CN103937830B - A kind of recombinant bacterium of efficient secretory expression Nattokinase - Google Patents

Abstract

The invention discloses a recombinant bacterium capable of efficiently secreting and expressing nattokinase, which can significantly increase the output of nattokinase. The recombinant bacterium of the present invention is a recombinant Pichia pastoris containing more than two copies of the nattokinase Pro-NK gene and the Hac1p regulatory factor gene. Up to 470IU/mL.

Description

A recombinant bacterium that efficiently secretes and expresses nattokinase

technical field

The invention relates to a recombinant bacterium, in particular to a recombinant bacterium capable of efficiently secreting and expressing nattokinase.

Background technique

Nattokinase (Nattokinase, NK) is a serine protease produced by Bacillus natto or Bacillus subtilisnatto during the fermentation of natto. In 1980, Professor Sumi Yoko of Japan first discovered that the enzyme could dissolve artificial thrombus, then studied the mechanism of action of the enzyme, and named it nattokinase.

Since the first report on natto and nattokinase in China in 1995, many articles have been published so far, and research and development have gone hand in hand. At present, the properties of nattokinase (nattokinase gene sequence, protein amino acid sequence, active center and catalytic center, pI, temperature and pH stability, optimum reaction temperature and pH, inhibitor and activator, and the solubility of nattokinase Thrombosis mechanism includes ①direct thrombolytic effect of nattokinase; ②activation of prourokinase into urokinase; ③activation of vascular endothelial cells to produce t-PA; ④indirect thrombolytic effect of nattokinase, through degradation and inactivation of PAI- 1. The effect of increasing fibrinolysis has been elucidated, and the enzyme has been separated and purified, and a lot of research work has been done in vivo and in vitro thrombolytic animal experiments, acute toxicity experiments and preliminary clinical trials for the treatment of cerebral infarction.

At present, nattokinase has been extensively and deeply researched in Japan, and more than 10 large companies in Japan have produced a variety of products with nattokinase as the main component. Soybean kinase capsules are also on the market, and similar products are also available in South Korea and North Korea. In my country, many manufacturers have produced nattokinase capsule products, but due to the low yield of the production strains, compared with the products imported from Japan, the activity units of nattokinase health products sold in the market are very different. Therefore, it is necessary to transform the nattokinase producing strain to greatly increase the yield of the strain and improve the quality of the final product.

Compared with traditional breeding, relying on genetic engineering and using model hosts such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Pichia pastoris to produce recombinant nattokinase is a shortcut to increase the production of nattokinase in a short period of time. So far, the cases of expressing nattokinase in Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae have not been very successful. In recent years, Pichia pastoris has been used more and more in the expression of medicine and important food enzyme preparations due to its safe and efficient secretion and expression. Therefore, the use of Pichia pastoris to produce nattokinase will be the most applicable One of the foreground methods. At present, there have been some reports on the expression of nattokinase by Pichia pastoris. Luo Lixin et al. successfully expressed the mature peptide gene of nattokinase in Pichia pastoris GS115 and KM71, and the enzyme activity in the fermentation supernatant was 120U/mL ( Huang Zhili, Luo Lixin, Ling Junjian, etc. Construction and stability of nattokinase gene recombinant expression vector［J］. Journal of Guangdong Pharmaceutical University, 2000, 16(4):265-267). Wang Jiangbo et al. successfully expressed and compared the full peptide gene of nattokinase in Pichia pastoris GS115, KM71, and SMD1168 (Wang Jiangbo, Xu Fang, Zhang Jingfang. Secretion and expression of nattokinase progene in Pichia pastoris[ J]. China Brewing, 2008, 19(196):40-41). Cai Litao and others cloned the NK gene into the expression vector pHBM905A of Pichia pastoris, and induced expression with methanol. The results of SDS-PAGE showed that NK was successfully expressed, and the fibrin plate experiment showed that the expressed product had good fibrinolytic activity (Cai Litao, Xu Xiang, Wang Tingting et al. Expression, purification and antibody preparation of nattokinase gene in Pichia pastoris［J］. Chinese Journal of Biochemical Medicine, 2010(1)). However, the expression level of nattokinase in the above article still cannot meet the needs of industrialization, and further optimization and improvement of the production strains are still needed.

Contents of the invention

The purpose of the present invention is to provide a recombinant bacterium capable of efficiently secreting and expressing nattokinase.

In order to achieve the above object, the technical scheme of the present invention is as follows:

The recombinant vector of nattokinase Pro-NK gene is obtained by inserting more than two nattokinase Pro-NK genes into the eukaryotic expression vector.

A further preferred recombinant vector is obtained by inserting two or more nattokinase Pro-NK genes into the eukaryotic expression vector in the same direction.

The coding sequence of the above nattokinase Pro-NK gene is shown in SEQ ID NO.3 in the sequence listing.

Each nattokinase Pro-NK gene has a corresponding promoter.

The aforementioned promoter is alcohol oxidase I promoter ( _PAOX1 ) or formaldehyde dehydrogenase promoter ( _PFLD1 ).

The preferred recombinant vector of nattokinase Pro-NK gene, the selected eukaryotic expression vector is the pAO815 expression vector; the pAO815 expression vector is a 1000bp fragment obtained by double-digesting the pPICZα vector with SacI and EcoRI and similarly using SacI and EcoRI It is obtained by ligating the pAO815 vector digested by EcoRI; each nattokinase Pro-NK gene is expressed under a corresponding alcohol oxidase I promoter, and alcohol is connected in series between adjacent nattokinase Pro-NK genes Oxidase I terminator, alcohol oxidase I promoter and α-mating factor signal peptide sequence.

The recombinant vector is constructed as follows:

(1) Artificially synthesize or PCR amplify the nattokinase Pro-NK gene, add an XhoI restriction site at the upstream of its 5' end, and add an EcoRI restriction site at the downstream of the 3' end; After enzyme digestion, it was ligated with the pAO815 vector that was also digested with XhoI and EcoRI double enzymes to obtain a recombinant vector pAOα-PNK containing a single copy of nattokinase Pro-NK gene;

The above synthesis method is preferably PCR amplification method, and its specific steps are as follows: extract the chromosomal DNA of Bacillus subtilis subspnatto (Bacillus subtilis subspnatto), use it as a template, and use the upper and lower streams shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence table PCR with primers to obtain the target DNA fragment;

(2) Digest and digest the recombinant expression vector pAOα-PNK obtained in step (1) with BamHI and BglII, recover a 2589bp fragment, and connect it to the BamHI site of the recombinant expression vector pAOα-PNK to obtain Recombinant expression vector pAOα-PNK-2 containing two copies of nattokinase Pro-NK gene;

(3) By the same method and so on, the recombinant vector pAOα-PNK-n containing more than two copies of the nattokinase Pro-NK gene was obtained, wherein n represents the copy number of the nattokinase Pro-NK gene in the recombinant vector, n>2.

A recombinant bacterium efficiently secreting and expressing nattokinase is a recombinant Pichia pastoris containing more than two copies of nattokinase Pro-NK gene and Hac1p regulatory factor gene.

The Pichia pastoris mentioned above is Pichia pastoris GS115.

The above-mentioned nattokinase Pro-NK gene is introduced into the described Pichia pastoris through the recombinant vector of the above-mentioned nattokinase Pro-NK gene, so as to obtain a recombination containing more than two copies of the nattokinase Pro-NK gene Pichia pastoris.

Continue to introduce the Hac1p regulatory factor gene into the recombinant Pichia pastoris containing more than two copies of the nattokinase Pro-NK gene through a constitutive expression vector, and the resulting recombinant bacteria can produce the Hac1p regulatory factor, further improving increased the secretion of nattokinase;

The above-mentioned constitutive expression vector is preferably the constitutive expression vector pGAPZ-Hac1p, which is constructed by inserting the Hac1p regulatory factor gene into the pGAPZA vector.

The "constitutive expression vector" in this article means that the exogenous gene in it can not be regulated by regulatory factors, and can continuously express the exogenous gene under suitable conditions. The above-mentioned vector pGAPZ-Hac1p adopts the constitutive promoter _PGAP , and the target gene can be expressed without induction.

The aforementioned Hac1p regulatory factor gene is derived from yeast or mold.

The coding sequence of the aforementioned Hac1p regulatory factor gene is shown in SEQ ID NO.5 in the sequence listing.

The above-mentioned method for introducing the recombinant vector into Pichia pastoris is an electroporation method.

The present invention also provides the application of the above-mentioned recombinant bacteria highly secreting and expressing nattokinase in the processing of health care products and the application in the preparation of medicines for treating cerebral infarction.

The beneficial effects of the present invention are: through the recombinant vector constructed by the present invention, the yield level of the obtained recombinant bacteria secreting and expressing nattokinase in shake flasks can reach 470 IU per milliliter (measured according to the detection method of urokinase activity), which is higher than the current There are significant improvements in technology. The nattokinase prepared by the recombinant bacterium of the invention can be processed into a health care product or used as an active ingredient of a medicine for treating cerebral infarction.

Description of drawings

Figure 1 is a schematic diagram of the construction of the secretory expression vector pAO815alpha.

Fig. 2 is a map of the recombinant expression vector pAOα-PNK.

Fig. 3 is a map of the recombinant expression vector pAOα-PNK-2.

Fig. 4 is a map of the constitutive expression vector pGAPZ-Hac1p.

detailed description

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereby.

The formulations of the culture medium and buffer used in the following examples are as follows:

LB (Luria-Bertani) medium: 10g/L sodium chloride, 10g/L peptone, 5g/L yeast powder, pH7.4-7.6;

LLB medium: 5g/L sodium chloride, 10g/L peptone, 5g/L yeast powder, pH7.4-7.6;

BMGY liquid medium: 10g/L yeast extract, 20g/L peptone, after sterilizing at 121°C for 20 minutes, add 13.4g/LYNB, 4×10 ^-4 g/L biotin, 10g/L when the temperature dropped to room temperature L glycerol;

BMMY induction medium: 10g/L yeast extract, 20g/L peptone, after sterilizing at 121°C for 20 minutes, add 13.4g/LYNB, 4×10 ^-4 g/L biotin and 0.5% when the temperature drops to room temperature (V/V) Methanol;

YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose;

YPD plate: 10g/L yeast powder, 20g/L peptone, 20g/L glucose, 15g/L agarose;

MD plate: 13.4g/LYNB, 4×10 ^-4 g/L biotin, 20g/L glucose, 15g/L agarose;

200mmol/L trichloroacetic acid: Weigh 3.268g of trichloroacetic acid, add water to dissolve and dilute to 100mL;

STE buffer: 10mmol/LTris-Cl, pH8.0, 0.1mmol/LNaCl, 1mmol/LEDTA, pH8.0;

TE buffer: 10mmol/LTris-Cl, pH8.0, 0.1mmol/LNaCl, 1mmol/LEDTA, pH8.0;

50mmol/L borax buffer solution: add 9g sodium chloride to 19.07g sodium tetraborate, dissolve and add water to make the volume to 1000mL, adjust the pH to 8.5 with boric acid.

The experimental materials in the following examples can be purchased through conventional commercial channels unless otherwise specified. The methods adopted in the following examples are conventional methods unless otherwise specified.

Embodiment 1, the construction of the recombinant expression vector containing nattokinase Pro-NK gene

(1) Extraction of chromosomal DNA from Bacillus subtilis subspnatto

Inoculate the spore suspension or slant mycelia of Bacillus subtilis natto (preservation number CGMCC1.1086, purchased from the China General Microorganism Culture Collection Management Center) stored at -70°C into 5mL LB medium, and shake culture at 37°C 48 hours. Take 3 mL of the bacterial liquid and centrifuge to collect the mycelia, and wash the mycelium twice with 500 μL STE buffer. Then use 500μL STE to fully suspend the mycelium, add lysozyme to its final concentration of 2mg/mL, at this time, the suspension of the bacteria becomes translucent gel, and then add 250μL of 2% SDS (Sodiumdodecylsulfate, hexadecylsulfate sodium), vortexed to mix. Then add 250 μL of neutral phenol/chloroform mixed solution and mix well, wherein the volume ratio of phenol and chloroform is 25:24. After centrifuging at 10000r/min for 5 minutes, transfer the supernatant to a new Eppendorf tube, and add RNase to its The final concentration is 20-40 μg/mL, and incubated at 37°C for 30 minutes. Then use the same method, repeat the extraction with neutral phenol/chloroform 3-4 times, until there is no denatured protein at the interface between the two phases, and then extract twice with chloroform to remove the phenol in the aqueous solution. Add 1/10 volume of 3mol/L sodium acetate and an equal volume of isopropanol to the aqueous solution, mix well and place at room temperature for 30 minutes. Centrifuge at 10000r/min for 5 minutes, wash with 70% ethanol, precipitate once, dry the precipitate in vacuum and redissolve in 100-200 μLTE buffer to obtain Bacillus subtilis natto chromosomal DNA, which is used as the synthetic nattokinase Pro- PCR reaction template used for NK gene.

(2) Construction of intermediate carrier

As shown in Figure 1, the pPICZα vector (purchased from Invitrogen) was double digested with SacI/EcoRI to obtain a 1000bp fragment, and the small fragment was connected to the pAO815 vector (purchased from Invitrogen) that was also digested with SacI/EcoRI. The secretory vector pAO815alpha based on pAO815 is obtained. The _PAOX1 strong promoter in the expression vector can regulate the expression of the inserted target gene, and contains the α-mating signal peptide sequence, which can make the inserted target gene expressed as an extracellular secreted protein.

(3) PCR amplification of Nattokinase Pro-NK gene

Design PCR primers according to the Nattokinase Pro-NK gene (GenBank number: AF368283.1) derived from Natto Bacillus subtilis in GenBank, and the upstream and downstream primers are shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence table, respectively. Natto The nucleotide sequence of the kinase Pro-NK gene is shown in SEQ ID NO.3 in the sequence listing.

Upstream primer: 5'-GACT CTCGAG AAAAGAGCCGGAAAAAGCAGTACA-3'

Downstream primer: 5'-GGTC GAATTC TATTATTGTGCAGCTGCTT-3'

Downstream of the α-mating factor signal peptide coding sequence of the secretory vector pAO815alpha, there is an XhoI restriction site (CTCGAG, corresponding to the amino acid Leu-Glu), followed by a Kex2 protease recognition site (AAAAGA, corresponding to the amino acid sequence Lys -Arg), therefore, the upstream primer was designed to introduce the XhoI site and Kex2 protease recognition site sequence before the nattokinase Pro-NK gene. Through this design, the nattokinase Pro-NK gene can be directly connected to the signal peptide sequence, so that after protein translation, under the action of Pichia pastoris’ own Kex2 protease, it can finally express a protein with different amino acids than the target protein. become a mature protein without introducing additional amino acid residues due to the addition of enzyme cleavage sites. The downstream primer contains the stop codon TAT and introduces an EcoRI restriction site (GAATTC).

Using the chromosomal DNA of Bacillus subtilis natto obtained in step (1) as a template, add upstream primers, downstream primers, Primestar DNA polymerase (purchased from TaKaRa Company) and dNTP mixture to carry out PCR reaction. The reaction conditions are as follows: Deformation for 3 minutes; cycle amplification 30 times: 98°C for 15s, 55°C for 15s, 72°C for 70s; finally extend at 72°C for 90s. The amplified product was sequenced and identified to obtain a gene fragment with a size of 1080bp, which contained the coding sequence of nattokinase Pro-NK gene, and there was an XhoI restriction site upstream of the 5' end of the sequence, and the 3' end There is an EcoRI restriction site downstream.

(4) Construction of a recombinant expression vector containing a single copy of nattokinase Pro-NK gene

After the PCR product obtained in step (3) was digested with XhoI and EcoRI, it was connected to the secretory vector pAO815alpha which was also digested with XhoI and EcoRI to obtain a recombinant gene containing a single copy of nattokinase Pro-NK The expression vector pAOα-PNK, the map of the vector is shown in Figure 2.

The obtained recombinant expression vector pAOα-PNK was transformed into Escherichia coli DH5α competent cells, and the required recombinant was screened out with LB medium containing 10 μg/mL ampicillin and sequenced and analyzed. After sequencing and identification, the inserted fragment in the obtained recombinant vector was The sequence and structure of the recombinant expression vector were named pAOα-PNK.

(5) Construction of recombinant expression vector containing two copies of nattokinase gene

Digest the recombinant expression vector pAOα-PNK obtained in step (4) with BamHI and BglII, recover the 2589bp BamHI-PNK-BglII fragment, and connect it into the BamHI site of the expression vector pAOα-PNK to obtain two The expression vector pAOα-PNK-2 for copying the nattokinase gene is shown in FIG. 3 .

The obtained recombinant expression vector pAOα-PNK-2 was transformed into Escherichia coli DH5α competent cells, and the required recombinant was screened out with LB medium containing 10 μg/mL ampicillin and sequenced and analyzed. After sequencing and identification, the recombinant vector obtained was The sequence and structure of the inserted fragment were correct, and the recombinant expression vector was named pAOα-PNK-2.

(6) Construction of constitutive expression vector pGAPZ-Hac1p

First, artificially synthesize the Hac1p regulatory factor gene, its coding sequence is shown in the sequence table SEQ ID NO.5, and an EcoRI and NotI site are introduced at the 5' end and 3' end of the sequence respectively, and the Hac1p gene is connected after enzyme digestion On the pGAPZA vector (purchased from Invitrogen) that was also digested with EcoRI and NotI, a constitutive expression vector of pGAPZ-Hac1p was obtained. The map of the vector is shown in FIG. 4 .

The obtained recombinant expression vector pGAPZ-Hac1p was transformed into Escherichia coli DH5α competent cells, and the required recombinants were screened out with LLB medium containing 25 μg/mL bleomycin (Zeocin) and subjected to sequencing analysis. After sequencing identification, the obtained The sequence and structure of the inserted fragment in the recombinant vector were correct, and the recombinant expression vector was named pGAPZ-Hac1p.

Embodiment 2, the preparation of the yeast strain expressing nattokinase and its functional detection

1. Preparation of Strains Expressing Nattokinase

(1) Preparation of electrotransformed Pichia pastoris cells

Pichia pastoris strain GS115 (purchased from Invitrogen, CA.18100) was inoculated in 500 mL of LYPD medium and cultured at 30° C. and 200 r/min until OD ₆₀₀ =1.3-1.5. Centrifuge at 4°C and 4500r/min for 5 minutes to collect the bacterial cells, and wash once with 500 mL, 250 mL of pre-cooled sterilized water and 20 mL of pre-cooled 1mol/L sorbitol, respectively. After each wash, the cells were collected by centrifugation at 4°C and 4500 r/min for 5 minutes, and finally suspended with 1 mL of pre-cooled 1 mol/L sorbitol to obtain electroshock competent cells.

(2) Preparation of recombinant Pichia pastoris strain containing recombinant expression vector pAOα-PNK

Take about 10 μg of the recombinant expression vector pAOα-PNK obtained in Example 1, digest and linearize it with StuI, precipitate with ethanol, recover the linear DNA and dissolve it in 10 μL sterile water. Mix the above-mentioned linearized DNA with 80 μL GS115 competent cells, and transfer them into a pre-cooled 0.2 cm electroshock cuvette. Electric shock was performed according to the electroporation instrument (GenepulserXCell ^TM , purchased from BIO-RAD Company) used. Immediately after the electric shock, add 1 mL of pre-cooled 1mol/L sorbitol to the electric shock cup, then transfer all the solution in the electric shock cup to a sterile centrifuge tube, and directly take 200-300 μL of the bacterial solution to coat the MD plate. Inverted culture at 30°C for 2-4 days until colonies appeared, and a single-copy strain expressing nattokinase was obtained, which was named GSPNK.

(3) Preparation of recombinant Pichia pastoris strain containing recombinant expression vector pAOα-PNK-2

According to the method provided in step (2), the recombinant expression vector pAOα-PNK-2 obtained in Example 1 was linearized and electrotransformed to obtain recombinant Pichia pastoris containing two copies of nattokinase gene. After plate screening A two-copy strain expressing nattokinase was obtained and named GS2PNK.

(4) Preparation of recombinant Pichia pastoris strains containing expression vector pAOα-PNK-2 and constitutive expression vector pGAPZ-Hac1p

Take about 10 μg of the constitutive expression vector pGAPZ-Hac1p obtained in Example 1, digest and linearize it with AvrII, and transform it into the recombinant strain GS2PNK according to the method provided in step (2), and immediately add 1 mL of pre-cooled 1mol/L sorbitol into the electric shock cup, then transfer all the solution in the electric shock cup to a sterile centrifuge tube, incubate at 30°C for 2 hours, and then take 500 μL of the bacterial solution and spread it on 50 μg/mL Bleomycin On the YPD plate of Zeocin. Inverted culture at 30°C for 2-4 days until colonies appear, and through colony PCR identification, a recombinant Pichia pastoris strain containing Hac1p protein regulatory factors can be obtained, named GS2PNK-H.

2. Function detection

(1) Fermentation of recombinant bacteria

Inoculate the recombinant strains GSPNK, GS2PNK and GS2PNK-H obtained above into 25mL BMGY liquid medium respectively, set up three groups in parallel, and cultivate them on a shaker at 30°C and 200r/min until OD ₆₀₀ =10-20, 3000r/min The cells were collected by centrifugation for 5 minutes, the supernatant was discarded, and the cells were washed 1-2 times with sterile purified water. Dilute the bacteria with BMMY induction medium to OD ₆₀₀ =10, replace the cotton plug with 4 layers of gauze, culture on a shaker at 30°C and 200r/min, and add methanol every day to a final concentration of 0.5% (V/V) .

(2) Determination of extracellular enzyme activity

After the recombinant bacteria were induced and cultured at 30°C for 4 days, 1 mL of the fermentation broth was drawn into a centrifuge tube, centrifuged at 10,000 r/min for 10 minutes, and the supernatant was drawn into a test tube for later use. Add 1.4mL of 50mmol/L borax buffer solution and 0.4mL of fibrinogen solution (Fibrinogen, purchased from Sigma, product number T8630, specification: 100mg/tube) to another test tube, heat in a constant temperature water bath at 37°C for 5 Minutes later, 0.1 mL of thrombin (Thrombin, purchased from Sigma, product number T4648, specification 1000 U/cartridge) was added and mixed evenly. Incubate in a water bath at 37°C for 10 minutes, then add 0.1mL sample solution, mix for 5 seconds, and incubate for 60 minutes. Take out and shake at the 20th and 40th minutes of incubation. After the incubation, add 2mL of 200mmol/L trichloroacetic acid into the test tube, put it in a water bath for 20 minutes, and centrifuge at 15000r/min for 5 minutes. The supernatant was taken to measure the absorbance at 275 nm. Add the same amount of inactivated enzyme solution as a blank control, and the treatment method of the blank control is the same as that of the sample. Three parallel samples were set up for each group, and the final measurement result was the average value of the three measurement values.

The enzyme activity unit of nattokinase is defined as: at 37°C, in borax buffer solution with pH 8.5, the amount of enzyme needed to decompose 1mg of fibrin is one enzyme activity unit IU.

The test results are shown in Table 1. After 96 hours of induced fermentation, the average enzyme activities of the shake flask fermentation broth of GSPNK, GS2PNK and GS2PNK-H recombinant strains can reach 180, 230 and 470IU/mL respectively (Urokinase is used as the measurement standard) , indicating that the increase in copy number significantly increased the expression level of nattokinase, and after the Hac1p regulatory factor gene from Saccharomyces cerevisiae was introduced, the enzyme activity was further improved, which proved that this energy factor can significantly increase the secretion expression level of nattokinase strengthen.

Table 1 Nattokinase activity of different recombinant yeast strains (IU/ml)

Claims (8)

1. A recombinant bacterium that efficiently secretes and expresses nattokinase, is characterized in that, described recombinant bacterium is the recombinant Pichia pastoris containing more than two copies of nattokinase Pro-NK gene and Hac1p regulatory factor gene simultaneously (Pichia pastoris); The nattokinase Pro-NK gene is introduced into the recombinant Pichia pastoris through the recombinant vector of the nattokinase Pro-NK gene; The recombination vector of the nattokinase Pro-NK gene is obtained by inserting more than two nattokinase Pro-NK genes into the eukaryotic expression vector. 2. The recombinant bacterium for efficiently secreting and expressing nattokinase according to claim 1, wherein each nattokinase Pro-NK gene is expressed under a corresponding promoter. 3. the recombinant bacterium that efficiently secretes and expresses nattokinase according to claim 2, is characterized in that, described promoter is alcohol oxidase I promoter or formaldehyde dehydrogenase promoter. 4. the recombinant bacterium of efficiently secreting and expressing nattokinase according to claim 3 is characterized in that, each nattokinase Pro-NK gene is expressed under a corresponding alcohol oxidase I promoter, adjacent The alcohol oxidase I terminator, alcohol oxidase I promoter and α-mating factor signal peptide sequence are serially connected in series between the nattokinase Pro-NK gene. 5. the recombinant bacterium of efficiently secreting and expressing nattokinase according to claim 1, is characterized in that, described eukaryotic expression carrier is pAO815 expression carrier; Described pAO815 expression carrier is to carry out double by pPICZα carrier with SacI and EcoRI The 1000bp fragment obtained by enzyme digestion was ligated with the pAO815 vector digested with SacI and EcoRI. 6. the recombinant bacterium of efficiently secreting and expressing nattokinase according to claim 1, is characterized in that, described Hac1p regulator gene is to import into the nattokinase Pro-NK gene that has more than two copies by constitutive expression carrier recombinant Pichia pastoris. 7. the recombinant bacterium of efficiently secreting and expressing nattokinase according to claim 6, is characterized in that, described constitutive expression carrier is constitutive expression carrier pGAPZ-Hac1p, and this constitutive expression carrier is by Hac1p regulator gene Inserted into the pGAPZA vector to construct. 8. The recombinant bacterium for efficiently secreting and expressing nattokinase according to claim 6, wherein the Hac1p regulatory factor gene is derived from yeast or mold.