGCGACCTGTTTGGTCTGTCCCTGATTGAAAAGCATCTGAGCTAGATGACGCGGGCACGAAAGAAGTTCTGACCCGCCTGCGCGACCTGTTTGGTCTGTCCCTGATTGAAAAGCATCTGAGCT

GGTATCTGATGAATGGTCGTCTGTCCATGCAACGTGGTCGTACCGTTGGCACGTATATCAACCGTCTGCTGGTTAAAATCGGTATCTGATGAATGGTCGTCTGTCCATGCAACGTGGTCGTACCGTTGGCACGTATATCAACCGTCTGCTGGTTAAAATC

CGTCCGCACGCGCTGGATCTGGTAGATGCATTCGGCTACGGTGCTGAACATCTGCGTGCTGCTATCGCAACTGGTGCCGACGTCCGCACGCGCTGGATCTGGTAGATGCATTCGGCTACGGTGCTGAACATCTGCGTGCTGCTATCGCAACTGGTGCCGA

AGCAACCCGTCAGGACGAAGCGCGTACTTACTTCCGTCAGCAACGCGCGTCTGGTTCTGCACCGGCTGATGAAAAAACTCAGCAACCCGTCAGGACGAAGCGCGTACTTACTTCCGTCAGCAACGCGCGTCTGGTTCTGCACCGGCTGATGAAAAAACTC

TGCTGGCGATCAAAGCAGGCAAATCTCGCTGCTGGCGATCAAAGCAGGCAAAATCTCGC

SEQ ID NO.2SEQ ID NO.2

ATGAACCCGGACCTGAAACGCGAACGTAAAGGCGCATCCTTTGATGCAACCGCAACTCGTGAACGTTTTCAGAAACTGGTATGAACCCGGACCTGAAACGCGAACGTAAAGGCGCATCCTTTGATGCAACCGCAACTCGTGAACGTTTTCAGAAACTGGT

AGCGGATGAACCGGTGTTTGCGAATGATGACAAATACTTCCTGTCTCGTGTGCAGAACTATGAACGTGTTCTGCAGAAGGAGCGGATGAACCGGTGTTTGCGAATGATGACAAATACTTCCTGTCTCGTGTGCAGAACTATGAACGTGTTCTGCAGAAGG

TTGTGCGCGGCATCCAAATGTGCATCGAACATAACATCACTCCGGAAGATGCGGAATACCTGTTCCACTTCATCGGTGAATTGTGCGCGGCATCCAAATGTGCATCGAACATAACATCACTCCGGAAGATGCGGAATACCTGTTCCACTTCATCGGTGAA

GAGTTCATGGTTGCGCTGCACTGGTCCATGTTCATCCCGACCCTGCAGGGTCAGGCTACCAACGACCAGAAACTGCAGTGGAGTTCATGGTTGCGCTGCACTGGTCCATGTTCATCCCGACCCTGCAGGGTCAGGCTACCAACGACCAGAAACTGCAGTG

GCTGCCGCTGGCGCAGACCTTCCAGATCATTGGCTGTTATGCCCAGACGGAGATGGGTCATGGCTCCAACGTGCGTGGTCGCTGCCGCTGGCGCAGACCTTCCAGATCATTGGCTGTTATGCCCAGACGGAGATGGGTCATGGCTCCAACGTGCGTGGTC

TGGAAACGACCGCGACCTACGACAAAGCGACGCAGGAATTCGTTCTGCATTCCCCTACCCTGACTTCCACCAAATGGTGGTGGAAACGACCGCGACCTACGACAAAGCGACGCAGGAATTCGTTCTGCATTCCCCCTACCCTGACTTCCACCAAATGGTGG

CCGGGCGGCCTGGGCAAAACGTCCACTCACTGCGTCACTCACGCACGTCTGCTGGTGGAAGGCAAGGACCATGGTGTGGCCCGGGCGGCCTGGGCAAAACGTCCACTCACTGCGTCACTCACGCACGTCTGCTGGTGGAAGGCAAGGACCATGGTGTGGC

CACCTTCATCGTTCAGATTCGCTCTACCGATGACCATGCGCCGATGCCGGGCGTAACTGTCGGCGATATCGGTCCTAAATCACCTTCATCGTTCAGATTCGCTCTACCGATGACCATGCGCCGATGCCGGGCGTAACTGTCGGCGATATCGGTCCTAAAT

TTGGCTACGACACCCAGGATAACGGTTTCCTGCGTTTTGACCACGTTCGTATCCCACGTGATCAAATGCTGATGAAATACTTGGCTACGACACCCAGGATAACGGTTTCCTGCGTTTTGACCACGTTCGTATCCCACGTGATCAAATGCTGATGAAATAC

AAGCAGGTCTCCCCGGAAGGTGTTGTGACCGAAGCACCGAAAAAGCTGTCTAAACTGAGCTATGGCACCATGATGTACATAAGCAGGTCTCCCCGGAAGGTGTTGTGACCGAAGCACCGAAAAAGCTGTCTAAACTGAGCTATGGCACCATGATGTACAT

TCGCTCCCGCATCGTTGGTGGTGCATCCTCTACGCTGGCACGTGCGTGCACCATTGCAGTACGTTATTCCGCTGTACGTCTCGCTCCCGCATCGTTGGTGGTGCATCCTCTACGCTGGCACGTGCGTGCACCATTGCAGTACGTTATTCCGCTGTACGTC

GTCAGTTCAGCGACGCAGACAACGAACCGGAAAAACAGGTACTGGACTACCGTATGCAGCAGTATCGTCTGCTGCCGCTGGTCAGTTCAGCGACGCAGACAACGAACCGGAAAAACAGGTACTGGACTACCGTATGCAGCAGTATCGTCTGCTGCCGCTG

CTGGCCACTGCATACGCATTCCATTTCACCGGTCGTTACATGCGTAACATCTACGATGAGCTGATGCGTAACATCCAGTCCTGGCCACTGCATACGCATTCCATTTCACCGGTCGTTACATGCGTAACATCTACGATGAGCTGATGCGTAACATCCAGTC

CGACGACGTGTCTGCGCTGCCGGAAGTTCACGCGACCTCTGCGGGCCTGAAAGCGGTTACCACTTGGATGACCGCGGATGCGACGACGTGTCTGCGCTGCCGGAAGTTCACGCGACCTCTGCGGGCCTGAAAGCGGTTACCACTTGGATGACCGCGGATG

GTATTGAGGAATGCCGTAAATGCTGCGGTGGCCACGGCTACAGCAAGTTTGCGGGTATTTCTGACATTTACGTAAACTATGTATTGAGGAATGCCGTAAATGCTGCGGTGGCCACGGCTACAGCAAGTTTGCGGGTATTTCTGACATTTACGTAAACTAT

GTACCAGCCTGCACTTATGAAGGCGATAACGTTGTCATGTGCCTGCAGACCGCGCGTTACCTGGTTAAGACCGCACGTGGGTACCAGCCTGCACTTATGAAGGCGATAACGTTGTCATGTGCCTGCAGACCGCGCGTTACCTGGTTAAGACCGCACGTGG

CGCTGCAAAGGGTGAACCTCTGGTGGGCAGCGTGCAGTGTCCGGCGCAGAAAGTTGCGGATTTCCTGTGCCCGCGTACCTCGCTGCAAAGGGTGAACCTCTGGTGGGCAGCGTGCAGTGTCCGGCGCAGAAAGTTGCGGATTTCCTGTGCCCGCGTACCT

GGGTTGATGCTTTTGCGCTGCGTGCACGCTTCTGCGTTTTCGAGACCGTTAAAAAACTGGACGCTCTGAAGGGTCGTGGTGGGTTGATGCTTTTGCGCTGCGTGCACGCTTCTGCGTTTTCGAGACCGTTAAAAAACTGGACGCTCTGAAGGGTCGTGGT

CTGAACGATAAACAGGTTTGGAACGAAGCTCAGATCGATCTGGTAAAGATGACCAAAGCGCATTGTTATTACACCATCGTCTGAACGATAAACAGGTTTGGAACGAAGCTCAGATCGATCTGGTAAAGATGACCAAAGCGCATTGTTATTACACCATCGT

GCGTAATTTCGCAAACGCGGTAGAAAAGGTTGAGGATAAACAGCTGCAGGCCGTTCTGCACAAACTGTGCATGCTGTTCGGCGTAATTTCGCAAACGCGGTAGAAAAGGTTGAGGATAAACAGCTGCAGGCCGTTCTGCACAAACTGTGCATGCTGTTCG

CGCTGTACCAGGTGCAGCGTGACCTGGGCGATTTTACTTGTTCTGGTTACCTGGCTCAGGAACAGGTCCCGCTGCTGAACCGCTGTACCAGGTGCAGCGTGACCTGGGCGATTTTACTTGTTCTGGTTACCTGGCTCAGGAACAGGTCCCGCTGCTGAAC

GAAGCTGTGGAAGTGCTGCTGAGCGAACTGCGTAAAGACGCTGTACCGCTGGTAGACTCTTTCGACTTCTCTGATCACTTGAAGCTGTGGAAGTGCTGCTGAGCGAACTGCGTAAAGACGCTGTACCGCTGGTAGACTCTTTCGACTTCTCTGATCACTT

TCTGAACAGCAGCCTGGGTCGTTACAACGGTGACGTCTATGAGCACATGTATAAATGGGCGCAGAAGGAACCACTGAACCTCTGAACAGCAGCCTGGGTCGTTACAACGGTGACGTCTATGAGCACATGTATAAATGGGCGCAGAAGGAACCACTGAACC

AGGCGCCGTACGCTACCCAGCCTCCAGGCTATGAAAAATACCTGAAACGCCTGCTGAACGGCGAGGTACTGCAGGAAGCCAGGCGCCGTACGCTACCCAGCCTCCAGGCTATGAAAAATACCTGAAACGCCTGCTGAACGGCGAGGTACTGCAGGAAGCC

ATCCAGAACAAAATGACTAAAGCTAACCTGATCCAGAACAAAATGACTAAAGCTAACCTG

SEQ ID NO.3SEQ ID NO.3

ATGACTGACAGCTCTATTCCTGGCGCTGATGCACTGAAGGCACGTGATGAACTGCGCGATGTTCTGTTTGGCGGTACCTTATGACTGACAGCCTCTATTCCTGGCGCTGATGCACTGAAGGCACGTGATGAACTGCGCGATGTTCTGTTTGGCGGTACCTT

CGAATCTCACCATCAGTCCATTCGCAAAGTGCTGCTGGACCCGATCTTCGATCCGCAGAGCGGTCTGAACATGGAACAAGCGAATCTCACCATCAGTCCATTCGCAAAGTGCTGCTGGACCCGATCTTCGATCCGCAGAGCGGTCTGAACATGGAACAAG

CTGGTCGTCTGGCTTACGCTCGTAGCCGTCATGTGCACGGTGCGCTGGAACGTCCGCTGGAAATTCTGGCGAACCCGCGTCTGGTCGTCTGGCTTACGCTCGTAGCCGTCATGTGCACGGTGCGCTGGAACGTCCGCTGGAAATTCTGGCGAACCCGCGT

CGTCTGTTCGCTCTGGCAGAATGGCCGTCTCTGCTGGATGTTGCATCTTTTAGCCTGCTGATGGTGCACTACAACCTGTGCGTCTGTTCGCTCTGGCAGAATGGCCGTCTCTGCTGGATGTTGCATCTTTTAGCCTGCTGATGGTGCACTACAACCTGTG

CCTGGGTACCGTGTTCGACCATGCGCGCGACCGTTCCGATATCGCTGATCTGACCGAAGCCCTGGACGGCCTGACCTCTTCCTGGGTACCGTGTTCGACCATGCGCGCGACCGTTCCGATATCGCTGATCTGACCGAAGCCCTGGACGGCCTGACCTCTT

TCGGTCCTTACATGGCTACCGAACTGGGTTTCGGTAACAATGTCGCTGCTCTGCAAACTGAAGCAGTGTACGATCGTCAGTCGGTCCTTACATGGCTACCGAACTGGGTTTCGGTAACAATGTCGCTGCTCTGCAAACTGAAGCAGTGTACGATCGTCAG

AGCCAGACCTTCACCCTGAACACCCCATCTGTTTCTGCGCAGAAATACATGAGCTACAGCGGCTTCGGCGACATCCCGAAAGCCAGACCTTCACCCTGAACACCCCATCTGTTTCTGCGCAGAAATACATGAGCTACAGCGGCTTCGGCGACATCCCGAA

AGTGGCAACCGTTATGGCGCGTCTGAAAATCGAAGGTAAAGATTATGGCGTCTTCCCGTTCCTGGTTCGCCTGTCTACTGAGTGGCAACCGTTATGGCGCGTCTGAAAATCGAAGGTAAAGATTATGGCGTCTTCCCGTTCCTGGTTCGCCTGTCTACTG

AGGCTGGTCTGTGCCCAGGTATCCGTGCTGCACTGTGTCCGGAAAAACCGGTTCAGGGCCTGGATAACGGTCTGACCTGGAGGCTGGTCTGTGCCCAGGTATCCGTGCTGCACTGTGTCCGGAAAAACCGGTTCAGGGCCTGGATAACGGTCTGACCTGG

TTTGACAACGTGCGCGTACCGCGTTCTAGCCTGCTGCATGGTGATATGGGTCACTTCGCCGAAGATGGTCACTTCGTAGTTTTGACAACGTGCGCGTACCGCGTTCTAGCCTGCTGCATGGTGATATGGGTCACTTCGCCGAAGATGGTCACTTCGTAGT

TGGTGCAGGCAACGCTCGTTCTCGTTTCCTGCGCGCAATGAGCCGCATTGTTCCAGGTCGTCTGTGCGTTGCATCTGCAGTGGTGCAGGCAACGCTCGTTCTCGTTTCCTGCGCGCAATGAGCCGCATTGTTCCAGGTCGTCTGTGCGTTGCATCTGCAG

CGCAGGGCGCATCTCGTGCATCTCTGTACATCGCGCTGCGTTATGGTCAACAGCGTCTGACTAACGCTCCGGGTACTAACCGCAGGGCGCATCTCGTGCATCTCTGTACATCGCGCTGCGTTATGGTCAACAGCGTCTGACTAACGCTCCGGGTACTAAC

GATATGCCGGTTATTGAATATCGTTCTTACCAGGTCCCACTGTTCTCTGCACTGGCGTCTACCTACGCCATGACCCTGCTGATATGCCGGTTATTGAATATCGTTCTTACCAGGTCCCACTGTTCTCTGCACTGGCGTCTACCTACGCCATGACCCTGCT

GCTGAACGAAGCTAAGGCGCGTTTCCTGGCAAACACCACTGAACCGGCTGTTGACGTTGTGTCTCTGATCAACATCACCAGCTGAACGAAGCTAAGGCGCGTTTCCTGGCAAACACCACTGAACCGGCTGTTGACGTTGTGTCTCTGATCAACATCACCA

AAGCTCTGGCGACTTGGGACGCGTCTGCAGTCATCGCAGAGTGTCGCGAACGTTGTGGTGCACAGGGCATCTTCTCTGCCAAGCTCTGGCGACTTGGGACGCGTCTGCAGTCATCGCAGAGTGTCGCGAACGTTGTGGTGCACAGGGCATCTTCTCTGCC

AACCGTATCGCTGACTACGGTTCTCTGCTGCAGGGCCTGGTCACCGCAGAAGGTGATAACCTGGTCCTGCTGGCTACCGTAACCGTATCGCTGACTACGGTTCTCTGCTGCAGGGCCTGGTCACCGCAGAAGGTGATAACCTGGTCCTGCTGGCTACCGT

TGCAGGCCAGCTGCTGGCTCAGGTATGGCAGGGTCCAGAACCTCTGCGTCCGGTTCGTGCTCGTCGTCTGGCTGAACCTGTGCAGGCCAGCTGCTGGCTCAGGTATGGCAGGGTCCAGAACCTCTGCGTCCGGTTCGTGCTCGTCGTCTGGCTGAACCTG

AGTGGCTGATCGCCGCTATTGCGTTCCGCGAACATCAACTGTGGCTGACCATCCGCGAAGAAATGAACACTGATGAGCGTAGTGGCTGATCGCCGCTATTGCGTTCCGCGAACATCAACTGTGGCTGACCATCCGCGAAGAAATGAACACTGATGAGCGT

GGCTATTTCGAAGTGTGGAACGACGCAATGAACCCTGGCCTGGAACTGGCTCGTCTGCGTGGTGAACGTCTGGCACTGGAGGCTATTTCGAAGTGTGGAACGACGCAATGAACCCTGGCCTGGAACTGGCTCGTCTGCGTGGTGAACGTCTGGCACTGGA

ACAGCTGTGGTCTGCCTCTCTGCACGCACAGCAAGACGAGGCTAAAGCGGCGCTGAACTGTCTGGCAAGCCTGTACGGTCACAGCTGTGGTCTGCCTCTCTGCACGCACAGCAAGACGAGGCTAAAGCGGCGCTGAACTGTCTGGCAAGCCTGTACGGTC

TGAACCTGCTGCGTCGTGACGCGGCTTGGTACCTGGCACACGAACTGATTGATGCTGGTCAAGCACTGTCTCTGCCGGGCTGAACCTGCTGCGTCGTGACGCGGCTTGGTACCTGGCACACGAACTGATTGATGCTGGTCAAGCACTGTCTCTGCCGGGC

CGTATCGATCAACAGTGCGTTGCACTGCGTCCGCATGTTTCCATGCTGATTGACGGTTTTGGTCTGAGCCCTGAACTGCTCGTATCGATCAACAGTGCGTTGCACTGCGTCCGCATGTTTCCATGCTGATTGACGGTTTTGGTCTGAGCCCTGAACTGCT

GCGTGCTCCGATCGCTCAGGACGATTACATCCAGGCCTTCTGCAAACAGGTTAATGCGAACGTAGACGCGTGCTCCGATCGCTCAGGACGATTACATCCAGGCCTTCTGCAAACAGGTTAATGCGAACGTAGAC

实施例2Example 2

本实施例中,取纯化后的巴纳氏杆菌和假单胞菌来源酰基辅酶A氧化酶进行后续的酶活检测实验。具体步骤如下:In this example, the purified acyl-CoA oxidases from Barnerella and Pseudomonas were used for subsequent enzyme activity detection experiments. Specific steps are as follows:

(1)溶液配制(1) Solution preparation

1M Tris-HCl pH 8.0:称取Tris粉末121.14g,倒入1L烧杯中并加灭菌纯化水至800ml。待搅拌均匀后使用浓盐酸在25℃下调pH为8.0,然后定容至1L。0.22um过滤,4℃保存;1M Tris-HCl pH 8.0: Weigh 121.14g of Tris powder, pour it into a 1L beaker and add sterilized purified water to 800ml. After stirring evenly, use concentrated hydrochloric acid to adjust the pH to 8.0 at 25°C, and then adjust the volume to 1L. Filter at 0.22um, store at 4°C;

5mM棕榈酰辅酶A:称取10mg棕榈酰辅酶A粉末溶于1.99mL去离子水中。5mM palmitoyl-CoA: Weigh 10mg of palmitoyl-CoA powder and dissolve in 1.99mL deionized water.

5mM FAD:称取0.0041g FAD粉末溶于1mL去离子水中,避光保存。。5mM FAD: Weigh 0.0041g FAD powder, dissolve it in 1mL deionized water, and store in the dark. .

0.1%4-氨基安替比林:称取0.05g 4-氨基安替比林粉末溶于50mL去离子水中,避光保存。0.1% 4-aminoantipyrine: Weigh 0.05g 4-aminoantipyrine powder, dissolve it in 50mL deionized water, and store in the dark.

0.1%苯酚:称取0.05g苯酚粉末溶于50mL去离子水中,避光保存。0.1% phenol: Weigh 0.05g of phenol powder and dissolve in 50mL of deionized water, and store in the dark.

5mL工作液配制参考表2。Refer to Table 2 for the preparation of 5mL working solution.

表2Table 2

试剂Reagent 加入体积add volume 终浓度Final concentration 1M Tris-HCl1M Tris-HCl 0.1mL0.1mL 20mM20mM 0.1%4-AA0.1% 4-AA 1.56mL1.56mL 1.5mM1.5mM 0.1%苯酚0.1% phenol 1mL1mL 2.1mM2.1mM 过氧化物酶(5U/μL)Peroxidase (5U/μL) 5μL5μL 25U25U FAD(5mM)FAD (5mM) 10μL10μL 10μM10μM 棕榈酰辅酶A(5mM)Palmitoyl-CoA (5mM) 500μL500μL 0.5mM0.5mM ddH2OddH2O 1795μL1795μL  the

阳性酶的配制:将200U阳性酶(市售的酰基辅酶A氧化酶对照品)溶于0.2mL PBSpH 7.4缓冲液中,制成1U/μL的酶液,按照梯度再逐步稀释,稀释液为PBS pH 7.4缓冲液。Preparation of positive enzyme: Dissolve 200U positive enzyme (commercially available acyl-CoA oxidase reference substance) in 0.2mL PBSpH 7.4 buffer solution to make 1U/μL enzyme solution, and gradually dilute it according to the gradient. The diluent is PBS pH 7.4 buffer.

(2)仪器检测(2) Instrument testing

酶标仪预热30min,加入反应液100μL,在500nm处检测吸光值,空白组加入1μL酶稀释液,在500nm处检测吸光值A1,实验组加入各浓度的酶液1μL,震荡混匀5s,反应5分钟,在500nm处检测吸光值A2,绘制标准曲线见图6。计算样品与空白的OD差值A2-A1,并根据差值求出样品浓度。结果见表3。Preheat the microplate reader for 30 minutes, add 100 μL of the reaction solution, and measure the absorbance at 500 nm. Add 1 μL of enzyme diluent to the blank group, and measure the absorbance A1 at 500 nm. Add 1 μL of enzyme solutions of various concentrations in the experimental group, shake and mix for 5 seconds. After reacting for 5 minutes, the absorbance value A2 was detected at 500 nm, and a standard curve was drawn as shown in FIG. 6 . Calculate the OD difference A2-A1 between the sample and the blank, and calculate the sample concentration based on the difference. The results are shown in Table 3.

表3table 3

Figure BDA0004026028410000141
Figure BDA0004026028410000141

Figure BDA0004026028410000151
Figure BDA0004026028410000151

经纯化的巴纳氏杆菌源重组酰基辅酶A氧化酶原液浓度为1.917mg/mL,平均活力为0.2611U/μL,则比活力为(0.4628*1000)/1.917=241.4U/mg。而假单胞菌来源的重组酰基辅酶A氧化酶经检测,无酶活。The concentration of the stock solution of the purified Barnerella-derived recombinant acyl-CoA oxidase is 1.917mg/mL, and the average activity is 0.2611U/μL, so the specific activity is (0.4628*1000)/1.917=241.4U/mg. However, the recombinant acyl-CoA oxidase derived from Pseudomonas has no enzymatic activity after testing.

实施例3Example 3

本实施例中,选择纯化产物酶活性好的巴纳氏杆菌源酰基辅酶A氧化酶,对其编码基因进行不同方式的大肠杆菌同义密码子偏好性优化,筛选获得可溶性表达量较高的优化密码子。In this example, the Barnerella-derived acyl-CoA oxidase with good enzymatic activity of the purified product was selected, and its coding gene was optimized for Escherichia coli synonymous codon preference in different ways, and the optimized one with higher soluble expression was screened. a.

选择巴纳氏杆菌源的酰基辅酶A氧化酶基因序列,经过不同于实施例1的同义密码子偏好性优化,得到大量优化密码子,示例性地如SEQ ID NO.4-7所示的优化密码子,并按照实施例1相同方式合成重组质粒并在大肠杆菌内进行培养,并测可溶性表达量,结果见表4。Select the acyl-CoA oxidase gene sequence derived from Barnerella, and optimize the synonymous codon preference different from Example 1 to obtain a large number of optimized codons, exemplarily as shown in SEQ ID NO.4-7 The codon was optimized, and the recombinant plasmid was synthesized in the same manner as in Example 1 and cultured in Escherichia coli, and the soluble expression level was measured. The results are shown in Table 4.

表4Table 4

优化密码子optimized codon 是否存在可溶性表达Whether there is soluble expression 纯化产量Purification Yield 优化密码子ⅠOptimized codon Ⅰ 可溶性表达Soluble expression 6.23mg/g6.23mg/g 优化密码子ⅣOptimized codon Ⅳ 包涵体表达Inclusion body expression // 优化密码子ⅤOptimized codon Ⅴ 可溶性表达Soluble expression 4.58mg/g4.58mg/g 优化密码子ⅥOptimized codon Ⅵ 包涵体表达Inclusion body expression // 优化密码子ⅦOptimize codon VII 包涵体表达Inclusion body expression //

SEQ ID NO.4:SEQ ID NO.4:

ATGACTGAAGTAGTGGATAGAGCTAGTTCCCCAGCATCCCCTGGATCAACTACCGCTGCTGCTGATGGTGCCAAAGTTGCAGTTGAACCAAGAGTTGACGTCGCTGCTCTGGGAGAACAGTTACTAGGAAGATGGGCTGATATAAGGTTGCATGCTAGAGATCTGGCTGGTAGAGAGGTCGTTCAAAAGGTCGAGGGTCTTACTCATACCGAACACAGATCCAGAGTCTTTGGACAACTTAAATACTTGGTCGACAATAACGCCGTTCATAGAGCTTTTCCTAGTAGGTTAGGAGGATCTGATGACCACGGTGGAAACATTGCTGGCTTTGAAGAATTGGTAACAGCTGATCCTTCCCTTCAGATTAAGGCTGGCGTCCAATGGGGCCTTTTTGGATCCGCTGTTATGCACCTTGGTACACGTGAGCACCATGATAAGTGGCTGCCAGGAATTATGAGTCTGGAGATCCCCGGTTGTTTTGCTATGACAGAAACAGGACATGGTTCCGACGTTGCTAGTATTGCAACAACCGCTACTTACGATGAAGAAACTCAGGAGTTCGTCATTGACACTCCATTCCGTGCTGCATGGAAGGACTATATTGGAAACGCTGCTAATGACGGACTTGCTGCAGTGGTATTTGCCCAACTAATAACACGAAAAGTTAACCATGGCGTTCATGCCTTCTATGTCGATTTGAGAGACCCCGCCACAGGTGACTTTTTGCCAGGTATAGGAGGTGAGGATGATGGTATAAAGGGAGGTTTGAACGGTATAGATAATGGAAGGTTGCACTTCACATGACTGAAGTAGTGGATAGAGCTAGTTCCCCAGCATCCCCTGGATCAACTACCGCTGCTGCTGATGGTGCCAAAGTTGCAGTTGAACCAAGAGTTGACGTCGCTGCTCTGGGAGAACAGTTACTAGGAAGATGGGCTGATATAAGGTTGCATGCTAGAGATCTGGCTGGTAGAGAGGTCGTTCAAAAGGTCGAGGGTCTTACTCATACCGA ACACAGATCCAGAGTCTTTGGACAACTTAAATACTTGGTCGACAATAACGCCGTTCATAGAGCTTTTCCTAGTAGGTTAGGAGGATCTGATGACCACGGTGGAAACATTGCTGGCTTTGAAGAATTGGTAACAGCTGATCCTTCCCTTCAGATTAAGGCTGGCGTCCAATGGGGCCTTTTTGGATCCGCTGTTATGCACCTTGGTACACGTGAG CACCATGATAAGTGGCTGCCAGGAATTATGAGTCTGGAGATCCCCGGTTGTTTTGCTATGACAGAAACAGGACATGGTTCCGACGTTGCTAGTATTGCAACAACCGCTACTTACGATGAAGAAACTCAGGAGTTCGTCATTGACACTCCATTCCGTGCTGCATGGAAGGACTATATTGGAAACGCTGCTAATGACGGACTTGCTGCAGTGGT ATTTGCCCAACTAATAACACGAAAAGTTAACCATGGCGTTCATGCCTTCTATGTCGATTTGAGAGACCCCGCCACAGGTGACTTTTTGCCAGGTATAGGAGGTGAGGATGATGGTATAAAGGGAGGTTTGAACGGTATAGATAATGGAAGGTTGCACTTCAC

CAACGTAAGAATTCCACGTACAAACCTGCTGAACAGATATGGCGATGTGGCTGTCGACGGTACATACAGTTCAACTATTGCAACGTAAGAATTCCACGTACAAACCTGCTGAACAGATATGGCGATGTGGCTGTCGACGGTACATACAGTTCAACTATTG

AAAGTCCTGGTCGAAGATTCTTTACTATGCTGGGAACCTTAGTTCAGGGACGTGTTAGTTTGGATGGTGCCGCTGTTGCTAAAGTCCTGGTCGAAGATTCTTACTATGCTGGGAACCTTAGTTCAGGGACGTGTTAGTTTGGATGGTGCCGCTGTTGCT

GCTAGTAAGGTTGCTTTACAGTCTGCTATCCACTATGCCGCTGAAAGACGTCAGTTCAACGCCACTTCTCCTACCGAAGAGCTAGTAAGGTTGCTTTACAGTCTGCTATCCACTATGCCGCTGAAAGACGTCAGTTCAACGCCACTTCTCTCTACCGAAGA

AGAGGTTTTGTTAGACTACCAAAGGCACCAGAGAAGACTATTCACAAGATTGGCAACAACGTACGCTGCTTCTTTCGCTCAGAGGTTTTGTTAGACTACCAAAGGCACCAGAGAAGACTATTCACAAGATTGGCAACAACGTACGCTGCTTCTTTCGCTC

ATGAACAACTTTTGCAGAAGTTTGATGATGTTTTTTCTGGAGCACACGACACTGACGCAGACAGACAGGATCTAGAGACCATGAACAACTTTTGCAGAAGTTTGATGATGTTTTTTCTGGAGCACACGACACTGACGCAGACAGACAGGATCTAGAGACC

CTTGCTGCTGCTCTGAAACCACTATCCACTTGGCACGCACTTGACACCTTGCAAGAATGTAGAGAAGCTTGTGGTGGTGCCTTGCTGCTGCTCTGAAACCACTATCCACTTGGCACGCACTTGACACCTTGCAAGAATGTAGAGAAGCTTGTGGTGGTGC

TGGTTTCTTGATTGAAAATAGATTTGCTTCCTTAAGAGCAGATTTAGATGTCTATGTCACTTTTGAAGGAGACAATACCGTGGTTTCTTGATTGAAAATAGATTTGCTTCCTTAAGAGCAGATTTAGATGTCTATGTCACTTTTGAAGGAGACAATACCG

TTCTGCTTCAACTTGTCGCTAAGCGATTGTTAGCCGATTATGCTAAGGAGTTCAGAGGAGCTAATTTTGGCGTACTGGCATTCTGCTTCAACTTGTCGCTAAGCGATTGTTAGCCGATTATGCTAAGGAGTTCAGAGGAGCTAATTTTGGCGTACTGGCA

CGATACGTTGTTGATCAGGCTGCTGGAGTTGCTCTTCATAGGACTGGCCTAAGACAGGTTGCTCAATTCGTAGCCGATTCCGATACGTTGTTGATCAGGCTGCTGGAGTTGCTCTTCATAGGACTGGCCTAAGACAGGTTGCTCAATTCGTAGCCGATTC

CGGAAGTGTGCAAAAGTCTGCCTTGGCACTACGTGATGAAGAAGGTCAAAGAACATTGTTGACCGACCGAGTTCAATCCACGGAAGTGTGCAAAAGTCTGCCTTGGCACTACGTGATGAAGAAGGTCAAAGAACATTGTTGACCGACCGAGTTCAATCCA

TGGTCGCTGAGGTGGGAGCCGCTTTGAAGGGAGCTGGAAAGCTACCCCAACATCAAGCAGCTGCCCTATTCAACCAACATTGGTCGCTGAGGTGGGAGCCGCTTTGAAGGGAGCTGGAAAGCTACCCCAACATCAAGCAGCTGCCCTATTCAACCAACAT

CAAAACGAACTTATTGAAGCAGCACAGGCTCATGCAGAATTATTGCAGTGGGAGGCTTTCACAGAAGCTTTGGCTAAGGTCAAAACGAACTTATTGAAGCAGCACAGGCTCATGCAGAATTATTGCAGTGGGAGGCTTTTCACAGAAGCTTTGGCTAAGGT

TGACGATGCCGGAACTAAGGAAGTGTTGACTAGACTGAGAGACTTGTTTGGTCTATCTCTGATTGAAAAGCACCTATCCTTGACGATGCCGGAACTAAGGAAGTGTTGACTAGACTGAGAGACTTGTTTGGTCTATCTCTGATTGAAAAGCACCTATCCT

GGTACTTAATGAACGGTCGACTGTCTATGCAGAGAGGAAGAACTGTCGGAACTTATATTAATAGACTTCTAGTGAAGATCGGTACTTAATGAACGGTCGACTGTCTATGCAGAGAGGAAGAACTGTCGGAACTTATATTAATAGACTTCTAGTGAAGATC

AGGCCACACGCCCTTGATTTGGTGGATGCTTTCGGATACGGTGCCGAACATCTTAGGGCTGCTATTGCAACTGGTGCTGAAGGCCACACGCCCTTGATTTGGTGGATGCTTTCGGATACGGTGCCGAACATCTTAGGGCTGCTATTGCAACTGGTGCTGA

GGCTACAAGACAGGACGAAGCCCGTACTTACTTTAGACAACAGCGAGCTTCCGGATCCGCACCAGCAGATGAAAAAACATGGCTACAAGACAGGACGAAGCCCGTACTTACTTTTAGACAACAGCGAGCTTCCGGATCCGCACCAGCAGATGAAAAAACAT

TATTAGCTATCAAAGCTGGTAAATCTAGGTATTAGCTATCAAAGCTGGTAAATCTAGG

SEQ ID NO.5:SEQ ID NO.5:

ATGACGGAAGTAGTAGACCGAGCATCATCTCCAGCTTCACCAGGATCCACAACAGCTGCTGCAGATGGCGCAAAGGTTGCATGACGGAAGTAGTAGACCGAGCATCATCTCCAGCTTCACCAGGATCCACAACAGCTGCTGCAGATGGCGCAAAGGTTGC

GGTGGAACCTAGAGTTGATGTCGCGGCTCTTGGAGAACAGCTTCTCGGCAGATGGGCAGATATTCGTCTGCATGCAAGAGGGTGGAACCTAGAGTTGATGTCGCGGCTCTTGGAGAACAGCTTCTCGGCAGATGGGCAGATATTCGTCTGCATGCAAGAG

ACCTTGCGGGGCGCGAAGTCGTTCAGAAAGTGGAAGGACTGACACATACCGAACATCGCTCTCGTGTCTTTGGACAGCTGACCTTGCGGGGCGCGAAGTCGTTCAGAAAGTGGAAGGACTGACACATACCGAACATCGCTCTCGTGTCTTTGGACAGCTG

AAATACCTGGTCGATAACAATGCTGTGCATCGTGCTTTTCCGAGCAGATTGGGCGGATCTGATGACCACGGGGGAAACATAAATACCTGGTCGATAACAATGCTGTGCATCGTGCTTTTCCGAGCAGATTGGGCGGATCTGATGACCACGGGGGAAACAT

CGCCGGCTTCGAAGAACTGGTGACGGCAGATCCTTCACTTCAGATTAAAGCGGGAGTGCAGTGGGGCCTTTTCGGATCAGCGCCGGCTTCGAAGAACTGGTGACGGCAGATCCTTCACTTCAGATTAAAGCGGGAGTGCAGTGGGGCCTTTTCGGATCAG

CTGTGATGCATCTGGGTACCAGAGAACATCATGACAAATGGCTTCCGGGAATCATGTCACTTGAAATTCCTGGATGTTTCCTGTGATGCATCTGGGTACCAGAGAACATCATGACAAATGGCTTCCGGGAATCATGTCACTTGAAATTCCTGGATGTTTC

GCTATGACGGAGACCGGTCACGGGTCTGACGTTGCCTCAATTGCCACAACAGCGACATACGATGAGGAAACACAAGAATTGCTATGACGGAGACCGGTCACGGGTCTGACGTTGCCTCAATTGCCACAACAGCGACATACGATGAGGAAACACAAGAATT

TGTCATAGACACACCTTTTCGAGCTGCTTGGAAAGATTATATAGGAAACGCTGCGAACGATGGCCTTGCAGCGGTAGTTTTGTCATAGACACACCTTTTCGAGCTGCTTGGAAAGATTATAGGAAACGCTGCGAACGATGGCCTTGCAGCGGTAGTTT

TTGCGCAACTTATAACAAGAAAAGTTAACCATGGGGTCCACGCCTTCTACGTTGACCTTAGAGATCCTGCCACAGGTGATTTGCGCAACTTATAACAAGAAAAGTTAACCATGGGGTCCACGCCTTCTACGTTGACCTTAGAGATCCTGCCACAGGTGAT

TTCTTACCAGGTATCGGGGGCGAAGATGATGGTATTAAAGGAGGTTTAAACGGGATCGATAATGGTCGTTTACATTTTACTTCTTACCAGGTATCGGGGGCGAAGATGATGGTATTAAAGGAGGTTTAAACGGGATCGATAATGGTCGTTTACATTTTAC

TAACGTTCGCATTCCGCGCACTAACTTGTTAAACCGCTATGGCGACGTGGCAGTCGATGGAACATATTCCTCTACGATTGTAACGTTCGCATTCCGCGCACTAACTTGTTAAACCGCTATGGCGACGTGGCAGTCGATGGAACATATTCCTCTACGATTG

AAAGCCCGGGACGTCGGTTTTTCACAATGTTAGGCACACTTGTACAAGGTAGAGTCTCGTTAGATGGCGCGGCAGTAGCAAAAGCCCGGGACGTCGGTTTTTCACAATGTTAGGCACACTTGTACAAGGTAGAGTCTCGTTAGATGGCGCGGCAGTAGCA

GCAAGCAAAGTTGCTCTGCAGTCTGCCATTCATTATGCTGCGGAACGACGGCAGTTTAATGCGACCAGCCCGACAGAAGAGCAAGCAAAGTTGCTCTGCAGTCTGCCATTCATTATGCTGCGGAACGACGGCAGTTTAATGCGACCAGCCCCGACAGAAGA

AGAGGTCCTTCTCGATTACCAGAGACATCAGAGAAGACTTTTCACTCGTCTTGCAACGACTTATGCGGCTTCCTTTGCTCAGAGGTCCTTCTCGATTACCAGAGACATCAGAGAAGACTTTTCACTCGTCTTGCAACGACTTATGCGGCTTCCTTTGCTC

ATGAACAGCTTTTGCAAAAATTCGACGATGTCTTTTCTGGCGCGCATGATACTGACGCTGACCGTCAGGATCTGGAGACAATGAACAGCTTTTGCAAAAATTCGACGATGTCTTTTCTGGCGCGCATGATACTGACGCTGACCGTCAGGATCTGGAGACA

TTGGCCGCCGCCCTTAAACCGTTATCCACCTGGCATGCTTTAGACACGCTTCAAGAATGCCGAGAAGCGTGCGGCGGAGCTTGGCCGCCGCCCTTAAACCGTTATCCACCTGGCATGCTTTAGACACGCTTCAAGAATGCCGAGAAGCGTGCGGCGGAGC

AGGTTTTTTGATTGAGAATAGATTTGCGTCCTTGCGCGCGGACTTAGATGTTTATGTTACATTTGAGGGCGACAACACAGAGGTTTTTTGATTGAGAATAGATTTGCGTCCTTGCGCGCGGACTTAGATGTTTTATGTTACATTTGAGGGCGACAACACAG

TACTGTTACAATTAGTTGCTAAACGCCTGCTTGCCGATTATGCGAAGGAATTCCGAGGCGCTAATTTTGGCGTGCTGGCATACTGTTACAAATTAGTTGCTAAACGCCTGCTTGCCGATTATGCGAAGGAATTCCGAGGCGCTAATTTTGGCGTGCTGGCA

CGCTATGTTGTTGATCAAGCTGCGGGTGTTGCATTGCATCGGACAGGGCTCAGACAGGTTGCTCAATTTGTTGCCGACTCCGCTATGTTGTTGATCAAGCTGCGGGTGTTGCATTGCATCGGACAGGGCTCAGACAGGTTGCTCAATTTGTTGCCGACTC

AGGCTCCGTGCAAAAGTCCGCATTGGCACTGCGGGATGAAGAAGGACAGCGAACGCTGCTGACAGACCGGGTACAGTCTAAGGCTCCGTGCAAAAGTCCGCATTGGCACTGCGGGATGAAGAAGGACAGCGAACGCTGCTGACAGACCGGGTACAGTCTA

TGGTAGCAGAAGTCGGCGCTGCGCTTAAAGGTGCAGGAAAACTTCCGCAGCACCAGGCCGCTGCTCTCTTCAACCAGCATTGGTAGCAGAAGTCGGCGCTGCGCTTAAAGGTGCAGGAAAACTTCCGCAGCACCAGGCCGCTGCTCTCTTCAACCAGCAT

CAGAATGAACTGATCGAAGCTGCCCAAGCGCACGCCGAACTGTTACAATGGGAAGCATTCACAGAAGCGCTTGCTAAGGTCAGAATGAACTGATCGAAGCTGCCCAAGCGCACGCCGAACTGTTACAATGGGAAGCATTCACAGAAGCGCTTGCTAAGGT

AGATGATGCGGGTACCAAAGAAGTGTTAACCCGTCTGAGAGACCTGTTTGGGCTGTCACTGATAGAGAAACATTTATCATAGATGATGCGGGTACCAAAGAAGTGTTAACCCGTCTGAGAGACCTGTTTGGGCTGTCACTGATAGAGAAACATTTATCAT

GGTATCTCATGAACGGACGGCTCAGCATGCAGCGTGGACGTACTGTTGGGACCTACATTAATAGACTTCTGGTAAAAATCGGTATCTCATGAACGGACGGCTCAGCATGCAGCGTGGACGTACTGTTGGGACCTACATTAATAGACTTCTGGTAAAAATC

CGCCCTCATGCTCTTGATCTCGTAGATGCTTTTGGCTACGGCGCAGAACATCTTAGAGCTGCAATTGCGACAGGAGCGGACGCCCTCATGCTCTTGATCTCGTAGATGCTTTTGGCTACGGCGCAGAACATCTTAGAGCTGCAATTGCGACAGGAGCGGA

AGCTACTCGCCAAGACGAAGCACGGACATATTTTCGGCAGCAGCGAGCAAGTGGTTCAGCCCCAGCAGACGAAAAAACATAGCTACTCGCCAAGACGAAGCACGGACATATTTTTCGGCAGCAGCGAGCAAGTGGTTCAGCCCCAGCAGACGAAAAAACAT

TGCTTGCGATTAAAGCCGGCAAGTCAAGATGCTTGCGATTAAAGCCGGCAAGTCAAGA

SEQ ID NO.6:SEQ ID NO.6:

ATGACTGAAGTCGTTGATAGAGCATCCTCACCCGCATCTCCAGGTTCAACGACAGCCGCTGCTGATGGTGCTAAGGTTGCATGACTGAAGTCGTTGATAGAGCATCCTCACCCGCATCTCCAGGTTCAACGACAGCCGCTGCTGATGGTGCTAAGGTTGC

TGTGGAACCAAGAGTTGATGTCGCGGCACTGGGTGAACAATTATTAGGTCGTTGGGCTGATATCCGTCTTCATGCTAGGGTGTGGAACCAAGAGTTGATGTCGCGGCACTGGGTGAACAATTATTAGGTCGTTGGGCTGATATCCGTCTTCATGCTAGGG

ATTTGGCTGGTAGAGAGGTAGTTCAAAAGGTGGAGGGGTTGACCCACACAGAACATAGATCTAGGGTTTTTGGTCAGTTGATTTGGCTGGTAGAGAGGTAGTTCAAAAGGTGGAGGGGTTGACCCACACAGAACATAGATCTAGGGTTTTTGGTCAGTTG

AAATACTTGGTGGATAATAATGCTGTACATAGGGCTTTTCCATCCAGGTTGGGTGGATCAGACGACCATGGTGGTAACATAAATACTTGGTGGATAATAATGCTGTACATAGGGCTTTTCCATCCAGGTTGGGTGGATCAGACGACCATGGTGGTAACAT

CGCAGGTTTTGAAGAACTAGTAACCGCTGATCCATCTCTTCAAATCAAAGCTGGAGTCCAATGGGGCCTGTTCGGGAGTGCGCAGGTTTTGAAGAACTAGTAACCGCTGATCCATCTCTTCAAATCAAAGCTGGAGTCCAATGGGGCCTGTTCGGGAGTG

CTGTTATGCATCTAGGTACAAGAGAGCATCATGATAAGTGGTTGCCTGGAATAATGAGTTTGGAAATTCCCGGTTGTTTTCTGTTATGCATCTAGGTACAAGAGAGCATCATGATAAGTGGTTGCCTGGAATAATGAGTTTGGAAATTCCCGGTTGTTTT

GCTATGACCGAGACGGGTCACGGCTCTGATGTCGCCTCCATCGCTACAACTGCAACCTACGATGAAGAAACCCAAGAATTGCTATGACCGAGACGGGTCACGGCTCTGATGTCGCCTCCATCGCTACAACTGCAACCTACGATGAAGAAACCCAAGAATT

TGTTATAGATACCCCTTTTAGAGCTGCATGGAAAGATTATATCGGTAATGCTGCAAACGATGGTCTTGCTGCTGTGGTTTTGTTATAGATACCCCTTTTAGAGCTGCATGGAAAGATTATATCGGTAATGCTGCAAACGATGGTCTTGCTGCTGTGGTTT

TCGCGCAATTGATTACTCGTAAAGTTAATCATGGTGTTCACGCTTTCTACGTAGATTTGCGTGACCCAGCTACTGGGGATTCGCGCAATTGATTACTCGTAAAGTTAATCATGGTGTTCACGCTTTCTACGTAGATTTGCGTGACCCAGCTACTGGGGAT

TTCTTGCCAGGTATCGGTGGTGAAGACGATGGTATTAAAGGTGGTTTGAATGGGATTGATAATGGCAGATTACATTTTACTTCTTGCCAGGTATCGGTGGTGAAGACGATGGTATTAAAGGTGGTTTGAATGGGATTGATAATGGCAGATTACATTTTAC

AAACGTGAGAATCCCTAGAACAAACCTTCTAAACAGATATGGAGATGTAGCAGTTGATGGTACCTACAGTTCAACAATAGAAACGTGAGAATCCCTAGAACAAACCTTCTAAACAGATATGGAGATGTAGCAGTTGATGGTACCTACAGTTCAACAATAG

AATCACCTGGTAGAAGATTCTTCACCATGTTGGGTACTCTGGTTCAAGGAAGAGTGAGTTTGGATGGGGCAGCAGTTGCTAATCACCTGGTAGAAGATTCTTCACCATGTTGGGTACTCTGGTTCAAGGAAGAGTGAGTTTGGATGGGGCAGCAGTTGCT

GCTTCAAAAGTGGCTTTGCAAAGCGCCATTCATTATGCAGCAGAACGTAGACAATTTAATGCCACTTCTCCGACTGAGGAGCTTCAAAAGTGGCTTTGCAAAGCGCCATTCATTATGCAGCAGAACGTAGACAATTTAATGCCACTTCTCCGACTGAGGA

AGAGGTATTATTGGATTACCAAAGGCATCAGAGGAGGTTATTTACGAGATTAGCAACTACTTACGCTGCATCTTTTGCACAGAGGTATTATTGGATTACCAAAGGCATCAGAGGAGGTTATTTACGAGATTAGCAACTACTTACGCTGCATCTTTTGCAC

ACGAACAATTGCTGCAAAAATTTGACGACGTTTTTTCAGGTGCACATGATACAGATGCCGATAGGCAAGATTTGGAGACTACGAACAATTGCTGCAAAAATTTGACGACGTTTTTTTCAGGTGCACATGATACAGATGCCGATAGGCAAGATTTGGAGACT

CTTGCCGCCGCACTTAAACCACTTAGTACTTGGCATGCTCTAGACACTCTTCAAGAGTGCAGAGAGGCATGCGGTGGAGCCTTGCCGCCGCACTTAAACCACTTAGTACTTGGCATGCTCTAGACACTCTTCAAGAGTGCAGAGAGGCATGCGGTGGAGC

GGGATTTCTGATAGAAAATAGATTTGCAAGCTTAAGAGCCGATTTGGATGTCTACGTTACCTTTGAAGGTGACAATACTGGGGATTTCTGATAGAAAATAGATTTGCAAGCTTAAGAGCCGATTTGGATGTCTACGTTACCTTTGAAGGTGACAATACTG

TTCTTTTGCAATTGGTTGCTAAGAGGTTGTTGGCCGATTACGCAAAGGAATTCAGAGGAGCTAATTTTGGTGTTTTGGCCTTCTTTTGCAATTGGTTGCTAAGAGGTTGTTGGCCGATTACGCAAAGGAATTCAGAGGAGCTAATTTTGGTGTTTTGGCC

AGATACGTAGTCGATCAAGCTGCCGGGGTTGCACTGCATAGAACAGGTCTTAGACAAGTGGCACAGTTCGTCGCGGATTCAGATACGTAGTCGATCAAGCTGCCGGGGTTGCACTGCATAGAACAGGTCTTAGACAAGTGGCACAGTTCGTCGCGGATTC

AGGTAGCGTACAAAAGTCAGCCCTAGCCCTTAGGGATGAAGAAGGCCAGAGAACCTTGCTAACTGATAGAGTACAATCTAAGGTAGCGTACAAAAGTCAGCCTAGCCCTTAGGGATGAAGAAGGCCAGAGAACCTTGCTAACTGATAGAGTACAATCTA

TGGTAGCTGAAGTTGGAGCAGCTTTGAAGGGTGCCGGGAAATTACCACAGCATCAAGCCGCAGCGTTGTTTAACCAGCATTGGTAGCTGAAGTTGGAGCAGCTTTGAAGGGTGCCGGGAAATTACCACAGCATCAAGCCGCAGCGTTGTTTAACCAGCAT

CAAAATGAATTAATCGAAGCTGCTCAAGCTCACGCAGAATTGTTGCAATGGGAAGCTTTTACTGAAGCTCTAGCTAAGGTCAAAATGAATTAATCGAAGCTGCTCAAGCTCACGCAGAATTGTTGCAATGGGAAGCTTTTACTGAAGCTCTAGCTAAGGT

CGATGATGCGGGCACGAAAGAGGTTTTGACTAGATTACGTGATCTGTTCGGATTGTCTTTGATTGAAAAACATTTATCTTCGATGATGCGGGCACGAAAGAGGTTTTGACTAGATTACGTGATCTGTTCGGATTGTCTTTGATTGAAAAACATTTATCTT

GGTATTTAATGAATGGTCGTTTAAGTATGCAGAGGGGAAGAACAGTTGGTACATATATTAACCGTTTGCTTGTAAAGATAGGTATTTAATGAATGGGTCGTTTAAGTATGCAGAGGGGAAGAACAGTTGGTACATATATTAACCGTTTGCTTGTAAAGATA

AGGCCTCATGCGTTGGATCTTGTTGACGCCTTTGGATATGGGGCAGAACATTTAAGGGCTGCTATCGCAACCGGTGCTGAAGGCCTCATGCGTTGGATCTTGTTGACGCCTTTGGATATGGGGCAGAACATTTAAGGGCTGCTATCGCAACCGGTGCTGA

GGCAACTAGACAAGATGAAGCAAGAACATATTTCAGACAGCAAAGAGCAAGTGGAAGCGCCCCAGCTGACGAAAAGACCTGGCAACTAGACAAGATGAAGCAAGAACATATTTCAGACAGCAAAGAGCAAGTGGAAGCGCCCCAGCTGACGAAAAGACCT

TGTTGGCTATCAAAGCGGGAAAGTCCAGATGTTGGCTATCAAAGCGGGAAAGTCCAGA

SEQ ID NO.7:SEQ ID NO.7:

ATGACTGAGGTAGTGGACAGGGCTTCTAGTCCTGCATCTCCTGGATCTACAACTGCAGCTGCCGACGGTGCAAAGGTGGCATGACTGAGGTAGTGGACAGGGCTTCTAGTCCTGCATCTCCTGGATCTACAACTGCAGCTGCCGACGGTGCAAAGGTGGC

AGTTGAGCCTAGAGTGGACGTTGCTGCCCTCGGAGAACAACTGCTGGGTCGGTGGGCCGATATTAGGTTGCATGCCAGAGAGTTGAGCCTAGAGTGGACGTTGCTGCCCTCGGAGAACAACTGCTGGGTCGGTGGGCCGATATTAGGTTGCATGCCAGAG

ATCTGGCCGGTAGGGAAGTCGTTCAGAAGGTCGAAGGACTGACCCACACCGAACATAGGAGCAGAGTTTTCGGCCAGCTTATCTGGCCGGTAGGGAAGTCGTTCAGAAGGTCGAAGGACTGACCCACACCGAACATAGGAGCAGAGTTTTCGGCCAGCTT

AAATACCTGGTTGATAATAATGCAGTCCACAGAGCCTTTCCCAGCCGACTTGGAGGAAGCGACGACCACGGAGGCAATATAAATACCTGGTTGATAATAATGCAGTCCACAGAGCCTTTCCCAGCCGACTTGGAGGAAGCGACGACCACGGAGGCAATAT

CGCAGGCTTTGAAGAGCTGGTTACCGCCGATCCCTCCCTTCAAATTAAAGCCGGGGTGCAGTGGGGTCTGTTTGGTAGTGCGCAGGCTTTGAAGAGCTGGTTACCGCCGATCCCTCCCTTCAAATTAAAGCCGGGGTGCAGTGGGGTCTGTTTGGTAGTG

CAGTAATGCACCTGGGCACAAGGGAACACCACGACAAGTGGCTCCCAGGGATCATGAGCCTGGAAATCCCGGGGTGTTTTCAGTAATGCACCTGGGCACAAGGGAACACCACGACAAGTGGCTCCCAGGGATCATGAGCCTGGAAATCCCGGGGTGTTTT

GCCATGACCGAAACCGGACATGGCTCTGACGTGGCGTCTATTGCTACAACGGCAACTTACGACGAGGAAACCCAAGAGTTGCCATGACCGAAACCGGACATGGCTCTGACGTGGCGTCTATTGCTACAACGGCAACTTACGACGAGGAAACCCAAGAGTT

TGTTATAGATACCCCCTTCAGAGCCGCCTGGAAGGACTATATCGGTAATGCAGCAAACGATGGTCTCGCCGCCGTTGTGTTGTTATAGATACCCCCTTCAGAGCCGCCTGGAAGGACTATATCGGTAATGCAGCAAACGATGGTCTCGCCGCCGTTGTGT

TTGCCCAGCTGATTACAAGAAAGGTGAACCACGGGGTTCACGCATTTTACGTGGATCTCAGAGACCCTGCAACCGGTGATTTGCCCAGCTGATTACAAGAAAGGTGAACCACGGGGTTCACGCATTTTACGTGGATCTCAGAGACCCTGCAACCGGTGAT

TTCTTGCCTGGCATCGGAGGGGAAGATGACGGGATCAAGGGTGGCCTGAATGGGATTGACAACGGTAGGCTGCATTTCACTTCTTGCCTGGCATCGGAGGGGAAGATGACGGGATCAAGGGTGGCCTGAATGGGATTGACAACGGTAGGCTGCATTTCAC

AAACGTGAGGATCCCCAGGACCAACCTGCTCAATAGATACGGTGATGTGGCCGTAGATGGTACATACTCCAGCACCATCGAAACGTGAGGATCCCCAGGACCAACCTGCTCAATAGATACGGTGATGTGGCCGTAGATGGTACATACTCCAGCACCATCG

AGTCCCCCGGACGAAGGTTTTTTACCATGTTGGGTACGCTCGTCCAGGGCCGGGTGTCTCTTGATGGAGCAGCGGTGGCAAGTCCCCCGGACGAAGGTTTTTTTACCATGTTGGGTACGCTCGTCCAGGGCCGGGTGTCTCTTGATGGAGCAGCGGTGGCA

GCTTCAAAGGTGGCCCTCCAGTCTGCCATTCACTACGCTGCTGAGCGAAGGCAGTTCAACGCCACCTCTCCAACCGAGGAGCTTCAAAGGTGGCCCTCCAGTCTGCCATTCACTACGCTGCTGAGCGAAGGCAGTTCAACGCCACCTCTCCAACCGAGGA

GGAGGTGCTGTTGGATTACCAAAGGCACCAAAGACGCCTTTTCACCCGGCTCGCAACCACATACGCAGCAAGCTTCGCGCGGAGGTGCTGTTGGATTACCAAAGGCACCAAAGACGCCTTTTCACCCGGCTCGCAACCACATACGCAGCAAGCTTCGCGC

ACGAGCAGCTGCTCCAGAAGTTCGATGACGTTTTCTCTGGAGCCCATGATACCGATGCCGATAGGCAAGACCTGGAGACCACGAGCAGCTGCTCCAGAAGTTCGATGACGTTTTCTCTGGAGCCCATGATACCGATGCCGATAGGCAAGACCTGGAGACC

TTGGCAGCAGCACTGAAACCTCTCTCAACCTGGCACGCTTTGGACACTTTGCAGGAATGCAGGGAGGCATGCGGAGGTGCTTGGCAGCAGCACTGAAACCTCTCTCAACCTGGCACGCTTTGGACACTTTGCAGGAATGCAGGGAGGCATGCGGAGGTGC

TGGCTTCCTGATCGAGAACCGCTTTGCTTCACTTCGCGCCGATCTTGACGTATATGTCACTTTCGAAGGGGACAATACCGTGGCTTCCTGATCGAGAACCGCTTTGCTTCACTTCGCGCCGATCTTGACGTATATGTCACTTTCGAAGGGGACAATACCG

TGCTTCTCCAGCTTGTCGCAAAAAGACTGCTGGCAGACTACGCCAAGGAATTCAGAGGAGCAAATTTTGGAGTATTGGCCTGCTTCTCCAGCTTGTCGCAAAAAGACTGCTGGCAGACTACGCCAAGGAATTCAGAGGAGCAAATTTTGGAGTATTGGCC

AGATACGTCGTGGATCAGGCAGCGGGAGTTGCACTGCACAGGACTGGCTTGCGCCAGGTTGCTCAGTTTGTGGCCGACAGAGATACGTCGTGGATCAGGCAGCGGGAGTTGCACTGCACAGGACTGGCTTGCGCCAGGTTGCTCAGTTTGTGGCCGACAG

TGGAAGCGTACAGAAGAGTGCATTGGCTCTGCGGGATGAGGAAGGGCAGAGAACGCTGCTGACCGATCGGGTGCAGTCAATGGAAGCGTACAGAAGAGGTGCATTGGCTCTGCGGGATGAGGAAGGGCAGAGAACGCTGCTGACCGATCGGGTGCAGTCAA

TGGTGGCAGAGGTGGGCGCAGCTCTGAAAGGAGCAGGGAAACTGCCTCAGCATCAGGCTGCTGCTCTGTTCAACCAGCACTGGTGGCAGAGGTGGGCGCAGCTCTGAAAGGAGCAGGGAAACTGCCTCAGCATCAGGCTGCTGCTCTGTTCAACCAGCAC

CAGAACGAGCTGATCGAGGCCGCACAAGCCCATGCAGAGTTGCTGCAGTGGGAGGCGTTTACAGAGGCCCTGGCTAAAGTCAGAACGAGCTGATCGAGGCCGCACAAGCCCATGCAGAGTTGCTGCAGTGGGAGGCGTTTACAGAGGCCCTGGCTAAAGT

TGATGATGCTGGCACTAAAGAGGTCCTCACTAGGTTGCGGGATCTCTTCGGACTGTCTCTGATTGAAAAACATCTGTCATTGATGATGCTGGCACTAAAGAGGTCCTCACTAGGTTGCGGGATCTCTTCGGACTGTCTCTGATTGAAAAACATCTGTCAT

GGTATCTGATGAATGGACGACTTTCCATGCAGCGGGGGAGAACAGTGGGAACTTATATTAACCGGCTGCTGGTAAAAATTGGTATCTGATGAATGGACGACTTTCCATGCAGCGGGGGAGAACAGTGGGAACTTATATTAACCGGCTGCTGGTAAAAATT

CGACCCCATGCTCTCGACCTGGTGGATGCCTTTGGTTACGGCGCTGAACACCTCCGGGCTGCTATAGCCACTGGAGCAGACGACCCCATGCTCTCGACCTGGTGGATGCCTTTGGTTACGGCGCTGAACACCTCCGGGCTGCTATAGCCACTGGAGCAGA

AGCCACAAGGCAGGACGAGGCCAGGACATACTTTAGACAACAGAGAGCCAGTGGATCTGCCCCCGCTGATGAAAAGACCCAGCCACAAGGCAGGACGAGGCCAGGACATACTTTAGACAACAGAGAGCCAGTGGATCTGCCCCCGCTGATGAAAAGACCC

TGCTCGCAATTAAAGCAGGCAAGTCCAGGTGCTCGCAATTAAAGCAGGCAAGTCCAGG

本领域的普通技术人员可以理解,上述各实施方式是实现本发明的具体实施例,而在实际应用中,可以在形式上和细节上对其作各种改变,而不偏离本发明的精神和范围。Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made to it in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (10)

1. An isolated polynucleotide encoding an acyl-coa oxidase, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of the following:
(i) A polynucleotide as shown in SEQ ID NO. 1;
(ii) A polynucleotide having a homology of greater than 95% with the sequence shown in SEQ ID NO. 1; and
(iii) A polynucleotide complementary to the sequence set forth in (i) or (ii).
2. An expression vector comprising the polynucleotide of claim 1.
3. The expression vector according to claim 3, characterized in that it is an E.coli expression vector, preferably pET-28a (+).
4. A host cell comprising the expression vector of claim 2 or 3; or alternatively
The polynucleotide of claim 1 integrated into the genome of the host cell.
5. A method for preparing an acyl-coa oxidase, the method comprising the steps of:
transforming a host cell with a vector comprising the polynucleotide of claim 1;
culturing the host cell to express the acyl-coa oxidase.
6. The method of claim 5, wherein the host cells are cultured using TB medium.
7. The method of claim 5, wherein the host cell is cultured at a temperature of 16 to 19 ℃.
8. The method according to claim 5, wherein the medium used in culturing the host cell comprises a kanamycin resistance gene.
9. The method of claim 5, wherein the host cell is cultured to express the protein of interest by IPTG induction.
10. A kit, comprising: the polynucleotide of claim 1; or alternatively
The expression vector of claim 2 or 3; or alternatively
The host cell of claim 4; or alternatively
An acyl-coa oxidase prepared according to the method of any one of claims 5 to 9.
CN202211704989.5A 2022-12-29 2022-12-29 Preparation method and application of acyl-CoA oxidase Pending CN116042664A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190112622A1 (en) * 2016-03-30 2019-04-18 Basf Se Fermentative production of n-butylacrylate using alcohol acyl transferase enzymes
US20200224231A1 (en) * 2018-09-17 2020-07-16 Levadura Biotechnology, Inc. Production of cannabinoids in yeast using a fatty acid feedstock

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190112622A1 (en) * 2016-03-30 2019-04-18 Basf Se Fermentative production of n-butylacrylate using alcohol acyl transferase enzymes
US20200224231A1 (en) * 2018-09-17 2020-07-16 Levadura Biotechnology, Inc. Production of cannabinoids in yeast using a fatty acid feedstock

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MIKIO BAKKE 等: "N-Ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: Molecular cloning, gene expression and characterization of the recombinant enzyme", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1774, no. 1, 25 October 2006 (2006-10-25), pages 66 *

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