CN112626099A - Method for fermenting and expressing angiotensin converting enzyme 2 by using prokaryotic cells - Google Patents

Method for fermenting and expressing angiotensin converting enzyme 2 by using prokaryotic cells Download PDF

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CN112626099A
CN112626099A CN202011049030.3A CN202011049030A CN112626099A CN 112626099 A CN112626099 A CN 112626099A CN 202011049030 A CN202011049030 A CN 202011049030A CN 112626099 A CN112626099 A CN 112626099A
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ace2
leu
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plasmid
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邢新会
卢元
王怡
苏楠
李璐
张翀
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Tsinghua University
Shenzhen International Graduate School of Tsinghua University
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Shenzhen International Graduate School of Tsinghua University
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    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)

Abstract

The application relates to a plasmid containing ACE2, a prokaryotic cell containing the plasmid, and a method for fermenting and expressing angiotensin converting enzyme 2(ACE2) by using the prokaryotic cell as a host cell. The invention also relates to a method for producing angiotensin converting enzyme 2(ACE2) using a prokaryote, comprising: constructing a plasmid, wherein the plasmid contains a natural ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out as an exogenous gene; introducing the plasmid into a host prokaryote; culturing a host prokaryote, and expressing ACE2 in the culture; ACE2 was purified from the culture.

Description

Method for fermenting and expressing angiotensin converting enzyme 2 by using prokaryotic cells
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for expressing angiotensin converting enzyme 2(ACE2) by using prokaryotic cells as host cells.
Background
Currently, the specific cell receptor of 2019 novel coronavirus SARS-CoV-2 is known to be ACE2 (Angiotensin-converting enzyme 2, Angiotensin converting enzyme 2) protein, so that a novel coronavirus control protein drug and a rapid drug screening platform are developed on the basis of ACE2 receptor protein, and the novel coronavirus control protein drug and the rapid drug screening platform can be used as a novel and effective novel coronavirus control strategy. First, the receptor protein ACE2 can specifically bind to the new coronavirus, and the function similar to "antibody" realizes neutralization of the virus and rapid excretion out of the body, so as to realize the effect of the new coronavirus treatment. Secondly, the receptor protein ACE2 is relatively conservative and not easy to mutate in cells of different populations, so that the receptor protein ACE2 serving as a protein drug can solve the problem of mutation of a new coronavirus, and is long-acting and stable, thereby avoiding the 'failure' problem of the traditional vaccine and antibody strategy. Thirdly, the rational design and the large-scale production and manufacture of the recombinant protein ACE2 medicament can be rapidly realized by combining the means of synthetic biology and the like which are rapidly developed at present. Fourthly, due to the biological safety requirement and the development of animal models, the research and development process of vaccines, antibodies and small molecular chemical drugs is greatly limited, a novel coronavirus drug screening platform can be developed on the basis of the receptor protein ACE2, the limitation required by a biological safety laboratory is removed, and drug screening can be carried out in a common laboratory, so that the screening and the accuracy of drugs are greatly accelerated, and the research and development cycle of 'design-establishment-test-learning' of the drugs is accelerated. Furthermore, based on ACE2, the mechanism and rule of interaction between novel coronavirus surface S protein, research and development of drugs and the receptor ACE2 can be rapidly and accurately explored, and drug synthesis and clinical experimental research can be guided. Meanwhile, the receptor protein ACE2 platform based on the emerging paradigm can break away from biological safety and animal model constraint, and rapidly screen vaccines, antibodies and chemical small molecule drugs. The combination rule of the surface protein, the medicine and the receptor ACE2 of the new coronavirus is explored, so that the basic research of the action and the development mechanism of the new coronavirus is guided, and the synthesis and the later clinical experimental research of the prevention and treatment medicine of the new coronavirus are further guided.
The above applications all require the ability to economically and stably produce angiotensin-converting enzyme 2(ACE2) for research use; however, to the best of the applicant's knowledge, there is currently no prior art approach to the expression of angiotensin converting enzyme 2(ACE2) using fermentation techniques.
Disclosure of Invention
The technical scheme of this application provides:
1. a plasmid comprising a native ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out further as a foreign gene.
2. The plasmid of clause 1, further comprising a sequence of a tag protein downstream of the ACE2 sequence of clause 1.
3. The plasmid according to item 2, wherein the tag protein is selected from any one of HIS, Flag, HA, Myc, Strep-II, most preferably HIS or Strep-II tag protein.
4. The plasmid according to item 3, wherein the plasmid is selected from any one of pET series, pGEX series, pQE series, pBAD series and pGAD series plasmids, and most preferably the plasmid is pET-30a (+) plasmid having a sequence shown in SEQ ID NO. 7.
5. The plasmid according to item 4, wherein the natural ACE2 has an amino acid sequence shown in SEQ ID NO.1 or is expressed by a nucleotide sequence shown in SEQ ID NO.2, the ACE2 with a transmembrane region and an intracellular region knocked out has an amino acid sequence shown in SEQ ID NO.3 or is expressed by a nucleotide sequence shown in SEQ ID NO.4, and the ACE2 with a part of extracellular region knocked out has an amino acid sequence shown in SEQ ID NO.5 or a nucleotide sequence shown in SEQ ID NO. 6.
6. A prokaryote into which the plasmid according to any one of items 1 to 5 has been introduced.
7. The prokaryote according to item 6, said prokaryote being a bacterium selected from the group consisting of Bacillus subtilis, Streptomyces, Corynebacterium glutamicum or Escherichia coli, most preferably Escherichia coli.
8. The prokaryote according to item 7, wherein the E.coli strain is selected from any one of TB1, JM109, TOP10, BL21(DE3), BL21(DE3) pLysS or Rosetta (DE3), most preferably BL21(DE3) or BL21(DE3) pLysS.
9. A method for producing angiotensin converting enzyme 2(ACE2) using a prokaryote, comprising:
-constructing a plasmid containing a natural ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out as a foreign gene;
-introducing the plasmid into a host prokaryote;
-culturing a host prokaryote and expressing ACE2 in the culture;
-extraction and purification of ACE2 from the culture.
10. The production method according to claim 9, wherein the host prokaryote is introduced with the plasmid according to any one of claims 1 to 5.
11. The production method according to item 9 or 10, wherein the prokaryote is a bacterium, preferably Escherichia coli.
12. The process according to item 11, wherein the culturing and expression are carried out under conditions in which the seed solution is subjected to shake cultivation, then transferred to M9YE medium, and IPTG is added at a temperature lower than room temperature to induce expression of the target protein; and collecting the thallus.
13. The production method according to item 12, wherein the extraction purification uses at least the following steps: high pressure crushing to extract intracellular protein; ACE2 was extracted using affinity chromatography.
Technical effects of the invention
By adopting the production method, the angiotensin converting enzyme 2(ACE2) can be obtained economically and stably by using a fermentation technology. The technical scheme of the application makes full use of the economical and practical property of a prokaryotic expression system (such as an escherichia coli expression system), and establishes a method for expressing ACE2 derived from eukaryotes in the prokaryotic system; in particular, a particularly excellent, economical production is achieved by the following several optimization measures: 1. BL21(DE3) and BL21(DE3) pLysS of escherichia coli are selected as the preferred strains for expression, and the optimal effect of expression in prokaryotic cells is achieved; 2, the HIS is selected as the optimal label to obtain excellent technical effect, and the main effects are low price and large protein extraction amount; 3 particular truncated forms, in particular 740 and 615 amino acids, are also advantageous for increasing expression in prokaryotic cells and, by knocking out the transmembrane region, increase the water solubility of ACE 2.
Drawings
FIG. 1 is a schematic diagram of the structure of pPET-30a (+) plasmid;
FIG. 2 SDS-PAGE analysis of the fractions of intracellular expression products using E.coli for expression of ACE2(ACE2(18-805aa) -10 × HIS);
FIG. 3 Western Blot analysis of fractions of intracellular expression products using E.coli for ACE2(ACE2(18-805aa) -10 × HIS) expression;
FIG. 4. ACE2(ACE2(18-805aa) -10 × HIS) expression using E.coli, interaction signal of intracellular expression product with S protein (ELISA assay);
FIG. 5 shows the interaction signals of the target protein component and S protein obtained by purifying the intracellular expression product (ELISA assay) using E.coli for ACE2 expression (ACE2(18-805aa) -10 × HIS);
FIG. 6. expression of ACE2(ACE2(18-615aa) -Strep-II and ACE2(18-740aa) -Strep-II), SDS-PAGE analysis and Western Blot analysis of the fractions of the intracellular expression product using E.coli;
FIG. 7. expression of ACE2(ACE2(18-805aa) -10 × HIS) using E.coli, signals of interaction of intracellular expression products with S protein (ELISA assay);
FIG. 8 is a schematic diagram of the construction of pXMJ19 plasmid (four construction modes);
FIG. 9 Western Blot analysis of fractions of intracellular expression products from ACE2 expression using C.glutamicum;
FIG. 10 ACE2 expression using C.glutamicum, signals of interaction of intracellular expression products with S protein (ELISA assay).
Detailed Description
It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
The present application relates in a first aspect to a plasmid.
In one embodiment, a plasmid is provided, which comprises a native ACE2 sequence, a ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out as a foreign gene.
In the context of the present specification, angiotensin converting enzyme (ACE2, EC 3.4.15.1) is an exopeptidase. Its main functions in the body are the following two: catalyzes the conversion of angiotensin I to angiotensin II; inactivating bradykinin. The angiotensin converting enzyme is an ideal target for treating diseases such as hypertension, heart failure, diabetes and hypertension. Angiotensin converting enzyme inhibitors reduce the production of angiotensin II and increase the activity of bradykinin. In addition, ACE also catalyzes the conversion of angiotensin (1-9) to angiotensin (1-7). Through research, ACE2 is also a target of the action of a novel coronavirus (SARS-CoV-2), and the purpose of the application is how to economically and rapidly produce ACE2, so that the ACE can be used for researching and developing antiviral drugs based on the target effect. In the context of the present specification, rhACE2-740/615 refers to recombinant human ACE 2. Herein, rhACE2-850 refers to a natural ACE2 sequence with an amino acid sequence length of 850, while rhACE2-740 is an ACE2 sequence with a transmembrane region and an intracellular region knocked out, and rhACE2-615 is an ACE2 sequence with a partial extracellular region knocked out further. It should be noted that the protein contained in the biological membrane is called membrane protein, and is a main undertaker of the function of the biological membrane. Membrane proteins can be divided into three main groups, based on the ease of protein separation and the location of distribution in the membrane: the extrinsic membrane proteins or peripheral membrane proteins, the intrinsic membrane proteins or integral membrane proteins, and the lipocalins. The membrane protein includes glycoprotein, carrier protein and enzyme. The ACE2 protein to which this application relates belongs to the integral membrane protein of the enzyme class. In general, the use of a particular truncated form of ACE2 may be beneficial for production using prokaryotes because the presence of a transmembrane region may affect the aqueous solubility of membrane proteins.
His, Flag, HA, Myc, Strep-II are all commonly used protein tags in the context of the present specification. The Strap-tag-II is formed by 8 amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). the principle of the Strap-tag technology is to utilize the binding reaction between biotin and streptavidin (streptavidin), which is also designed as the Strap-tag, so that the affinity of Strap-tag-II can be increased by nearly one hundred times. At present, the Strap-tag-II/Strep-Tactin system has become one of the most widely used affinity systems.
For example, His10 refers to a fusion tag consisting of ten histidine residues, which can be inserted at the C-terminus or N-terminus of a protein of interest. When the epitope tag is used as a tag, firstly, the epitope can be formed to facilitate detection; secondly, unique structural characteristics (binding ligand) are formed, which is beneficial to purification. The side chain of histidine residue has strong attraction with solid nickel, and can be used for immobilized metal chelating chromatography (IMAC) to separate and purify recombinant protein. The use of His-tag has the following technical advantages: 1. the molecular weight of the label is small and is only-0.84 KD, and GST and protein A are respectively-26 KD and-30 KD, so that the function of the target protein is not influenced generally; his-tag fusion protein can be purified under the condition of the existence of non-ionic surfactant or under the denaturation condition, the His-tag fusion protein is applied to the purification of protein with strong hydrophobicity, and the His-tag fusion protein is particularly useful for the purification of inclusion body protein; his-tag fusion proteins have also been used in protein-protein, protein-DNA interaction studies; 4, the immunogenicity of the His label is relatively low, and the purified protein can be directly injected into animals for immunization to prepare antibodies; 5. the method can be applied to various expression systems, and the purification condition is mild; 6. the parent and tag may be constructed together with other affinity tags.
The Flag tag protein is a hydrophilic polypeptide (DYKDDDDK) for encoding 8 amino acids, and a Kozak sequence constructed in the vector enables the fusion protein with the FLAG to be higher in expression efficiency in a eukaryotic expression system. The FLAG as a tag protein has the following advantages after fusion expression of target protein: FLAG as a fusion expression tag that does not normally interact with and affect the function, properties of the protein of interest, thus allowing for downstream studies of fusion proteins by researchers. 2. The target protein fused with FLAG can be directly subjected to affinity chromatography through FLAG, the chromatography is non-denaturing purification, active fusion protein can be purified, and the purification efficiency is high. And 3, FLAG is used as a tag protein and can be recognized by an anti-FLAG antibody, so that the detection and identification of the FLAG-containing fusion protein can be conveniently carried out by Western Blot, ELISA and other methods. 4. FLAG fused to the N-terminus, which can be cleaved by enterokinase (DDDK), resulting in a specific protein of interest. Therefore, the FLAG tag is widely applied to the related fields of protein expression, purification, identification, functional research, protein interaction and the like.
The amino acid sequence of the HA tag is YPYDVPDYA, is derived from hemagglutinin surface antigenic determinant of influenza virus, HAs 9 amino acids, HAs little influence on the spatial structure of exogenous target protein, and is easy to construct tag protein fused to the N end or the C end. Easy to detect with Anti-HA antibody and ELISA.
The C-Myc tag protein is a small tag containing 11 amino acids, the amino acid sequence of the C-Myc tag protein is Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, and the 11 amino acids are expressed as epitope and can still identify corresponding antibodies in different protein frameworks. The C-Myc tag has been successfully applied to Western-blot hybridization technology, immunoprecipitation and flow cytometry, and can be used for detecting the expression of recombinant protein in target cells.
In the context of the present specification, "plasmid" refers to a closed circular double-stranded DNA molecule that is present in the cytoplasm of a DNA molecule other than chromosomes (or a nucleomimetic) in organisms such as bacteria, yeasts and actinomycetes (except yeast, a 2 μm plasmid of yeast is present in the nucleus of a cell), has an autonomous replication ability, can maintain a constant copy number in daughter cells, and expresses genetic information carried thereby. The plasmid is not necessary for the growth and reproduction of bacteria, and can be automatically lost or eliminated by artificial treatment, such as high temperature, ultraviolet ray, etc. The genetic information carried by the plasmid can endow the host bacteria with certain biological characters, and is beneficial to the survival of the bacteria under specific environmental conditions. Bacterial plasmids are commonly used vectors in DNA recombination technology. The vector is a tool for introducing a useful foreign gene into a recipient cell by genetic engineering means for proliferation and expression. A certain target gene segment is recombined into a plasmid to form a recombinant gene or a recombinant. Then the recombinant is transferred into a receptor cell (such as Escherichia coli) by a microbiological transformation technology, so that the target gene in the recombinant can be propagated or expressed in the receptor cell, thereby changing the original character of the host cell or generating new substances.
The pET system has been the most powerful system for expression of recombinant proteins in E.coli, and is now the most widely used system for prokaryotic expression. In the system, a target gene is cloned to a pET plasmid vector and is controlled by a strong bacteriophage T7 transcription and translation signal; expression is induced by T7RNA polymerase provided by the host cell. The T7RNA polymerase mechanism is very efficient: when fully induced, almost all cellular resources are used to express the protein of interest; the protein of interest can usually account for more than 50% of the total cellular protein only a few hours after induction of expression. Although this system is extremely powerful, expression levels can be readily reduced by varying the concentration of the inducer. Lowering the expression water is routinely used to increase the soluble fraction yield of certain proteins of interest. Another important advantage of this system is that under non-inducing conditions, the gene of interest can be completely silenced without transcription. The cloning of target gene with host bacteria without T7RNA polymerase can solve the problem of plasmid instability caused by the toxicity of target protein expression to host cells. The two T7 promoters and the multiple host cells with different levels of background-suppressed expression together constitute a very flexible and efficient system for optimal expression of various proteins of interest. In particular, pET-30a in the pET series has a T7lac promoter; the N end is provided with a His.tag/S.tag fusion tag, the His.tag can be used for carrying out metal ion chelation chromatography purification expression protein, and the S.tag fusion tag can also be used for carrying out affinity purification and high-sensitivity quantitative detection; contains Thrombin (Thrombin) and Enterokinase (Enterokinase) protease cutting sites. In the application, plasmid pET-30a (+) was obtained from Nanjing King-Spirie Biotech Ltd. pGEX series, pQE series, pBAD series and pGAD series are all common prokaryotic expression vectors.
It is to be noted that, in the context of the present specification, the sequences identified as 18-805aa, 18-710aa, 18-615aa inserted into the plasmid represent the full-length amino acid sequence (i.e., the native signal peptide from which amino acids 1 to 17 are planed), the truncated form of amino acids 18 to 710 (i.e., the transmembrane region and the intracellular region are knocked out), and the truncated form of amino acids 18 to 615 (i.e., the transmembrane region, the intracellular region and a part of the extracellular region are knocked out), respectively, of ACE 2.
The present application relates in a second aspect to a prokaryotic cell.
In one embodiment, a prokaryote is provided into which the above plasmid has been introduced.
In the context of the present specification, a prokaryote refers to a unicellular organism composed of prokaryotic cells (prokaryotic cells). The main characteristics of the cells are: there is no nucleus bounded by the nuclear envelope, nor nucleolus, only the pseudokaryoid; they have cellular membranes but differ in their composition from eukaryotic cells. They are small in size, have no shaped nuclei, no chromosomes, and DNA does not bind to proteins. Escherichia coli (Escherichia coli) and Corynebacterium glutamicum (Corynebacterium glutamicum) related to the technical scheme belong to bacteria, and the bacteria belong to prokaryotes.
In the context of the present specification, "Escherichia coli", also known as Escherichia coli, was discovered by Escherichia in 1885. Escherichia coli is a conditional pathogen, and can cause gastrointestinal tract infection or urinary tract infection of various local tissues and organs of human and various animals under certain conditions. Coli is often used as a genetically engineered bacterium, and its advantages are: (1) is the most thoroughly studied prokaryotic bacterium; (2) is a mature gene clone expressing receptor cell; (3) the propagation is rapid, and the culture metabolism is easy to control; the disadvantages are: (1) some eukaryotic genes can only synthesize polypeptide chains without specific structures (2) lack of protein processing systems (3) endogenous protease and easily degrade foreign protein (4) endotoxin, and human pyrogen reaction can be caused. In the present application, Escherichia coli is used for expressing angiotensin-converting enzyme 2(ACE2) protein derived from eukaryotes.
In a specific embodiment, the E.coli strain is selected from any one of TB1, JM109, TOP10, BL21(DE3), BL21(DE3) pLysS or Rosetta (DE 3).
In the context of the present specification, Corynebacterium glutamicum (Corynebacterium glutamicum) is mainly characterized in that cells are short-rod to small-rod shaped, sometimes slightly bent, blunt-rounded at both ends, not branched, arranged singly or in a splayed pattern, and the cells are 0.7 to 0.9 × 1.0 to 2.5 μm, gram-positive, spore-free, motionless, moist in colony, and circular; the production and application aspects are as follows: the aerobic bacteria can be used for producing glutamic acid to prepare sodium glutamate (monosodium glutamate) in a microbial fermentation engineering, and the corynebacterium glutamicum needs to continuously introduce sterile air in the fermentation process, and the air forms fine bubbles by stirring and is quickly dissolved in a culture solution (dissolved oxygen); under the conditions that the temperature is 30-37 ℃ and the pH is 7-8, a large amount of glutamic acid can be generated in the culture solution after 28 hours to 32 hours, in the production of the glutamic acid, when the carbon-nitrogen ratio in the culture medium is 4:1, the glutamic acid generated by mass propagation of thalli is less; when the carbon-nitrogen ratio is 3:1, the thallus reproduction is inhibited, but the synthesis amount of the glutamic acid is greatly increased; during fermentation, when the pH is acidic, the Corynebacterium glutamicum can generate acetyl glutamine; when the dissolved oxygen is insufficient, the metabolite produced will be lactic acid or succinic acid. In the present application, C.glutamicum was also used for the production of angiotensin-converting enzyme 2(ACE2) protein from eukaryotes.
In a further embodiment, the strain of Corynebacterium glutamicum is Corynebacterium glutamicum with a accession number of CGMCC1.15647 (strain BZH 001).
In the context of the present specification, CGMCC is an abbreviation of China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center); the system is established in 1979, is a public welfare agency which is mainly used for providing professional technical services, is qualified by the Budapest treaty international collection center in 1995, and is the only national collection center which simultaneously provides general strain resource services and patent biological material preservation in China. The center is established at the institute of microbiology, academy of sciences of China.
In the context of the present specification, Streptomyces (Streptomyces) refers to a higher actinomycete. Which belongs to the family of actinomycete purposes. They have well-developed branched hyphae without transverse septa, and differentiate into vegetative hyphae, aerial hyphae and 65 spore filaments. The spore silk forms conidia again. The shape and color of spore silk and spore are different from species to species, and are one of the main identification traits of the species. There are thousands of species reported, mainly distributed in soil. The cultivation techniques for Streptomyces are also well established and can be carried out using common synthetic, semisynthetic, natural media. The Streptomyces expression system can also be selected to produce the ACE2 by fermentation culture expression.
In the context of the present specification, Bacillus subtilis refers to a species of Bacillus, CAS number 68038-70-0. The single cell is 0.7-0.8 multiplied by 2-3 microns and is uniformly colored. Without capsule, the perigenic flagellum can move. Gram-positive bacteria can form endogenous adversity resisting spores, the spores are 0.6-0.9 multiplied by 1.0-1.5 microns, the ellipse to the column are positioned in the center of the bacteria or slightly deviated, and the bacteria do not expand after the spores are formed. The growth and propagation speed is high, the surface of a colony is rough and opaque, and is white or yellowish, and when the colony grows in a liquid culture medium, the skin becomes always formed, so that the colony is an aerobic bacterium. Tryptophan can be decomposed to form indole by using protein, various sugars and starch. The application in genetic research is wide, and the research on the synthetic pathway of purine nucleotide and the regulation mechanism of the purine nucleotide in the bacterium is clear. Widely distributed in soil and putrefactive organic matters and is easy to propagate in the Sucus subtilis juice, so the method is named. Some strains are important production strains of alpha-amylase and neutral protease; some strains have enzyme systems which strongly degrade nucleotides, so that the strains are often used as parent strains for breeding nucleoside producing strains or strains for preparing 5' -nucleotidase. The study of Bacillus subtilis is relatively thorough. The application can also select a bacillus subtilis expression system to produce the ACE2 through fermentation culture expression.
The present application relates in a third aspect to a process for the production of angiotensin converting enzyme 2(ACE2) by fermentation expression using a prokaryotic cell as host cell.
In one embodiment, a method for producing angiotensin converting enzyme 2(ACE2) using a prokaryote is provided, comprising:
-constructing a plasmid containing a natural ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out as a foreign gene;
-introducing the plasmid into a host prokaryote;
-culturing a host prokaryote and expressing ACE2 in the culture;
-extraction and purification of ACE2 from the culture.
Herein, introduction of a plasmid into a host prokaryotic cell sometimes involves the preparation of competent cells. Methods for introducing a plasmid into a vector can be divided into transformation and electroporation. The former principle is based on the fact that the method is carried out by a plurality of special methods (such as CaCl)2Chemical agents such as RuCl), the permeability of the cell membrane changes, allowing the carrier molecule of the foreign DNA to pass through. The principle of the latter is that, without prior induction of bacterial competence, DNA is forced into the bacteria by means of a brief electric shock (creating a hole in the cell membrane). In the examples of the present application, for the step of introducing the vector into the host bacterium, transformation is used for E.coli, and electric transformation is used for C.glutamicum.
In this case, the expression of the target protein is usually induced by a specific method. IPTG induction is used in the present application. The principle is that when the lactose operon is used as a promoter for protein expression, an inducer is needed for induction (which is equivalent to ignition), but lactose can be used by cells, so that IPTG (isopropyl-beta-D-thiogalactoside) which is similar to lactose in structure can be used for promoting gene expression, but cannot be used by cells, thereby realizing continuous expression.
In the purification of proteins, chromatography is often used. On the premise of using the tag protein, the steps of protein purification are simplified due to the special structure of the tag protein. For example, when HIS tags are used, immobilized metal chelate chromatography (IMAC) can be used to isolate and purify recombinant proteins due to the strong affinity of the histidine residue side chains for solid-state nickel.
In the present application, in fermentation and expression using C.glutamicum, means for promoting production using a signal peptide is also used. The principle is as follows: the signal peptide reaches the lumen of endoplasmic reticulum through the pore canal formed by the protein in the membrane, and is then hydrolyzed by the signal peptidase located on the surface of the lumen, and the nascent polypeptide can enter the lumen through the endoplasmic reticulum membrane as a result of its guidance, and is finally secreted to the outside of the cell.
< example >
Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Example 1 design of expression of ACE2 using E.coli and analysis of the results thereof
In the first step, a recombinant expression vector is constructed, which carries the HIS tag or Strep-II tag.
Wherein, the ACE2(18-805aa) target protein sequence is a sequence of SEQ ID NO. 1;
the nucleotide sequence corresponding to the amino acid sequence SEQ ID NO.1 is the sequence of SEQ ID NO. 2;
ACE2(18-740aa) target protein sequence is the sequence of SEQ ID NO. 3;
the nucleotide sequence corresponding to the amino acid sequence SEQ ID NO.3 is the sequence of SEQ ID NO. 4;
ACE2(18-615aa) target protein sequence is the sequence of SEQ ID NO. 5;
the nucleotide sequence corresponding to the amino acid sequence SEQ ID NO.5 is the sequence of SEQ ID NO. 6;
the nucleotide sequence of the empty vector used (derived from pET-30a (+)) was set to SEQ ID NO. 7.
It should be noted that the sequences identified as 18-805aa, 18-710aa, and 18-615aa inserted into the plasmid represent the full-length amino acid sequence (excluding the native signal peptide of amino acids 1 to 17), the truncated form of amino acids 18 to 710, and the truncated form of amino acids 18 to 615, respectively, of ACE 2.
The specific sequences are respectively as follows:
SEQ ID NO.7：
ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTA GTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGT GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGATATCAGCCATGGCCTT GTCGTCGTCGTCGGTACCCAGATCTGGGCTGTCCATGTGCTGGCGTTCGAATTTAGCAGCAGCGGTTTCTTTCAT ACCAGAACCGCGTGGCACCAGACCAGAAGAATGATGATGATGATGGTGCATATGTATATCTCCTTCTTAAAGTT AAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGG ATCGAGATCGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTG GCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGC GTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGC GGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAG ATCCCGGACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAG GGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCG CGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAAT TACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCT GGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGG TGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGT GGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCG TTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGC GTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGT CTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTG GAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGC CAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGACATCTCGG TAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGC CTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTT GCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCG ATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAG TTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTG GGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGC AGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATT CGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCA TTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGG GGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTC TGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGC CCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAA CGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCAC AACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCA TTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATG GCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAG ACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTG AAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCC CGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAG TGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACC GCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGA TACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATT TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAA ACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGC CGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGT GCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGC CAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTT TATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAG AATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTT GTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTG ATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTG CCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTA ATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTC GGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTG CAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAA AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTT AAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAA ATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGG ACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGT TTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGG AAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGT AGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCA
SEQ ID NO.1：
MQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQ NLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLH AYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFL LRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIP KDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDIS NSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKV RISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRT EVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGE NPYASIDISKGENNPGFQNTDDVQTSF
SEQ ID NO.2：
ATGCAAAGCACCATTGAGGAGCAAGCGAAGACCTTTCTGGACAAGTTTAATCACGAAGCGGAGGACCTGTT TTATCAGAGCAGCCTGGCGAGCTGGAACTATAACACCAACATTACCGAGGAAAACGTGCAGAACATGAACAAC GCGGGTGACAAATGGAGCGCGTTCCTGAAGGAGCAAAGCACCCTGGCGCAGATGTACCCGCTGCAAGAAATCC AGAACCTGACCGTGAAACTGCAGCTGCAAGCGCTGCAGCAAAACGGCAGCAGCGTTCTGAGCGAGGATAAAAG CAAGCGTCTGAACACCATTCTGAACACCATGAGCACCATCTATAGCACCGGCAAGGTGTGCAACCCGGACAACC CGCAAGAATGCCTGCTGCTGGAGCCGGGCCTGAACGAAATCATGGCGAACAGCCTGGATTACAACGAACGTCTG TGGGCGTGGGAGAGCTGGCGTAGCGAAGTTGGCAAGCAGCTGCGTCCGCTGTACGAGGAATATGTGGTTCTGAA AAACGAGATGGCGCGTGCGAACCACTACGAAGACTATGGTGATTACTGGCGTGGCGACTACGAGGTGAACGGT GTTGACGGCTACGATTATAGCCGTGGCCAACTGATTGAGGATGTGGAACACACCTTCGAGGAAATCAAGCCGCT GTATGAACACCTGCACGCGTACGTTCGTGCGAAACTGATGAACGCGTATCCGAGCTACATTAGCCCGATTGGTT GCCTGCCGGCGCACCTGCTGGGTGACATGTGGGGCCGTTTCTGGACCAACCTGTACAGCCTGACCGTTCCGTTTG GCCAGAAACCGAACATTGACGTGACCGATGCGATGGTTGACCAAGCGTGGGATGCGCAGCGTATCTTCAAAGA GGCGGAAAAGTTCTTTGTGAGCGTTGGTCTGCCGAACATGACCCAAGGCTTTTGGGAGAACAGCATGCTGACCG ACCCGGGTAACGTGCAGAAAGCGGTTTGCCATCCGACCGCGTGGGACCTGGGCAAGGGCGATTTCCGTATTCTG ATGTGCACCAAAGTGACCATGGACGATTTTCTGACCGCGCACCACGAAATGGGCCACATCCAATATGATATGGC GTACGCGGCGCAGCCGTTCCTGCTGCGTAACGGTGCGAACGAGGGCTTTCACGAAGCGGTTGGCGAGATTATGA GCCTGAGCGCGGCGACCCCGAAACACCTGAAGAGCATCGGCCTGCTGAGCCCGGACTTCCAAGAGGATAACGA AACCGAAATTAACTTTCTGCTGAAACAGGCGCTGACCATCGTGGGTACCCTGCCGTTCACCTATATGCTGGAGA AGTGGCGTTGGATGGTTTTTAAAGGCGAAATTCCGAAGGACCAGTGGATGAAGAAATGGTGGGAAATGAAACG TGAGATCGTGGGTGTGGTTGAGCCGGTTCCGCACGACGAAACCTACTGCGATCCGGCGAGCCTGTTCCACGTGA GCAATGACTATAGCTTTATTCGTTACTATACCCGTACCCTGTACCAGTTCCAATTTCAGGAAGCGCTGTGCCAAG CGGCGAAGCACGAAGGTCCGCTGCACAAATGCGATATCAGCAACAGCACCGAGGCGGGTCAGAAGCTGTTCAA CATGCTGCGTCTGGGCAAAAGCGAGCCGTGGACCCTGGCGCTGGAAAACGTGGTTGGCGCGAAGAACATGAAC GTTCGTCCGCTGCTGAACTATTTCGAACCGCTGTTTACCTGGCTGAAAGACCAAAACAAGAACAGCTTTGTGGGT TGGAGCACCGACTGGAGCCCGTACGCGGATCAGAGCATTAAAGTTCGTATCAGCCTGAAGAGCGCGCTGGGCG ACAAAGCGTATGAGTGGAACGATAACGAAATGTACCTGTTCCGTAGCAGCGTGGCGTATGCGATGCGTCAATAC TTCCTGAAAGTTAAGAACCAGATGATTCTGTTTGGCGAGGAAGACGTGCGTGTTGCGAACCTGAAGCCGCGTAT CAGCTTTAACTTCTTTGTGACCGCGCCGAAAAACGTTAGCGATATCATTCCGCGTACCGAGGTGGAAAAGGCGA TTCGTATGAGCCGTAGCCGTATCAACGACGCGTTCCGTCTGAACGATAACAGCCTGGAGTTTCTGGGTATCCAAC CGACCCTGGGTCCGCCGAACCAGCCGCCGGTGAGCATTTGGCTGATCGTTTTCGGTGTGGTTATGGGCGTGATTG TGGTTGGTATCGTTATTCTGATCTTTACCGGCATCCGTGACCGTAAGAAAAAGAACAAAGCGCGTAGCGGCGAG AACCCGTACGCGAGCATTGATATCAGCAAGGGTGAAAACAACCCGGGCTTCCAAAATACCGACGATGTTCAAAC CAGCTTTTAA
SEQ ID NO.3
MQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQ NLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWE SWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLH AYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFL LRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIP KDQWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDIS NSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKV RISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRT EVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS
SEQ ID NO.4
ATGCAAAGCACCATTGAGGAGCAAGCGAAGACCTTTCTGGACAAGTTTAATCACGAAGCGGAGGACCTGTT TTATCAGAGCAGCCTGGCGAGCTGGAACTATAACACCAACATTACCGAGGAAAACGTGCAGAACATGAACAAC GCGGGTGACAAATGGAGCGCGTTCCTGAAGGAGCAAAGCACCCTGGCGCAGATGTACCCGCTGCAAGAAATCC AGAACCTGACCGTGAAACTGCAGCTGCAAGCGCTGCAGCAAAACGGCAGCAGCGTTCTGAGCGAGGATAAAAG CAAGCGTCTGAACACCATTCTGAACACCATGAGCACCATCTATAGCACCGGCAAGGTGTGCAACCCGGACAACC CGCAAGAATGCCTGCTGCTGGAGCCGGGCCTGAACGAAATCATGGCGAACAGCCTGGATTACAACGAACGTCTG TGGGCGTGGGAGAGCTGGCGTAGCGAAGTTGGCAAGCAGCTGCGTCCGCTGTACGAGGAATATGTGGTTCTGAA AAACGAGATGGCGCGTGCGAACCACTACGAAGACTATGGTGATTACTGGCGTGGCGACTACGAGGTGAACGGT GTTGACGGCTACGATTATAGCCGTGGCCAACTGATTGAGGATGTGGAACACACCTTCGAGGAAATCAAGCCGCT GTATGAACACCTGCACGCGTACGTTCGTGCGAAACTGATGAACGCGTATCCGAGCTACATTAGCCCGATTGGTT GCCTGCCGGCGCACCTGCTGGGTGACATGTGGGGCCGTTTCTGGACCAACCTGTACAGCCTGACCGTTCCGTTTG GCCAGAAACCGAACATTGACGTGACCGATGCGATGGTTGACCAAGCGTGGGATGCGCAGCGTATCTTCAAAGA GGCGGAAAAGTTCTTTGTGAGCGTTGGTCTGCCGAACATGACCCAAGGCTTTTGGGAGAACAGCATGCTGACCG ACCCGGGTAACGTGCAGAAAGCGGTTTGCCATCCGACCGCGTGGGACCTGGGCAAGGGCGATTTCCGTATTCTG ATGTGCACCAAAGTGACCATGGACGATTTTCTGACCGCGCACCACGAAATGGGCCACATCCAATATGATATGGC GTACGCGGCGCAGCCGTTCCTGCTGCGTAACGGTGCGAACGAGGGCTTTCACGAAGCGGTTGGCGAGATTATGA GCCTGAGCGCGGCGACCCCGAAACACCTGAAGAGCATCGGCCTGCTGAGCCCGGACTTCCAAGAGGATAACGA AACCGAAATTAACTTTCTGCTGAAACAGGCGCTGACCATCGTGGGTACCCTGCCGTTCACCTATATGCTGGAGA AGTGGCGTTGGATGGTTTTTAAAGGCGAAATTCCGAAGGACCAGTGGATGAAGAAATGGTGGGAAATGAAACG TGAGATCGTGGGTGTGGTTGAGCCGGTTCCGCACGACGAAACCTACTGCGATCCGGCGAGCCTGTTCCACGTGA GCAATGACTATAGCTTTATTCGTTACTATACCCGTACCCTGTACCAGTTCCAATTTCAGGAAGCGCTGTGCCAAG CGGCGAAGCACGAAGGTCCGCTGCACAAATGCGATATCAGCAACAGCACCGAGGCGGGTCAGAAGCTGTTCAA CATGCTGCGTCTGGGCAAAAGCGAGCCGTGGACCCTGGCGCTGGAAAACGTGGTTGGCGCGAAGAACATGAAC GTTCGTCCGCTGCTGAACTATTTCGAACCGCTGTTTACCTGGCTGAAAGACCAAAACAAGAACAGCTTTGTGGGT TGGAGCACCGACTGGAGCCCGTACGCGGATCAGAGCATTAAAGTTCGTATCAGCCTGAAGAGCGCGCTGGGCG ACAAAGCGTATGAGTGGAACGATAACGAAATGTACCTGTTCCGTAGCAGCGTGGCGTATGCGATGCGTCAATAC TTCCTGAAAGTTAAGAACCAGATGATTCTGTTTGGCGAGGAAGACGTGCGTGTTGCGAACCTGAAGCCGCGTAT CAGCTTTAACTTCTTTGTGACCGCGCCGAAAAACGTTAGCGATATCATTCCGCGTACCGAGGTGGAAAAGGCGA TTCGTATGAGCCGTAGCCGTATCAACGACGCGTTCCGTCTGAACGATAACAGCCTGGAGTTTCTGGGTATCCAAC CGACCCTGGGTCCGCCGAACCAGCCGCCGGTGAGCTAA
SEQ ID NO.5
MQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLT VKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWR SEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYV RAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGL PNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRN GANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQ WMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNST EAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYAD
SEQ ID NO.6
ATGCAAAGCACCATTGAGGAGCAAGCGAAGACCTTTCTGGACAAGTTTAATCACGAAGCGGAGGACCTGTTTTA TCAGAGCAGCCTGGCGAGCTGGAACTATAACACCAACATTACCGAGGAAAACGTGCAGAACATGAACAACGCG GGTGACAAATGGAGCGCGTTCCTGAAGGAGCAAAGCACCCTGGCGCAGATGTACCCGCTGCAAGAAATCCAGA ACCTGACCGTGAAACTGCAGCTGCAAGCGCTGCAGCAAAACGGCAGCAGCGTTCTGAGCGAGGATAAAAGCAA GCGTCTGAACACCATTCTGAACACCATGAGCACCATCTATAGCACCGGCAAGGTGTGCAACCCGGACAACCCGC AAGAATGCCTGCTGCTGGAGCCGGGCCTGAACGAAATCATGGCGAACAGCCTGGATTACAACGAACGTCTGTGG GCGTGGGAGAGCTGGCGTAGCGAAGTTGGCAAGCAGCTGCGTCCGCTGTACGAGGAATATGTGGTTCTGAAAA ACGAGATGGCGCGTGCGAACCACTACGAAGACTATGGTGATTACTGGCGTGGCGACTACGAGGTGAACGGTGTT GACGGCTACGATTATAGCCGTGGCCAACTGATTGAGGATGTGGAACACACCTTCGAGGAAATCAAGCCGCTGTA TGAACACCTGCACGCGTACGTTCGTGCGAAACTGATGAACGCGTATCCGAGCTACATTAGCCCGATTGGTTGCCT GCCGGCGCACCTGCTGGGTGACATGTGGGGCCGTTTCTGGACCAACCTGTACAGCCTGACCGTTCCGTTTGGCCA GAAACCGAACATTGACGTGACCGATGCGATGGTTGACCAAGCGTGGGATGCGCAGCGTATCTTCAAAGAGGCG GAAAAGTTCTTTGTGAGCGTTGGTCTGCCGAACATGACCCAAGGCTTTTGGGAGAACAGCATGCTGACCGACCC GGGTAACGTGCAGAAAGCGGTTTGCCATCCGACCGCGTGGGACCTGGGCAAGGGCGATTTCCGTATTCTGATGT GCACCAAAGTGACCATGGACGATTTTCTGACCGCGCACCACGAAATGGGCCACATCCAATATGATATGGCGTAC GCGGCGCAGCCGTTCCTGCTGCGTAACGGTGCGAACGAGGGCTTTCACGAAGCGGTTGGCGAGATTATGAGCCT GAGCGCGGCGACCCCGAAACACCTGAAGAGCATCGGCCTGCTGAGCCCGGACTTCCAAGAGGATAACGAAACC GAAATTAACTTTCTGCTGAAACAGGCGCTGACCATCGTGGGTACCCTGCCGTTCACCTATATGCTGGAGAAGTG GCGTTGGATGGTTTTTAAAGGCGAAATTCCGAAGGACCAGTGGATGAAGAAATGGTGGGAAATGAAACGTGAG ATCGTGGGTGTGGTTGAGCCGGTTCCGCACGACGAAACCTACTGCGATCCGGCGAGCCTGTTCCACGTGAGCAA TGACTATAGCTTTATTCGTTACTATACCCGTACCCTGTACCAGTTCCAATTTCAGGAAGCGCTGTGCCAAGCGGC GAAGCACGAAGGTCCGCTGCACAAATGCGATATCAGCAACAGCACCGAGGCGGGTCAGAAGCTGTTCAACATG CTGCGTCTGGGCAAAAGCGAGCCGTGGACCCTGGCGCTGGAAAACGTGGTTGGCGCGAAGAACATGAACGTTC GTCCGCTGCTGAACTATTTCGAACCGCTGTTTACCTGGCTGAAAGACCAAAACAAGAACAGCTTTGTGGGTTGG AGCACCGACTGGAGCCCGTACGCGGATTAA
the sequences in this application were all synthesized by Suzhou Jinzhi Biotechnology, Inc.
Second, selecting an expression host bacterium and introducing the vector into the host bacterium
Escherichia coli strains TB1, JM109, TOP10, BL21(DE3), BL21Star (DE3), BL21(DE3) pLysS, Rosetta (DE3) are all suitable. The present example is used for all of these strains, and it is noted here that:
1. BL21(DE3) and BL21(DE3) pLysS are preferred bacteria, and the evaluation criteria are protein expression levels.
2. The ACE2-805 sequence was sequence optimized according to the 18-805aa sequence of ACE2 and the codon usage bias of Escherichia coli, and then His10 sequence and stop codon sequence were added at the end of the sequence, synthesized by Jinzhi corporation and cloned into pPET-30a (+) engineering plasmid.
3. The steps of introducing the vector into the host bacterium are as follows:
e, E.coli transformation operation:
(1) the temperature of the thermostatic water bath was previously adjusted to 42 ℃.
(2) Taking out a tube (100 mu l) of the allelochemicals from a-70 ℃ ultralow temperature freezer, immediately heating and melting by fingers, inserting the tube into ice, and carrying out ice bath for 5-10 min.
(3) Add 5. mu.l of ligated plasmid mixture (DNA content not more than 100ng), shake gently and place on ice for 20 min.
(4) And (4) after shaking up lightly, inserting the mixture into a water bath at 42 ℃ for 1-2 min for heat shock, then quickly putting the mixture back into ice, and standing for 3-5 min.
(5) Add 500. mu.l LB medium (without antibiotics) into each tube in the clean bench, mix gently, then fix on the spring bracket of the shaking table, shake for 1h at 37 ℃.
(6) And (3) taking 100-300 mu l of the conversion mixed solution from a clean bench, respectively dripping the conversion mixed solution into solid LB plate culture dishes containing proper antibiotics, and uniformly coating the conversion mixed solution by using a glass coating rod burnt by an alcohol lamp (note that the alcohol on the glass coating rod is extinguished for a little moment, and then coating the conversion mixed solution after cooling).
(7) Marking on the coated culture dish, placing in a 37 deg.C constant temperature incubator for 30-60min until the surface liquid permeates into the culture medium, and then placing in the 37 deg.C constant temperature incubator overnight with the culture medium inverted.
Step three, the formula of the culture medium
Seed liquid culture: composition table of LB Medium
Composition (I) The dosage of L/g
NaCl
10
Yeast extract 5
Peptone 10
Agar-agar Adding Agar into solid culture medium according to 1.5%
Induced expression medium formulation (M9 YE):
Figure RE-GDA0002942552190000151
Figure RE-GDA0002942552190000161
step four, fermentation culture and expression conditions are as follows:
and carrying out shake culture on the seed solution at 37 ℃ for 14-18h, transferring the seed solution into an M9YE culture medium, and inducing the target protein expression for 24h by 15 ℃ IPTG. Collecting thallus, crushing under high pressure to extract intracellular protein for subsequent analysis. The specific operation is as follows:
1. plate activation
Taking out the preserved strain glycerol tube from a refrigerator at minus 80 ℃, placing the strain glycerol tube in an ice box, transferring the strain glycerol tube to an ultra-clean bench subjected to ultraviolet sterilization, scraping a ring of bacteria liquid by using a disposable inoculating ring, scratching the ring of bacteria liquid on an LB/Kan plate in a tail end crossing mode, sealing the plate with an EP film after the bacteria liquid on the plate is dried in the air, and inversely placing the plate in a constant-temperature incubator at 37 ℃ for culturing overnight (12-14 h).
5ul kanamycin (final concentration of 50 ug/ml) was added to an EP tube containing 5ml LB liquid medium, 3 to 4 large single colonies were picked up from the overnight-cultured plate with a disposable inoculating loop, placed in the above EP tube, and vigorously agitated 10 times to dissolve the cells in the liquid medium, and cultured on a constant temperature shaker at 37 ℃ and 180rpm for 8 to 10 hours until the medium became turbid.
2. Fermentation culture
Respectively adding 2ml of activated seed solution into 200ml of M9YE culture medium containing 1 ‰ kanamycin, culturing at 37 deg.C under 180rpm for about 3-3.5 hr, respectively adding 750ul of bacteria solution and 750ul of deionized water into quartz cuvette with optical path of 1cm, absorbing with ultraviolet and visible spectrophotometer to detect OD value of 600nm600To OD600Reaches-0.6.
When the bacterial liquid is pre-cultured to OD600When the pH was-0.6, IPTG was added, and the mixture was cultured at 15 ℃ and 200rpm for 24 hours, and the final OD600 was measured.
Fifth step, result analysis
The results of the SDS-PAGE analysis of the fractions of the intracellular expression product (ACE2(18-805aa) -10 × HIS) are shown in FIG. 2.
Results of Western Blot analysis (ACE2(18-805aa) -10 HIS) of the experiments performed in this example are shown in figure 3.
As can be seen from FIGS. 2 and 3, the results of SDS-PAGE and Western Blot showed that ACE2(18-805aa) -10 HIS was most efficiently expressed in two expression strains, BL21(DE3) and BL21(DE3) pLysS.
FIG. 4 shows the signal of interaction of the intracellular expression product with the S protein (ELISA assay) using E.coli for ACE2 expression;
FIG. 5 shows the interaction signals of the S protein and the protein component of interest (ELISA assay) obtained after purification of the intracellular expression product, using E.coli for ACE2 expression
FIG. 6 shows the expression of ACE2(ACE2(18-615aa) -Strep-II and ACE2(18-740aa) -Strep-II), SDS-PAGE analysis and Western Blot analysis of the fractions of the intracellular expression products using E.coli. This result indicates that when ACE2(18-615aa) and ACE2(18-740aa) truncated forms of ACE and Strep-II were used for expression as protein tags, the best expression was still achieved with BL21(DE3) and BL21(DE3) pLysS.
FIG. 7 shows the interaction signals of intracellular expression products and S protein (ELISA assay) using E.coli for ACE2(ACE2(18-805aa) -10 × HIS) expression; ELISA interaction experiments show that the purified protein component obtained by the expression strategy can generate obvious interaction signals with the S protein or the RBD structural domain.
Example 2 production Using Corynebacterium glutamicum to express ACE2 and analysis of the results thereof
First, construct recombinant expression vector (with signal peptide)
In order to improve the expression effect, a signal peptide sequence is particularly inserted before the ACE2 gene.
The plasmid constructed here is pXMJ19, the signal peptide referred to as the Sec signal peptide BZH001-cspB, which has the sequence SEQ ID NO. 8:
ATGTTCAACAACCGTATCCGTACCGCCGCCCTCGCAGGTGCCATCGCAA TCTCTACCGCAGCGTCTGGTCTGGTGGTCCCAGCATTCGCTCAGGAA, respectively; alternatively, the TAT signal peptide CGR0949 is used, having the sequence SEQ ID No. 9:
ATGCAAATAAACCGCCGAGGCTTCTTAAAAGCCACCGCAGGACTT GCCACTATCGGCGCTGCCAGCATGTTTATGCCAAAGGCCAACGCCCTT GGAGCA。
second, selecting an expression host bacterium and introducing the vector into the host bacterium
The selected strain is C.glutamicum CGMCC1.15647 (BZH 001). Similarly to example 1, the engineered plasmid (vector) from step one was introduced into the host bacterium by techniques common in the art, as detailed in the following steps:
1. preparation of competent Corynebacterium glutamicum:
-spreading glycerol bacteria stored at-80 ℃ on agar plates to obtain monoclonals; the single clones were collected and inoculated in 5mL of LBB medium, cultured overnight at 30 ℃ at 200 rpm;
transferring to 100mL of EPO medium (inoculum size 2%) to obtain initial cells (OD600 value 0.3), culturing at 200rpm at 30 ℃ for 3-5 hours until OD value reaches 0.6-0.9;
-placing the bacterial solution in a 50mL centrifuge tube for 15 minutes on ice, followed by centrifugation at 4000rpm for 10 minutes at 4 ℃;
-taking 30mL of pre-cooled 10% glycerol, resuspending the cells completely, centrifuging at 4000rpm for 10 minutes at 4 ℃;
-taking again 30mL of pre-cooled 10% glycerol, washing twice;
resuspend cells with 400mL of pre-cooled 10% glycerol, aliquote into 1.5mL centrifuge tubes, 100L per tube, and store at-80 ℃.
2. Electrotransformation method of corynebacterium glutamicum
-taking out at-80 ℃ and, immediately after thawing, placing on ice;
pipetting 5. mu.L of plasmid into competent cells, mixing and ice-cooling for 5-10 min;
-addition to a pre-cooled 0.1cm electric shock cup, 1.8kv, shock 5 ms;
add quickly to 500 μ Ι _ of recovery medium LBHIS and mix in a water bath at 46 ℃ (preheat recovery medium at 46 ℃);
-further incubation at 30 ℃ at 100rpm for 2 hours;
plates were plated on plates containing antibiotics and incubated overnight at 30 ℃.
Step three, the formula of the culture medium
LBB Medium (in g/L): 5.0 parts of yeast powder, 10.0 parts of peptone, 5.0 parts of sodium chloride, 10.0 parts of brain heart extract, 16.0 parts of agar powder (solid), and pH 6.8-7.0;
LBHIS medium (in g/L): 2.5 parts of yeast powder, 5.0 parts of peptone, 5.0 parts of sodium chloride, 18.5 parts of brain heart extract, 91.0 parts of sorbitol, 16.0 parts of agar powder (solid) and 6.8-7.0 parts of pH;
EPO Medium (units are g/L): 5.0 parts of yeast powder, 10.0 parts of peptone, 10.0 parts of sodium chloride, 25.0 parts of glycine, 5.0 parts of isoniazid, 801mL of tween and 6.8-7.0 parts of pH.
The fourth step, fermentation culture and expression conditions
Single colonies were picked from the electro-plated plates and cultured in LBB medium containing the desired antibiotic overnight, followed by 1-2% (starting OD 0.3-0.5) inoculation into LBB medium the next day, 1mM IPTG was added at a final concentration of 1H (OD 1), and further incubation continued for 22h at 30 ℃ and 250rpm throughout.
Fifth step, result analysis
Four plasmids, ACE2(740) -sec signal, ACE2(615) -sec signal peptide, ACE2(740) -tat signal and ACE2(615) -tat signal peptide, were constructed using pXMJ19 plasmid as a backbone, and they were successfully transferred into BZH001 strain, respectively. FIG. 8 shows a schematic diagram of the construction of pXMJ19 plasmid.
Two monoclonals were randomly selected from the four strains obtained above for parallel experiments, the results obtained from the supernatant of the fermentation broth are shown in the upper panel of FIG. 7, and the results obtained from the supernatant of the cell disruption solution are shown in the lower panel of FIG. 9; the exposure time for Western Blot analysis was 5 minutes.
The ACE2(740) -sec signals at different elution concentrations and different dilution ratios are shown in figure 10. 5% and 100% indicate the concentration of elution buffer during protein purification. The last value on the right side of the figure indicates the absorbance measured after the ELISA reaction.
The above results show that C.glutamicum has a weaker capacity to express ACE2, neither the truncated ACE2 nor the reset signal peptide, is able to improve the expression and secretion of ACE2 in C.glutamicum. Experiments prove that the effect of using the escherichia coli to express the ACE2 is better than that of corynebacterium glutamicum.
Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments and application fields, and the above-described embodiments are illustrative, instructive, and not restrictive. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> Qinghua university
Shenzhen International Graduate School of Tsinghua University
<120> method for expressing angiotensin-converting enzyme 2 by fermentation using prokaryotic cells
<130> PD01099
<141> 2020-09-29
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 789
<212> PRT
<213> Artificial Sequence
<400> 1
Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
1 5 10 15
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
20 25 30
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
35 40 45
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
50 55 60
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
65 70 75 80
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
85 90 95
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
100 105 110
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
115 120 125
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
130 135 140
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
145 150 155 160
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
165 170 175
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
180 185 190
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
195 200 205
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
210 215 220
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
225 230 235 240
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
245 250 255
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
260 265 270
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
275 280 285
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
290 295 300
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
305 310 315 320
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
325 330 335
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
340 345 350
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
355 360 365
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
370 375 380
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
385 390 395 400
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
405 410 415
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
420 425 430
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
435 440 445
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
450 455 460
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
465 470 475 480
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
485 490 495
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
500 505 510
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
515 520 525
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
530 535 540
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
545 550 555 560
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
565 570 575
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
580 585 590
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
595 600 605
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
610 615 620
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
625 630 635 640
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
645 650 655
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
660 665 670
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
675 680 685
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
690 695 700
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
705 710 715 720
Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val
725 730 735
Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg
740 745 750
Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile
755 760 765
Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp
770 775 780
Val Gln Thr Ser Phe
785
<210> 2
<211> 2370
<212> DNA
<213> Artificial Sequence
<400> 2
atgcaaagca ccattgagga gcaagcgaag acctttctgg acaagtttaa tcacgaagcg 60
gaggacctgt tttatcagag cagcctggcg agctggaact ataacaccaa cattaccgag 120
gaaaacgtgc agaacatgaa caacgcgggt gacaaatgga gcgcgttcct gaaggagcaa 180
agcaccctgg cgcagatgta cccgctgcaa gaaatccaga acctgaccgt gaaactgcag 240
ctgcaagcgc tgcagcaaaa cggcagcagc gttctgagcg aggataaaag caagcgtctg 300
aacaccattc tgaacaccat gagcaccatc tatagcaccg gcaaggtgtg caacccggac 360
aacccgcaag aatgcctgct gctggagccg ggcctgaacg aaatcatggc gaacagcctg 420
gattacaacg aacgtctgtg ggcgtgggag agctggcgta gcgaagttgg caagcagctg 480
cgtccgctgt acgaggaata tgtggttctg aaaaacgaga tggcgcgtgc gaaccactac 540
gaagactatg gtgattactg gcgtggcgac tacgaggtga acggtgttga cggctacgat 600
tatagccgtg gccaactgat tgaggatgtg gaacacacct tcgaggaaat caagccgctg 660
tatgaacacc tgcacgcgta cgttcgtgcg aaactgatga acgcgtatcc gagctacatt 720
agcccgattg gttgcctgcc ggcgcacctg ctgggtgaca tgtggggccg tttctggacc 780
aacctgtaca gcctgaccgt tccgtttggc cagaaaccga acattgacgt gaccgatgcg 840
atggttgacc aagcgtggga tgcgcagcgt atcttcaaag aggcggaaaa gttctttgtg 900
agcgttggtc tgccgaacat gacccaaggc ttttgggaga acagcatgct gaccgacccg 960
ggtaacgtgc agaaagcggt ttgccatccg accgcgtggg acctgggcaa gggcgatttc 1020
cgtattctga tgtgcaccaa agtgaccatg gacgattttc tgaccgcgca ccacgaaatg 1080
ggccacatcc aatatgatat ggcgtacgcg gcgcagccgt tcctgctgcg taacggtgcg 1140
aacgagggct ttcacgaagc ggttggcgag attatgagcc tgagcgcggc gaccccgaaa 1200
cacctgaaga gcatcggcct gctgagcccg gacttccaag aggataacga aaccgaaatt 1260
aactttctgc tgaaacaggc gctgaccatc gtgggtaccc tgccgttcac ctatatgctg 1320
gagaagtggc gttggatggt ttttaaaggc gaaattccga aggaccagtg gatgaagaaa 1380
tggtgggaaa tgaaacgtga gatcgtgggt gtggttgagc cggttccgca cgacgaaacc 1440
tactgcgatc cggcgagcct gttccacgtg agcaatgact atagctttat tcgttactat 1500
acccgtaccc tgtaccagtt ccaatttcag gaagcgctgt gccaagcggc gaagcacgaa 1560
ggtccgctgc acaaatgcga tatcagcaac agcaccgagg cgggtcagaa gctgttcaac 1620
atgctgcgtc tgggcaaaag cgagccgtgg accctggcgc tggaaaacgt ggttggcgcg 1680
aagaacatga acgttcgtcc gctgctgaac tatttcgaac cgctgtttac ctggctgaaa 1740
gaccaaaaca agaacagctt tgtgggttgg agcaccgact ggagcccgta cgcggatcag 1800
agcattaaag ttcgtatcag cctgaagagc gcgctgggcg acaaagcgta tgagtggaac 1860
gataacgaaa tgtacctgtt ccgtagcagc gtggcgtatg cgatgcgtca atacttcctg 1920
aaagttaaga accagatgat tctgtttggc gaggaagacg tgcgtgttgc gaacctgaag 1980
ccgcgtatca gctttaactt ctttgtgacc gcgccgaaaa acgttagcga tatcattccg 2040
cgtaccgagg tggaaaaggc gattcgtatg agccgtagcc gtatcaacga cgcgttccgt 2100
ctgaacgata acagcctgga gtttctgggt atccaaccga ccctgggtcc gccgaaccag 2160
ccgccggtga gcatttggct gatcgttttc ggtgtggtta tgggcgtgat tgtggttggt 2220
atcgttattc tgatctttac cggcatccgt gaccgtaaga aaaagaacaa agcgcgtagc 2280
ggcgagaacc cgtacgcgag cattgatatc agcaagggtg aaaacaaccc gggcttccaa 2340
aataccgacg atgttcaaac cagcttttaa 2370
<210> 3
<211> 724
<212> PRT
<213> Artificial Sequence
<400> 3
Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
1 5 10 15
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
20 25 30
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
35 40 45
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
50 55 60
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
65 70 75 80
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
85 90 95
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
100 105 110
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
115 120 125
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
130 135 140
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
145 150 155 160
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
165 170 175
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
180 185 190
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
195 200 205
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
210 215 220
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
225 230 235 240
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
245 250 255
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
260 265 270
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
275 280 285
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
290 295 300
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
305 310 315 320
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
325 330 335
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
340 345 350
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
355 360 365
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
370 375 380
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
385 390 395 400
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
405 410 415
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
420 425 430
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
435 440 445
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
450 455 460
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
465 470 475 480
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
485 490 495
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
500 505 510
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
515 520 525
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
530 535 540
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
545 550 555 560
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
565 570 575
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
580 585 590
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
595 600 605
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
610 615 620
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
625 630 635 640
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
645 650 655
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
660 665 670
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
675 680 685
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
690 695 700
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
705 710 715 720
Pro Pro Val Ser
<210> 4
<211> 2175
<212> DNA
<213> Artificial Sequence
<400> 4
atgcaaagca ccattgagga gcaagcgaag acctttctgg acaagtttaa tcacgaagcg 60
gaggacctgt tttatcagag cagcctggcg agctggaact ataacaccaa cattaccgag 120
gaaaacgtgc agaacatgaa caacgcgggt gacaaatgga gcgcgttcct gaaggagcaa 180
agcaccctgg cgcagatgta cccgctgcaa gaaatccaga acctgaccgt gaaactgcag 240
ctgcaagcgc tgcagcaaaa cggcagcagc gttctgagcg aggataaaag caagcgtctg 300
aacaccattc tgaacaccat gagcaccatc tatagcaccg gcaaggtgtg caacccggac 360
aacccgcaag aatgcctgct gctggagccg ggcctgaacg aaatcatggc gaacagcctg 420
gattacaacg aacgtctgtg ggcgtgggag agctggcgta gcgaagttgg caagcagctg 480
cgtccgctgt acgaggaata tgtggttctg aaaaacgaga tggcgcgtgc gaaccactac 540
gaagactatg gtgattactg gcgtggcgac tacgaggtga acggtgttga cggctacgat 600
tatagccgtg gccaactgat tgaggatgtg gaacacacct tcgaggaaat caagccgctg 660
tatgaacacc tgcacgcgta cgttcgtgcg aaactgatga acgcgtatcc gagctacatt 720
agcccgattg gttgcctgcc ggcgcacctg ctgggtgaca tgtggggccg tttctggacc 780
aacctgtaca gcctgaccgt tccgtttggc cagaaaccga acattgacgt gaccgatgcg 840
atggttgacc aagcgtggga tgcgcagcgt atcttcaaag aggcggaaaa gttctttgtg 900
agcgttggtc tgccgaacat gacccaaggc ttttgggaga acagcatgct gaccgacccg 960
ggtaacgtgc agaaagcggt ttgccatccg accgcgtggg acctgggcaa gggcgatttc 1020
cgtattctga tgtgcaccaa agtgaccatg gacgattttc tgaccgcgca ccacgaaatg 1080
ggccacatcc aatatgatat ggcgtacgcg gcgcagccgt tcctgctgcg taacggtgcg 1140
aacgagggct ttcacgaagc ggttggcgag attatgagcc tgagcgcggc gaccccgaaa 1200
cacctgaaga gcatcggcct gctgagcccg gacttccaag aggataacga aaccgaaatt 1260
aactttctgc tgaaacaggc gctgaccatc gtgggtaccc tgccgttcac ctatatgctg 1320
gagaagtggc gttggatggt ttttaaaggc gaaattccga aggaccagtg gatgaagaaa 1380
tggtgggaaa tgaaacgtga gatcgtgggt gtggttgagc cggttccgca cgacgaaacc 1440
tactgcgatc cggcgagcct gttccacgtg agcaatgact atagctttat tcgttactat 1500
acccgtaccc tgtaccagtt ccaatttcag gaagcgctgt gccaagcggc gaagcacgaa 1560
ggtccgctgc acaaatgcga tatcagcaac agcaccgagg cgggtcagaa gctgttcaac 1620
atgctgcgtc tgggcaaaag cgagccgtgg accctggcgc tggaaaacgt ggttggcgcg 1680
aagaacatga acgttcgtcc gctgctgaac tatttcgaac cgctgtttac ctggctgaaa 1740
gaccaaaaca agaacagctt tgtgggttgg agcaccgact ggagcccgta cgcggatcag 1800
agcattaaag ttcgtatcag cctgaagagc gcgctgggcg acaaagcgta tgagtggaac 1860
gataacgaaa tgtacctgtt ccgtagcagc gtggcgtatg cgatgcgtca atacttcctg 1920
aaagttaaga accagatgat tctgtttggc gaggaagacg tgcgtgttgc gaacctgaag 1980
ccgcgtatca gctttaactt ctttgtgacc gcgccgaaaa acgttagcga tatcattccg 2040
cgtaccgagg tggaaaaggc gattcgtatg agccgtagcc gtatcaacga cgcgttccgt 2100
ctgaacgata acagcctgga gtttctgggt atccaaccga ccctgggtcc gccgaaccag 2160
ccgccggtga gctaa 2175
<210> 5
<211> 599
<212> PRT
<213> Artificial Sequence
<400> 5
Met Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
1 5 10 15
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
20 25 30
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
35 40 45
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
50 55 60
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
65 70 75 80
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
85 90 95
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
100 105 110
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
115 120 125
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
130 135 140
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
145 150 155 160
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
165 170 175
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
180 185 190
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
195 200 205
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
210 215 220
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
225 230 235 240
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
245 250 255
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
260 265 270
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
275 280 285
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
290 295 300
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
305 310 315 320
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
325 330 335
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
340 345 350
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
355 360 365
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
370 375 380
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
385 390 395 400
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
405 410 415
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
420 425 430
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
435 440 445
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
450 455 460
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
465 470 475 480
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
485 490 495
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
500 505 510
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
515 520 525
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
530 535 540
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
545 550 555 560
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
565 570 575
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
580 585 590
Asp Trp Ser Pro Tyr Ala Asp
595
<210> 6
<211> 1800
<212> DNA
<213> Artificial Sequence
<400> 6
atgcaaagca ccattgagga gcaagcgaag acctttctgg acaagtttaa tcacgaagcg 60
gaggacctgt tttatcagag cagcctggcg agctggaact ataacaccaa cattaccgag 120
gaaaacgtgc agaacatgaa caacgcgggt gacaaatgga gcgcgttcct gaaggagcaa 180
agcaccctgg cgcagatgta cccgctgcaa gaaatccaga acctgaccgt gaaactgcag 240
ctgcaagcgc tgcagcaaaa cggcagcagc gttctgagcg aggataaaag caagcgtctg 300
aacaccattc tgaacaccat gagcaccatc tatagcaccg gcaaggtgtg caacccggac 360
aacccgcaag aatgcctgct gctggagccg ggcctgaacg aaatcatggc gaacagcctg 420
gattacaacg aacgtctgtg ggcgtgggag agctggcgta gcgaagttgg caagcagctg 480
cgtccgctgt acgaggaata tgtggttctg aaaaacgaga tggcgcgtgc gaaccactac 540
gaagactatg gtgattactg gcgtggcgac tacgaggtga acggtgttga cggctacgat 600
tatagccgtg gccaactgat tgaggatgtg gaacacacct tcgaggaaat caagccgctg 660
tatgaacacc tgcacgcgta cgttcgtgcg aaactgatga acgcgtatcc gagctacatt 720
agcccgattg gttgcctgcc ggcgcacctg ctgggtgaca tgtggggccg tttctggacc 780
aacctgtaca gcctgaccgt tccgtttggc cagaaaccga acattgacgt gaccgatgcg 840
atggttgacc aagcgtggga tgcgcagcgt atcttcaaag aggcggaaaa gttctttgtg 900
agcgttggtc tgccgaacat gacccaaggc ttttgggaga acagcatgct gaccgacccg 960
ggtaacgtgc agaaagcggt ttgccatccg accgcgtggg acctgggcaa gggcgatttc 1020
cgtattctga tgtgcaccaa agtgaccatg gacgattttc tgaccgcgca ccacgaaatg 1080
ggccacatcc aatatgatat ggcgtacgcg gcgcagccgt tcctgctgcg taacggtgcg 1140
aacgagggct ttcacgaagc ggttggcgag attatgagcc tgagcgcggc gaccccgaaa 1200
cacctgaaga gcatcggcct gctgagcccg gacttccaag aggataacga aaccgaaatt 1260
aactttctgc tgaaacaggc gctgaccatc gtgggtaccc tgccgttcac ctatatgctg 1320
gagaagtggc gttggatggt ttttaaaggc gaaattccga aggaccagtg gatgaagaaa 1380
tggtgggaaa tgaaacgtga gatcgtgggt gtggttgagc cggttccgca cgacgaaacc 1440
tactgcgatc cggcgagcct gttccacgtg agcaatgact atagctttat tcgttactat 1500
acccgtaccc tgtaccagtt ccaatttcag gaagcgctgt gccaagcggc gaagcacgaa 1560
ggtccgctgc acaaatgcga tatcagcaac agcaccgagg cgggtcagaa gctgttcaac 1620
atgctgcgtc tgggcaaaag cgagccgtgg accctggcgc tggaaaacgt ggttggcgcg 1680
aagaacatga acgttcgtcc gctgctgaac tatttcgaac cgctgtttac ctggctgaaa 1740
gaccaaaaca agaacagctt tgtgggttgg agcaccgact ggagcccgta cgcggattaa 1800
<210> 7
<211> 5422
<212> DNA
<213> Artificial Sequence
<400> 7
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180
cgacggagct cgaattcgga tccgatatca gccatggcct tgtcgtcgtc gtcggtaccc 240
agatctgggc tgtccatgtg ctggcgttcg aatttagcag cagcggtttc tttcatacca 300
gaaccgcgtg gcaccagacc agaagaatga tgatgatgat ggtgcatatg tatatctcct 360
tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta 420
tagtgagtcg tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca 480
tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca 540
ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta 600
tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc 660
ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt 720
cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt 780
atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg 840
ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac 900
caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg 960
aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc 1020
gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct 1080
cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa 1140
gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac 1200
tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg 1260
ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac 1320
ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta 1380
gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc 1440
actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt 1500
tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc 1560
aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt 1620
gcggacatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg 1680
ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg 1740
caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa 1800
agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 1860
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 1920
taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt 1980
caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 2040
tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg 2100
cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat 2160
cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa 2220
gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt 2280
gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg 2340
cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat 2400
ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat 2460
tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt 2520
attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac 2580
cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg 2640
tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc 2700
ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat 2760
cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc 2820
agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg 2880
tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 2940
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 3000
gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 3060
tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc 3120
acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact 3180
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3240
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3300
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3360
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3420
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3480
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 3540
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 3600
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 3660
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 3720
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 3780
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 3840
cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3900
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3960
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct 4020
tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct 4080
aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat 4140
aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag 4200
ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga 4260
ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct 4320
gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa 4380
gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg 4440
cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag 4500
gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat 4560
ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat 4620
tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta 4680
ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc 4740
ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat 4800
ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc 4860
taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 4920
ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt gtaaacgtta atattttgtt 4980
aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg 5040
caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg 5100
gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta 5160
tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg 5220
ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa 5280
gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct 5340
ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct 5400
acagggcgcg tcccattcgc ca 5422
<210> 8
<211> 96
<212> DNA
<213> Artificial Sequence
<400> 8
atgttcaaca accgtatccg taccgccgcc ctcgcaggtg ccatcgcaat ctctaccgca 60
gcgtctggtc tggtggtccc agcattcgct caggaa 96
<210> 9
<211> 99
<212> DNA
<213> Artificial Sequence
<400> 9
atgcaaataa accgccgagg cttcttaaaa gccaccgcag gacttgccac tatcggcgct 60
gccagcatgt ttatgccaaa ggccaacgcc cttggagca 99

Claims (10)

1. A plasmid comprising a native ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out further as a foreign gene.
2. The plasmid of claim 1 further comprising the sequence of a tag protein downstream of the ACE2 sequence of claim 1.
3. The plasmid according to claim 2, wherein the tag protein is selected from any one of HIS, Flag, HA, Myc, Strep-II, most preferably HIS or Strep-II tag protein.
4. The plasmid of claim 3, wherein the plasmid is selected from any one of pET series, pGEX series, pQE series, pBAD series and pGAD series plasmids, and most preferably the plasmid is pET-30a (+) plasmid having a sequence shown as SEQ ID No. 7.
5. The plasmid of claim 4, wherein the natural ACE2 has an amino acid sequence shown as SEQ ID No.1 or is expressed by a nucleotide sequence shown as SEQ ID No.2, the ACE2 with a transmembrane region and an intracellular region knocked out has an amino acid sequence shown as SEQ ID No.3 or is expressed by a nucleotide sequence shown as SEQ ID No.4, and the ACE2 with a partial extracellular region knocked out has an amino acid sequence and a nucleotide sequence shown as SEQ ID No.5 or is expressed by a nucleotide sequence shown as SEQ ID No. 6.
6. A prokaryote into which the plasmid according to any one of claims 1 to 5 has been introduced.
7. The prokaryote of claim 6, wherein the prokaryote is a bacterium selected from the group consisting of Bacillus subtilis, Streptomyces, Corynebacterium glutamicum, and Escherichia coli, most preferably Escherichia coli.
8. Prokaryotic organism according to claim 7, wherein the E.coli strain is selected from any one of TB1, JM109, TOP10, BL21(DE3), BL21(DE3) pLysS or Rosetta (DE3), most preferably BL21(DE3) or BL21(DE3) pLysS.
9. A method for producing angiotensin converting enzyme 2(ACE2) using a prokaryote, comprising:
-constructing a plasmid containing a natural ACE2 sequence, an ACE2 sequence with a transmembrane region and an intracellular region knocked out, or an ACE2 sequence with a partial extracellular region knocked out as a foreign gene;
-introducing the plasmid into a host prokaryote;
-culturing a host prokaryote and expressing ACE2 in the culture;
-extraction and purification of ACE2 from the culture.
10. The production method according to claim 9, wherein the host prokaryote is introduced with the plasmid according to any one of claims 1 to 5.
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