TWI310405B - - Google Patents

Description

1310405 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a shuttle plastid for enhancing the amount of enzyme. ^July/7曰 Revision | plastid stability and stable production [Prior Art] Bacillus subtilis is a well-known industrial strain that produces recombinant protein in cells, but is used in this strain. Gene expression plastids generally lack stability, which limits the production of recombinant proteins. Gram-positive bacteria (gram_p〇sitive) is one of the most well-known prokaryotes, and has a very important position in basic scientific research and industrial industries. Bacillus subtilis can secrete a variety of proteases, amylases and fats. Enzymes, etc., decompose foreign substances to provide nutrients for growth. In the industry, a variety of fungi are used to produce proteases, amylases and lipases (Harwood, c. R., price c/2„o/. , 1992, 1〇: p 247_ 256). Since B. subtilis has about 15~2% of cells at the end of logarithmic growth, it can transform foreign genes into cells (1) 汕(10)屯D, M/cr〇6 /〇/i?ev·, 1991. 55: ρ· 395-424.), therefore Bacillus subtilis can be used as a host cell for gene manipulation and production of recombinant proteins, and many heterologous protein genes have been transferred to Bacillus subtilis. Expression production is carried out, and examples thereof include staphylokinase, human interleukin-Ι, and antidig〇xin single-chain antibody (Ye R, KJ et Al Bi〇techn〇1 Bio Eng·, 1999. 62: p. 87-96; Bellini AV et al. "Method for the production of recombinant IL-1 beta by Bacillus subtilis: comparison between cells inside and outside 1310405 {Production processes of rec〇mbinant /L .; ^

B, s. Bacillus subtilis as a host cell for protein production has several advantages: (1) the cell itself is not pathogenic to the human body' (2) the gene is simple to operate, and there is no special preference for the gene codon (c〇d〇n)' 3) Rapid growth and easy culture 'No additional nutrient matrix is needed in the culture solution. (4) Strong and secreted secreted system can secrete extracellular proteins into the medium, which will help Purification of protein recovery, which is also a feature of Bacillus subtilis in genetic engineering applications (Wong, s, σ (9)e, 1989 83: Ρ · 215-223) » Bacillus subtilis can produce a variety of enzymes that have long been The study found that, but why the real mechanism is still in the process of research and exploration. According to the research, it is found that the terminal part of the sub-I1 protein contains 18 to 35 amino acids. This sequence is called the signal peptide. This amino acid sequence can secrete proteins from the cell. Go outside the cell. In the c-terminal part of the message peptide, there is a consensus Cleavage site. When the secreted protein is transported to the cell membrane, the signal peptidase (Spase) will cut off the message peptide. And send the protein out of the cell. The structure of the general peptide is a positive charge at the N-terminus, a stable pulse-spin structure in the middle, and a negative charge at the C-terminus. When the secreted protein gene is transcribed and translated to form a polypeptide, the N-terminal message peptide can be identified by the signal cytoplasmic recognition particle (SRP) 1310405, year, month // 曰 correction Replace the page and combine to form a complex that will lead to the SRP recptor junction to the location of the sin near cell membrane. Next, the translocase complex on the membrane is transported to the extracellular space to complete the translocation, and then the message peptide hydrolase excises the message peptide to form an active mature protein (Wely, KHMv·, subtilis The use of Bacillus subtilis as a protein cell, the primary problem is how to transform foreign genes into cells. The most commonly used transformation methods are about three types, including natural transformation, protoplast transformation, and electrotransformation (eletrop〇rati〇n). The natural transformation method is to use the ability of Bacillus subtilis to enter a stable phase at the end of the logarithmic growth phase to produce a foreign gene, and to transfer the desired target gene into the cell (Dubnau, DaD-A·, R·, "/. Mo/·(iv)/., 1971· 56: p. 209-221). In addition, if the natural transformation method is used to transform the plastid, it is necessary to become a polyploid because the plastid will recombine in the homologous region when it is transformed into a cell, and form a complete The plastid, if the plastid is a single _ body, this method cannot be used to transform the plastid into Bacillus subtilis. The protoplast transfer method mainly utilizes the cells in the early stage of logarithmic growth, and then uses lysozyme (lyS〇Zyme) to decompose the cell wall to a thin layer, and I7 becomes the indicated protoplast cell, and is highly sucrose, Gasification and PEG promote the transformation of plastids into cells, and then use the extremely rich nutrient matrix to culture cell wall regeneration and cell recovery (Chang s, cs,

Ge«ei·, 1979. 168(1 u_115)). Although using this method to transform the plastid, the mass 7 1310405 itself does not need to be a polyploid. Since the lysozyme is used to dissolve the cell wall first, it is easy to disintegrate the cell if it is accidentally manipulated. After the shape, the cells need to be cultured for a long time to restore the cell wall, so it is difficult to control in operation. The electrotransformation method uses a transient high-voltage discharge to transfer foreign genes into cells. It is currently the most commonly used gene transformation technique in various cells, and its main advantage is that the method is simple to operate (Chassy, BM < TIBTECH, 1988) 6: p. 303-310; Xue, G.-PJ et al, Journal 1999. 34: p. 183-191), but the use of electroporation in the haystack bacteria is likely to cause cell collapse, and the call transformation efficiency is poor. As described above, 'Because the transformation function of Bacillus subtilis is not as convenient as Escherichia coli, many studies have been conducted to construct Escherichia coli/Bacillus subtilis shuttle plastids (Kreft, j.) for the convenience of genetic manipulation. , et al., Gene, 1990. 87: p. 53-61; Enrlich, SD, Proc. Natl. lac/· 1978. 75: p_ 1433-1436.). The commonly used shuttle plastid is Escherichia coli. The original replication origin (origin 〇f repiicati〇n) type such as PMB1, PBR322, P15A, etc., and the B. subtilis plastid origin pattern are pUBl 10, pE194, pC194, etc. However, combining these origins of replication Constructed shuttle plastids, which are often used to produce recombinant proteins in Bacillus subtilis, often undergo plastid instability, mainly due to structural instability. This is mainly due to the application of hay The plastids of Bacillus are mostly in the form of rolling circle replication. If there are repeated gene sequences in the plastid, it is easy to cause homologous recombination during the replication process, and some genes are eliminated. 1310405 (Unlen, M. et al, Plasmid, 1981. 5: p. 161-169; Gryczan, T.J., and Dubnau, D., J. Bacteriol., 1978. 141: p. 246-253·

Primrose, S. B., and Ehrlich, S. D., Plasmid, 1981. 6: p. 193-201.). Although there are many studies that attempt to reduce the 'clear shape' of the stability of the shuttle, it still cannot solve this problem effectively. This is also the biggest loss of protein production using Bacillus subtilis. In view of this, there is still a need for further improvement using the Escherichia coli/Bacillus subtilis shuttle plastid. Φ [Summary of the Invention] The present invention relates to the construction of a shuttle plastid of Escherichia coli / Bacillus subtilis, which has a very high stability compared to the well-known shuttle plastids, and has been proved to be effective for stable production of a large number of natto. The kinase is extracellular. Past studies have revealed that the frequently used E. coli (pMB丨 replication origin)/Bacillus subtilis (pUB1 1〇 replication origin) shuttle plastids are transformed into B. subtilis to form transformed host cells. In the case of cell, it is easy to induce the phenomenon of plastid instability. Therefore, when the recombinant protein is produced in large quantities by this transformation, the production amount of the recombinant protein will be restricted. Based on this, the Applicant constructed in the present invention to combine the E. coli R6K plastid replication origin pattern and the Bacillus subtilis (1) plastid origin to produce a novel Escherichia coli/Bacillus subtilis plastid Improve the stability of the plastid to achieve the purpose of mass production of recombinant protein, and further enhance the industrial applicability of Bacillus subtilis. For the purpose of the invention, the present invention relates to a method for stably producing a 1310405 gene product in a recombinant host cell comprising the following steps (4) constructing a shuttle f body comprising a line from a large intestine rod (4) The origin of replication of the humic body (〇rigin) and the origin of replication of the pUB 110 plastid from the golden grapevine gp-cut; 〇&gt;) The gene fragment encoding the selected μ gene product is thinned ( Constructed by the step (4) to form a recombinant plastid, wherein the selected gene fragment is isolated, and is selected downstream of a promoter/information peptide; (C) formed by step (b) a recombinant plastid is transformed into a host cell, and the cells containing the recombinant plastid are screened by the medium; the recombinant plastid is obtained from the above-mentioned cells containing the 6 HM recombinant plastid, and then transformed into another host. 'in the cell' to produce a recombinant host cell; and (d) the recombinant cell obtained in step (c) is cultured in a medium to allow the selected gene to be expressed and secreted in the extracellular; and (e) harvested The selected gene product. Preferably, the step (a) constructed shuttle plasmid further comprises at least one of: a message peptide, at least two resistance genes, a enhancer sequence, a promoter sequence, a diverse selection site. And - a promoter sequence is preferred. Preferably, the promoter sequence is a P43 promoter. More preferably, the promoter sequence further comprises a promoter selected from the group consisting of a 〇8 promoter, a σΒ promoter, σ(:promoter , ctd promoter, σΕ promoter, σΡ promoter, promoter, σΗ promoter, (jL promoter, promoter, 10 1310405 spac promoter, T7 Kaihong 7 for steming PR promoter and XPL promoter The composition is suitable for the group of the (4) promoter. The 疋 message peptide sequence is a nattokinase message peptide sequence 〇蟑 better 疋 ' message peptide sequence and then contains - or more than one package: selected from the left The composition of the glycosidase message peptide, the neutral protein degrading enzyme message, and the test protein degrading enzyme message peptide are suitable for the group of the subtilis message peptide sequence. Preferably, the resistance gene system is suitable for large Anti-ampicillin gene of Bacillus and anti-connamycin gene suitable for Bacillus subtilis. More preferably, the resistance gene is one or more selected from the group consisting of ampicillin, chloramphenicol, kanamycin, tetracycline, More preferably, the gentamicin, the sulphate, the serotonin, the methylamine, the erythromycin, the lincomycin, the neomycin, and the phleomycin are integrated into the group. Among them, pneumomycin, connamycin, tetracycline, gentamicin, spectinomycin, streptomycin, and transaminyl methylamine biting resistance genes are suitable for Escherichia coli, and pneumomycin, erythromycin, and forest. Cocomycin, neomycin, phleomycin, tetracycline are suitable for Bacillus licheniformis. Preferably, the selected gene product of step (b) is nattokinase. Preferably, the selected gene product It can be a homologous or a heterologous peptide. Preferably, the selected gene fragment from step (b) is selected downstream of a nattokinase peptide. 1310405 / / 曰 correction replacement page is preferably, step (C) in the heavy cell transformation of the tissue system to a E. coli fine, preferably step (c) of the screening of the lid 迸 迸 养 基 LB A medium containing ampheicillin. Tetrabacillus. Preferably, the _heavy-host cell line formed by the step (4) is a grass. Preferably, the step (4) is carried out by culturing a recombinant cell in a LB containing Connor. :: Ming is also related to a shuttle plastid' which contains the origin of the paper from the large intestine rod and the origin of S. aureus. The origin and the golden grape ball. Preferably, the origin of the R6k strain of the Escherichia coli is reversed. Preferably, the plastid further comprises a P43 promoter, which cleaves the address. More than one resistance gene, the levansucrase message peptide gene, and the restriction enzyme gene = are more than one of the resistance gene lines being ampicillin resistance or a kalemycin resistance gene. The origin of the invention relating to a shuttle containing a nattokinase structural gene comprises the origin of the legs of the large intestine rod (four), the Staphylococcus aureus, the '' </ RTI> ugly kinase message peptide and the nattokinase gene. Promoter 0 preferably further comprises more than one resistance gene and p43-based dioxin::::...the gene system is An-I 12 1310405. The present invention is further related to a method comprising a R6K original comprising Escherichia coli Point and the microorganisms of the plastids of the S. aureus origin. Preferably, the microorganism is a bacterium. More preferably, the bacteria is Bacillus subtilis. The invention relates to a method for producing nattokinase which utilizes the aforementioned microorganism to produce nattokinase. [Embodiment] The present invention combines a shuttle plastid from Escherichia coli R6K with a new construct from the S. aureus PUB110 f-body replication region, compared to binding from the gut bar g PMB1 and from S. aureus pUBn 〇 plastid replication The shuttle of the region has a higher plastid structure in the strain of Bacillus licheniformis. In addition, the f-part separation stability was also higher in the strain of the stalk (4) than in the plastids from the E. coli ρΜΒι and the sputum sβ1 plastid replication region. Therefore, the use of the novel shuttle plastid of the present invention as an example of the production of nattokinase enables the Bacillus subtilis strain to efficiently and stably produce a large amount of extracellular proteins. The invention constructs a novel shuttle plastid of Escherichia coli/Bacillus subtilis, and proves that it has extremely high stability in Bacillus subtilis by an example, and can effectively produce a large amount of recombinant protein produced outside the cell to simplify the downstream protein. Purification procedure 'Deep industrial production of medical eggs: quality, antibody: peptide and peptide vaccine use value. Compared with the well-known Escherichia coli / Bacillus subtilis shuttle plastid, a new type of shuttle plastid is constructed, and it is used in Bacillus subtilis 13 1310405 8 to have extremely stable stability, so that mass production reorganization can be achieved. The purpose of protein outside the cell. The term "plastid" as used in the present invention means a genetic material other than the main chromosome in the ''cell body' (extrachr〇in〇s〇mai genetic materal), also known as a minichromosome. The large-part plastids are supercoiled double-stranded circular DNa. The term "shuttle plastid" as used in the present invention means a plastid having two origins of host cell replication, which can be transformed into two host cells for constructing or expressing a target gene fragment. The proper noun "Escherichia coli / Bacillus subtilis shuttle" used in the present invention means a plastid having a replication origin of both host cells of Escherichia coli and Bacillus subtilis. EXAMPLES The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

The experimental methods employed in the present invention, as well as for DNA cloning, are referenced to textbooks well known in the art: M〇lecular Cloning: a Laboratory Manual. 3rd ed. Cold Spring Harbor

Laboratory Press, New York, (Sambrook J, Russell DW. (2001)). For example, use restriction enzymes to cleave DNA fragment reactions, use T4 DNA ligase, bind sputum fragment reaction, polymerase chain reaction, agarose gel electrophoresis, sodium dodecyl sulfate-polyacrylamide electrophoresis, and plastid transformation And these technologies are all familiar to the technology and can be implemented according to existing technologies. For example, Escherichia coli used in the following examples 14 1310405

The plastid transformation method is carried out using competent cells treated with calcium chloride. The dry grass stem cell transformation method is carried out in an electroformed manner. Further, the cell density was measured using a spectrophotometer (V530, Jasco), and the measurement wavelength was 6 〇〇 nm, and the obtained absorbance was recorded as . The protein concentration analysis was performed by using a protein analysis reagent and analyzing the protein by gel electrophoresis using an image analyzer (&lt;3 VIII 89 〇〇〇, 1] ¥1^(〇). The casein activity test method was an activity test. The reaction volume is 0.9ml, and the reaction solution is 〇. 1M sodium phosphate buffer (ρΗ=7·5) contains 0.5% casein and 50μ1 cell supernatant, and after 37 minutes of reaction, ΙΟΟμΙ 2Μ hydrochloric acid solution is added. The reaction was terminated, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was taken and converted to tyrosine concentration by a spectrophotometer at a wavelength of 275 nm. Casein activity (cu) was defined as a periplasmic supernatant per unit volume. The breakdown of casein per minute produces a free exogenous acid concentration (mole/min/ml).

• QIApreP@ Spin MinipreP kit (called _ Co.) and Nucle 〇 Spin_ Acid Purification Kit (Clontech C〇 望 在 在 普.i , Bacterial chromosomes, plastids and DNA tablets Wizard® genomic DNA purification kit

The primers required for the polymerase chain reaction were synthesized by Mingxin Biotechnology Co., Ltd. (Taiwan).

Types of antibiotics, as needed, 15 1310405 Bacteria of ampicillin, chloramphenicol, kanamycin, tetracycline, gentamycin, spectinomycin ), streptomycin, sulfamerazine, etc., as well as chloramphenicol, kanamycin, erythromycin, lincomycin for Bacillus subtilis Lincomycin, neomycin, phleomycin

, tetracycline (tetracycline). The amount of antibiotic used, if desired, is 0.00 kg/mL for kanamycin and 50 pg/mL for ampicillin. Example 1: (A) Construction of Escherichia coli / Bacillus subtilis shuttle plastids pUBCII The shuttle plastid pUBCII contains the P43 promoter, multiple cloning sites, and the left sucrose enzyme peptide (仏Signal peptide) From E. coli pMB1 and from the Staphylococcus aureus (57//10) rew pUBllO plastid replication region (replicati〇n origin), anti-ampicillin (ampicillin) gene and anti-kanamycin (kanamycin) gene. The construction process is based on the nucleotide sequence of the plastid pWB980 to synthesize the following two primers: Forward primer: 5'-ACTGAATTC.CAGCTCAGCATTATTG-3, (SEQ id ΝΟ:1) Reverse primer: 5'-ATGCGCCATGACAGCCATG-3 '(SEQ ID NO: 2) The above forward primer was designed to have a cleavage site for the restriction enzyme c c RI (as indicated by the bottom line of 16 1310405). The plastid pWB980 purified by QIAprep® Spin Miniprep kit was used as a template, and the pCR reaction was carried out using the above two primers, thereby increasing the gene fragment containing the pUB11O plastid replication region (288).

Kb). Further, it was synthesized according to the nucleotide sequence of Escherichia coli plastid puc 18. The following two primers: Forward primer: 5'-CTCATGAGACAATAACCCTG-3, (SEQ ID NO: 3) Φ Reverse primer:

5'_GAAA〇AAXmGTGCCAGCTGCATTAATG-3, (SEQ ID NO: 4) The above reverse primer was designed to have a cleavage site for the restriction enzyme £c〇RI (as indicated by the bottom line). The plastid PUC18 purified by QIAprep® Spin Miniprep kh was used as a template, and the pcR reaction was carried out with the above two primers, thereby increasing the gene fragment containing the pMB1 plastid replication region (i93 Kb) and NucleoSpin® nucleic acid. Purification kits were used to purify the amplified dNA fragments, and the two gene fragments were separately cleaved with restriction enzymes &amp; gRI, and the resulting cleavage products were respectively ligated with ligase, according to the known known colon length (4) The law, transforming the ship's adhesive products into large

Enterobacteriaceae _, and the shuttle plastid pUBCII as shown in the first figure was obtained. The shuttle plastid PUBCII white A. Δr &gt;~ 匕3 · Ap 'anti-fetocillin gene; Kanr' anti-connamycin gene, 7. PMB1 state 'E. coli PMB1 origin of replication, pUBllO ori, present It t 金 金 勤 勤 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p It is marked in the MCS frame. The plastid pUBCII was sequenced by Yuanfu International Biotechnology Co., Ltd. (Taipei) to confirm the nucleotide sequence containing the P43 promoter contained in plastid pUBCII, diverse colonization sites, levansucra message peptide and pMB 1 and pUB 11 0 plastid replication region origin (replication origin).

(B) Construction of Escherichia coli / Bacillus subtilis shuttle plastids pUKVI The shuttle plastid pUKVI contains the P43 promoter, diverse colonization sites, levanase information peptide, E. coli R6K and Staphylococcus aureus pUB 110 Domain replication region, anti-ampicillin gene and anti-canemycin gene. The construction process is based on the nucleotide sequence of the shuttle plastid pUBCII to synthesize the following two primers: Forward primer: 5,-TGGTGAATmGCCGGATCAAGAGCTAC-3, (SEQ ID NO: 5) Reverse primer: 5'-AGATCCTTTGATCTTTTCTACG-3, (SEQ ID NO: 6) The above forward primer was designed to have a cleavage site for the restriction enzyme five coRI (as indicated by the bottom line). Using the plastid pUBCII purified by QIAprep® Spin Miniprep kit as a template, the PCR reaction was carried out with the above two primers, thereby increasing the gene fragment containing the pUB 110 plastid replication region (3.99 Kb), and The nucleotide sequence of Escherichia coli plastid pAH70 is synthesized from 18 1310405. The following two primers are synthesized: Forward primer: 5,-TAGG〇AAXXilGCGATAGGAACTAAGGAG-3, (SEQ ID NO: 7) Reverse primer: 5,-ATTCGCGATTTTTGCGGCCGCAAGATC-3, (SEQ 1〇NO: 8)

The above forward primer was designed to have a cleavage site for the restriction enzyme five coRI (as indicated by the bottom line). The plastid pAH70 purified using the QIAprep® Spin Miniprep kit was used as a template, and the PCR reaction was carried out using the above two primers, thereby increasing the gene fragment (0.46 Kb) containing the R6K plastid replication region. The amplified DNA fragment was purified by NucleoSpin® nucleic acid purification kit, and the two gene fragments were respectively cleaved with restriction enzyme five coRI, and the formed cleavage products were respectively bound to each other by ligase, according to the known large intestine. The plastid transformation method was used to transform the DNA-binding product into Escherichia coli BW25 142 to obtain the shuttle plastid pUKVI as shown in the second figure. The shuttle plastid pUKVI comprises: Apr, anti-ampicillin gene; KaV, anti-connamycin gene; R6K ori, E. coli R6K origin of replication, pUBllO ori, S. aureus pUBl 10 origin of replication, P43, promoter, The P43 promoter, *SP, the leuctosylase message peptide, and the restriction enzyme cutting sites are indicated in the MCS frame. The plastid body pUKVI was analyzed by the source of the International Biotechnology Corporation (Taipei) in 19 1310405, confirming the nucleotide sequence containing the P43 promoter contained in the plastid pUKVI, the multiple selection position left, and the polysucrosease message. Peptide and R6K and pUB 110 plastid replication regions. (C) Construction of a plastid pUBC-NAT2 containing a P43 promoter to control the expression of a nattokinase structure-gene. The plastid pUBC-NAT2 contains a P43 promoter for expression of a nattokinase structural gene, and its construction process is based on the nattokinase structural gene The nucleotide sequence to synthesize the following two #引引: Forward primer: 5,-ATCTGHTA££ATTAATGCACAGAATAG-3, (SEQ ID NO: 9) Reverse primer: S^CTTTGGATCCTTATTGTGCAGCTGCTTGTAC-3, (SEQ ID NO: 10 )

The above two primers are each designed to have a restriction site for the restriction enzyme and (as indicated by the bottom line). The natto bacteria (provided by Biostech) chromosome purified by the Wizard® genomic DNA purification kit was used as a template, and the above two primers were used for the PCR reaction, thereby increasing the gene fragment containing the nattokinase structure ( 1.23 Kb). The amplified DNA fragment was purified using a NucleoSpin® nucleic acid purification kit. Cleavage with a restriction enzyme, and the resulting cleavage product is incorporated into the cleavage plastid pUBCII, and the DNA adhesion product is transformed into the large intestine 20 1310405 bacillus DH5 α according to the known known E. coli plastid transformation method. In the middle, the plastid pUBC-NAT2 as shown in the third figure is obtained. The plastid PUBC-NAT2 comprises: Apr, anti-ampicillin gene; Kanr, anti-connamycin gene; pMBl ori, E. coli pMBl origin; pUBl 10 ori, S. aureus pUBllO origin; P43 promoter, P43 promoter; SP, nattokinase message peptide; aprN, nattokinase gene. The plastid PUBC-NAT2 was sequenced by Yuanfu International Biotechnology Co., Ltd. (Taipei) to confirm the nucleotide sequence of the P43 promoter contained in the plastid PUBC-NAT2 and the nucleotide sequence of the nattokinase structural gene. (D) Construction of a plastid PUKVI-NAT2 plastid containing the P43 promoter to control the expression of the nattokinase structural gene PUKVI-NAT2 contains the P43 promoter for expression of the nattokinase structural gene, and its construction procedure is as follows according to the nucleus of the nattokinase structural gene The nucleotide sequence is used to synthesize the following two primers = forward primer:

5,-ATCTGGTACCATTAATGCACAGAATAG-3, (SEQ ID NO: 11) Reverse primer: ^^ΓΤΤΤΓτΓτΑΤΓΓ.ΤΤΑΤΤΓτΤΓτΓ.ΑΓτΓΤΓτΓΤΤΓτΤΑΓ-3, (SEQ ID NO: 12) The above two primers were designed with restriction enzymes/7«1 And the cutting address (as indicated by the bottom line). The natto chromosome purified by the Wizard® genomic DNA purification kit was used as a template, and the above two primers were modified to replace the two primers for PCR reaction, thereby increasing the content of a natto containing natto _;,, · 〇 gene fragment (1.23 Kb). The amplified DNA fragment was purified using a NucleoSpin® nucleic acid purification kit. The cleavage of the restriction enzyme ρ Ι ΗΙ ΗΙ 并将 并将 并将 并将 并将 并将 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' The binding product was transformed into E. coli DH50C to obtain the plastid PUKVI-NAT2 as shown in Figure 4. The plastid PUKVI-NAT2 contained: A〆'anti-ampicillin gene, Kanr, anti-connamycin gene, R6K 〇ri , E. coli R6K origin of replication, pUBii〇ori, Staphylococcus aureus pUB1 1〇 origin of replication, p43 pr〇m〇ter, p43 promoter, SSp, nattokinase peptide, aprTV, nattokinase The plastid PUKVI-NAT2 was sequenced by Yuanzi International Biotechnology Co., Ltd. (Taipei) to confirm the nucleotide sequence containing the P43 promoter contained in the plastid pUKVI-NAT2 and the nucleotide of the nattokinase structural gene. (E) Construction of a plastid containing the P43 promoter to control the expression of the nattokinase-expressing gene pM43-NAT2 plastid PM43-NAT2 contains the P43 promoter for expression of the nattokinase structural gene, from Escherichia coli pMB1 and from Enterococcus faecalis ( EWerococcw/aec a/b) pAMpi plastid replication region, anti-ampicillin gene and anti-erythromycin gene. The construction process is as follows: The plastid PUBC-NAT2 is cleaved with restriction enzyme five coRI/p, and will be formed. The cleavage product containing the P43 promoter and the nattokinase gene is incorporated into the cleavage plastid pMTL5〇〇E, and the DNA adhesive product is transformed into the large intestine rod according to the known known E. coli plastid transformation method. In the DH5oc strain, the plastid pM43_NAT2 is obtained as shown in Fig. 5. The shell PM43-NAT2 contains: ΑρΓ 'anti-ampicillin gene, - Emr, anti-erythromycin gene, pMBl 〇ri, E. coli pMB1 rep Point, ρΑΜβΙ od, Enterococcus faecium ρΑΜβ1 origin of replication, ... and plant print five, bacillus 'PAM&quot; replication protein gene, Ρ43 pr〇m〇ter, Ρ43 promoter •, 叩rTVSP, nattokinase message peptide 'India riv, nattokinase gene. The plastid PM43-NAT2 was sequenced by Yuanfu International Biotechnology Co., Ltd. (Taipei) to confirm the nucleotide sequence containing the P43 promoter contained in the plastid PM43-NAT2 and nattokinase. Nuclear gene Acid sequence. Example 1. Production of nattokinase by recombinant strain WB700/pXJBC-NAT2 Firstly, in order to test the stability of the constructed plastid, the present invention selects nattokinase as a production mode 'according to the existing known Bacillus subtilis plastids The recombinant plasmid pBC-NAT2 was transformed into B. subtilis WB7(R), and the recombinant strain WB700/pUBC-NAT2 was obtained. _ WB700/pUBC-NAT2 recombinant strain was spotted from a solid culture dish and inoculated into 5 mL of lb medium containing l〇pg/L kanamycin, and cultured overnight at 37 C. Thereafter, the overnight cultured cells were inoculated into a 250 mL flask containing 2 mL of LB medium (containing 1 〇pg/mL Connorfulin) at a cell initial density of 〇〇8 (ο%. The inoculated bacterial culture was cultured in a constant temperature shaking culture at 37 ° C and 200 rpm. The cell sample was taken for 12 hours to perform plastid separation and structural stability test. The experimental procedure was as follows: 10 bacteria solution was taken. Dilute with 〇1M sodium phosphate buffer (pH 7.0) and spread it on the LB solid culture, then place it in a 37 ° C incubator for 14 hours. After the colony is formed, 100 individual colonies will be separated. Click on the LB solid culture dish containing i0jig/mL anti-conomycin and i% skim milk powder, place in 37. Incubate for 14 hours in a constant temperature incubator, and observe the number of colony formation on the solid culture dish, while the plastid The separation stability is expressed as the ratio of the number of colony formations to the number of individual colonies screened. In addition, the number of transparent rings produced around the colonies was examined, and the number of the bacteria was counted in the selected individual colonies. Proportion of The structural stability of the body. It was found from the experimental results that the recombinant strain WB700/PUBC-NAT2 was stable in the presence of plastids for 12 and 24 hours after culture, and there was no instability of plastid separation. About 35% and 77% of the strains produced unstable plastid structure. For further analysis, the colonies containing gluten-free milk powder on the LB solid culture dish were scraped and cultured without a transparent ring. The sequence analysis was carried out. The sequence analysis showed that the sequence of the nattokinase and the pMB 1 origin of replication has a sequence of GGCGGAGC, respectively. Please refer to the sixth figure, the homologous recombination phenomenon occurs in the two sequence regions. It leads to the loss of the ability to produce nattokinase in the plastid. The plastid PUBC-NAT2 shown in Figure 6 is a schematic diagram showing the region of plastid homologous recombination, which contains: A〆, ampicillin resistance gene; Kanr, Kanamycin resistance gene; pMB 1 ori, E. coli pMB 1 origin; pUBllO ori, S. aureus pUBn 〇 origin of replication, P43 Promoter, P43 promoter, apriV SP, natto Message peptide, apr# 'nattokinase gene, GGCGGAGC, gene homologous sequence region. 24 1310405 1 :丨y曰修正#懊页 i Example 3: Producing nattokinase with recombinant strain WB700/pUKVI-NAT2 -

It is known from the second example that the PUBC-NAT2 shuttle plastid is prone to structural instability, and the results of experimental results show that the instability of the plastid structure is related to the origin of pMB 1 replication of E. coli. Therefore, in order to solve this problem, we changed the pMB 1 of the Escherichia coli origin of replication on the plasmid to R6K, and produced a new expression plasmid pUKVI-NAT2. Thereafter, the expressed plastid PUKVI-NAT2 was transformed into B. subtilis WB700 according to the known Bacillus subtilis transformation method to obtain a recombinant strain WB700/pUKVI-NAT2. Recombinant strain culture and plastid stability were carried out in accordance with the procedure of Example 1. The WB700/pUKVI-NAT2 was cultured and tested. The results showed that all the strains tested on the LB solid culture dish containing the kanamycin and skim milk powder could form colonies and a transparent ring could be produced around the colonies. The results show that the plastid PUKVI-NAT2 has 100% structural stability in the host cells (as shown in Table 1).

Table 1. shows the process of expressing plastid WB700 fermentation to produce nattokinase, the structural stability of cellular endoplasmic plastids 12 hours 24 hours pUBC-NAT2 65% 23% pM43-NAT2 100% 100% pUKVI-NAT2 100% 100% In order to further test the stability of the new construct plastid PUKVI-NAT2 and the efficiency of producing recombinant protein, we will recombinase strain 25 respectively.

1310405 WB700/pUKVI-NAT2, WB700/pM43-NAT2 and natto bacteria were cultured in LB medium and placed in a constant temperature shaking incubator at 3 rpm and 2 rpm to compare the yield of each strain of nattokinase. , enzyme specific activity and plastid stability. The culture method and the plastid stability test were carried out in the same manner as in the first embodiment, and the nattokinase production and activity test was carried out by extracting the sputum culture solution from the culture broth after 24 hours of culture. (: The cells were centrifuged at 130 rpm for 1 minute to remove the cells, and then the supernatant was collected to carry out a yield analysis and activity test of nattokinase. The activity test was carried out in accordance with the aforementioned casein activity test method. The results showed that the recombinant plastids ρυκνι_ΝΑτ2 and PM43-NAT2 all have 100% plastid structural stability. Past studies have revealed that P. faecalis PAMP1 plastid replication is highly structurally stable, in a "copy mode, so this result This point is also confirmed and shows that the plastids from the E. coli PMB1 and the plastid-like region from the P. faecalis replication region have high plastid structure stability. However, after 24 hours of culture, please refer to Table- and Table 2, respectively. 'The former's f-separation stability is still maintained, while the latter's plastid separation stability is only 88%. Table 2 shows that the expressed plastids are produced in w_ + fermentation to produce nattokinase jgj, and the cell endoplasmic | Sexual flattening 12 hours 100% 95% 100% plasmid PUBC-NAT2 PM43-NAT2 PUKVI-NAT2 Same 'Please refer to Table 3 for 24 hours 100% 88% 100% based on enzyme activity test Fruit 26 1310405 Recombinant strain WB700/pUKVI-NAT2 can produce nattokinase activity up to 561 CU 'Compared with the recombinant strain wB700/pM43-NAT2 and natto bacteria, the enzyme activity is about 2. 丨 and 6.4 times Table 3 shows the casein activity of the nanokinase produced after 24 hours of fermentation using the natto and the recombinant strain. The CU protein yield (μΜ/min) (mg/L) Bacillus natto 76 15.5 WB7〇〇/pM43-NAT2 183 42 —WB700/PUKVI-NAT2 561 118 CU is the casein〇iytic activity unit 〇In addition, we also follow the aforementioned sodium dodecyl sulfate-polypropylene 醯

The monthly female electrophoresis and image analyzer were used for protein yield analysis. As described above, the supernatant after centrifugation of the bacterial sample was taken out for 4·8 μί, and then mixed with 1.2 μL of 5X-like buffer, and then loaded onto a 丨5% polyacrylamide gel in advance for electrophoresis. After the electrophoresis is completed, the gel is immersed in the coloring agent for 10 to 14 hours, and then the gel is washed with a decolorizing solution. When the protein block is clearly distinguishable, the film is put into the preservation solution for 15 to 30 minutes, and finally dried. The membrane will be stored in a dry state. The dried gel was placed on a human image analyzer for protein analysis and analysis. The recombinant strain WB700/pUKVI-NAT2 was produced and the kinase production was much higher than that of the recombinant strain WB700/pM43_ spear and endogenous bacteria. Moreover, the extracellular protein produced by the recombinant strain WB700/pUKVI-NAT2 is almost nattokinase, accounting for about 98% of the total extracellular protein' as shown in the seventh. The Fig. 7 shows the production of extracellular protein of nattokinase by natto and recombinant strain 27 1310405, respectively. Where diameter i: Protein molecular marker (Mbi)

Fermentas); Path 2: Use of natto bacteria to produce extracellular protein samples of nattokinase; Run 3: use of recombinant strain WB7〇〇/pM43_NAT2 to produce extracellular protein samples of nattokinase · Path 4: Use recombinant The strain 'WB70()/pUKVI-NAT2 was used to produce extracellular protein samples of nattokinase; the arrow indicates the setting of nattokinase M. Using the image judgment system, preliminary speculation, see Table 3, 'WB7〇〇/pUKVI_NAT2 The production of nano-kinase in lb medium &gt; Compared with the recombinant strain (4), the yield of 18 mg/L ‘(8)(10) _ NAT2 and natto was 18 times and 66 times higher, respectively. Taken together, the above results clearly show that 'the production of nattokinase as an example, the present invention combines the shuttle 胄ρυκνι from the E. coli R6K with the newly constructed plastid region from the S. aureus PUB11G plastid, compared to the binding from E. coli pMB1 The shuttle plastid pUBcii from the plastid replication region of S. aureus PUB110 has a higher plastid stability in the subtilisin, f; the other is compared to the pMBl from Escherichia coli and the phage β1 from E. faecalis In the plastid replication region, the shuttle plastid Ρ 'Π also has a higher plastid fraction in the B. subtilis strain = H. Therefore, the application of the novel shuttle plastid buckle of the present invention can make the B. subtilis strain effective and stable in mass production. Extracellular protein. It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. In the absence of the present invention, it is to be understood that the invention is not limited to the specific details. In fact, in the implementation of the mode of the presently known 28 1310405 s, the different amendments are also covered by the following materials. [Simplified illustration] It is an architectural diagram of the plastid pUBCII of the present invention. The second figure is an architectural diagram of the plastid pUKVI of the present invention. The third figure is an architectural diagram of the plastid pUBC_NAT2 of the present invention. The fourth figure is the architecture diagram of the plastid pUKVI-NAT2 of the present invention. The fifth figure is the architecture diagram of the plastid PM43-NAT2 of the present invention. The sixth figure is a schematic diagram of the plastid group region produced by the plastid pUBC-NAT2 of the present invention. Original weight The seventh figure is the analysis of the sodium dodecyl sulfate-polypropylene-branched amine knife of the present invention. ' [Main component symbol description] None 1310405

Sequence Listing &lt;110&gt; Feng Chia University &lt;120&gt; shuttle type plastid, its manufacturing method and application thereof &lt;130&gt; 94146121 &lt;160> 12 &lt;170&gt; Patentln version 3.1 &lt;210&gt; 1 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;400&gt; 1 actgaattcc agctcagcat tattg &lt;210> 2 &lt;211&gt; 19 &lt;212>DNA&lt;213&gt;Artificial sequence&lt;400&gt; 2 atgcgccatg acagccatg &lt; 210>3 &lt;211&gt; 20 &lt;212>DNA&lt;213&gt;Artificial sequence&lt;400&gt; 3 ctcatgagac aataaccctg &lt;210> 4 &lt;211&gt; 28 &lt;212>DNA &lt;213&gt; Artificial sequence &lt;4 (8) &gt; 4 gaaagaattc gtgccagctg cattaatg &lt;210&gt; 5 &lt;211&gt; 27 &lt;212>DNA &lt;213&gt; artificial sequence &lt;400&gt; 5 tggtgaattc gccggatcaa gagctac &lt;210&gt; 6 &lt;211&gt; 22 25 19 20 28 / 27 #鹣1310405 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;400&gt; 6 agatcctttg atcttttcta eg &lt;210&gt; 7 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;400&gt; 7 tagggaattc Gegataggaa ctaaggag &lt;210&gt; 8 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213 &gt;Artifical sequence &lt;400&gt; 8 attegegatt tttgcggccg caagatc &lt;210&gt; 9 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400 9 atctggtacc attaatgeae agaatag &lt;210&gt; 10 &lt;211&gt;&lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400&gt; 10 ctttggatcc ttattgtgca gctgcttgta c &lt;210&gt; 11 &lt;211&gt; 27

&lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;400&gt; 11 atctggtacc attaatgeae agaatag &lt;210> 12 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; artificial sequence. 1310405 1 &lt;400&gt; 12 ctttggatcc ttattgtgca gctgcttgta c

Claims (1)

I 1310405 gene product in one X. Patent application scope: 1. A method for stably producing a host cell, comprising the following steps: (4) constructing a shuttle plastid comprising at least a promoter sequence, at least one message peptide sequence, at least one-multiple selection Colonization position, i-deficiency gene = origin of replication of R6K plastid from E. coli and replication origin from Staphylococcus aureus ρυΒ110 # PUB110 plastid; (b) encode - The gene fragment of the selected gene product is selected (SUbCl_) in the shuttle plastid constructed by the step (4) to form a recombinant plastid, wherein the selected gene fragment is selected to be thin at the at least promoter/ Downstream of the message face peptide and wherein the promoter of the peptide is operably linked to the selected gene fragment; (c) transforming a recombinant plastid formed in step (b) into a host cell, screening the medium a fine run containing the recombinant plastid; the recombinant plastid is obtained from the cell containing the recombinant plastid, and then transformed into another host cell to produce a recombinant host cell; and (d) Recombinant cell step (C) obtained in the culture medium, expression of the gene serve to allow cloning of the extracellular secretory production; and (e) harvesting the selected gene product. 2. The method of claim i, wherein the shuttle plastid constructed in step (a) further comprises at least one of: a enhancer sequence and a regulatory promoter sequence. 3. The method of claim 2, wherein the promoter sequence 1 1310405 is a P43 promoter. 4. The method of claim 2, wherein the promoter sequence further comprises a member selected from the group consisting of a V promoter, a σΒ promoter, a y promoter, an aD promoter, a σΕ promoter, an aF promoter, a promoter, The σΗ promoter, promoter, σκ promoter, spac promoter, Τ7 promoter, XPR promoter and λρί promoter constitute a group suitable for promoters of Bacillus licheniformis. For example, the second column of the patent scope is the nattokinase message peptide sequence 6, as in the patent application scope of the "member" method, in which the message wins ι: 匕 contains - or more than one selected from the left polyesterase message Peptide, medium, == lyase message peptide, test protein degrading enzyme message peptide: suitable for the group of the message peptide sequence of bacillus. 7. The method of the second paragraph of the patent scope is applicable. An anti-connamycin gene against amphezicin base of Escherichia coli and a method suitable for use in a straw rod 8, such as the method of claim 2, a species or a species selected from the group consisting of amphibians and The middle resistance genes are, tetracycline, Qingda ", view «, chain ^ 素, konax erythromycin, lincomycin, neomycin and humus::: amine methyl one, 9, if applied In the group of the square of the patent scope, the group of natamycin, tetracycline, zymidine, spectrin, cytotoxic, chloramphenicol, and Kang·resistant gene are suitable for the large intestine rod I and 4 mycin 1 amine Base and gas ghrelin, erythromycin, autumn 1310405 ketomycin, neomycin, humicin, • often four The system is suitable for Bacillus subtilis. 100. For example, the method of applying for a patent for 笳圚 固 弟 ' 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中 其中The method of the present invention, wherein the selected, soil may be a homologous peptide fragment or a heterologous peptide fragment of Natkinase of Pentaphyllum. I2: The method of claim 1 of the patent scope, Wherein step (4) the recombinant shell system is transformed into an E. coli cell. 13. The method of claim 1, wherein the cultivating medium material LB of step (c) contains amphelimin medium. The method of the first aspect of the patent, wherein the step (C) forms one of the recombinant host cell lines is Bacillus subtilis. 15. The method of claim 2, wherein the step (4) is based on the inclusion of kanamycin in the LB The medium is cultured for recombinant cells. 16. A shuttle plastid comprising at least one promoter sequence, at least one diverse selection site, at least one resistance gene, and an origin of replication of a leg from Escherichia coli ( Origin) And a complex of Staphylococcus aureus. ', 17. The shuttle plastid as described in claim i, item 6, wherein the replication origin of the E. coli #R6K (Grigin) and Staphylococcus aureus The origin is reversed. 1 8. The shuttle plastid as described in claim 6 or 17 of the patent application, wherein the plastid further comprises more than one resistance gene, p43 promoter, one left sucrose The enzyme message peptide gene, and the restriction enzyme cleavage site 1310405, the shuttle plastid described in item 18, is an ampicillin resistance gene or a phytomycin 19, as in the patent application. Resistance-based factor resistance gene. 0. The shuttle plastid of claim 16 further comprising a nattokinase message peptide and a nattokinase gene, wherein the cyclin kinase message peptide is operably linked to the nattokinase gene. The shuttle plastid described in claim 20, further comprising one or more resistance genes and a P43 promoter. 22. The shuttle plastid of claim 2, wherein the one or more resistance genes are an ampicillin resistance gene or a oxymycin resistance gene. ', a microorganism containing a shuttle plastid containing the origin of R6K from Escherichia coli and the origin of replication of S. aureus. 2. The microorganism according to claim 23, wherein the microorganism is a bacterium. 2 5. The microorganism according to claim 24, wherein the bacterium is Bacillus subtilis. A method for producing nattokinase which produces nattokinase using a microorganism as described in claim 23 of the patent application. XI, schema: as the next page 4