CN109609481B

CN109609481B - Preparation method of escherichia coli beta galactosidase receptor

Info

Publication number: CN109609481B
Application number: CN201910039636.XA
Authority: CN
Inventors: 王贵利; 高秋峰; 龚俊; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2016-09-22
Filing date: 2016-09-22
Publication date: 2021-02-12
Anticipated expiration: 2036-09-22
Also published as: CN109797143B; CN109609535A; CN109666619B; CN109666619A; CN109609481A; CN109609535B; CN109613251A; CN106399278A; CN106399278B; CN109613251B; CN109797143A

Abstract

The application relates to a preparation method of an escherichia coli beta galactosidase receptor. The present application provides a polypeptide of SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 2, or a beta galactosidase receptor. Designing an enzyme receptor for deleting amino acids from 13 th to 33 th according to the structure and the function of the escherichia coli beta galactosidase and the complementary principle of an enzyme donor and an enzyme receptor; connecting the coding nucleotide of the enzyme receptor with a vector, and converting the coding nucleotide into escherichia coli to construct a recombinant expression host cell; (ii) mass expression of the enzyme receptor by fermentation; purifying the enzyme receptor by affinity chromatography, wherein the purity of the desalted enzyme receptor is more than 90%. Compared with the conventional enzyme receptor, the enzyme receptor has higher yield and stronger complementary activity, and can be applied to the development of CEDIA biochemical diagnostic reagents.

Description

Preparation method of escherichia coli beta galactosidase receptor

The application is a divisional application of Chinese patent application with the application number of 201610841474.8, the application date of 2016, 9, and 22, and the invention is named as 'an escherichia coli beta galactosidase receptor and a preparation method thereof'.

Technical Field

The present application relates to the field of biotechnology, and more specifically to clonal enzyme donor immunoassay technology. The application provides an enzyme receptor of escherichia coli beta galactosidase, and a coding nucleic acid, an expression vector, a host cell and a preparation method thereof.

Background

Coli beta galactosidase (GenBank: EDU66561.1) can be purified by hydrogen bromide CNBr₂Lysis or gene recombination techniquesTwo peptide stretches were obtained: large fragments lacking a small number of amino acids at the N-terminus, called Enzyme receptors (EA): a small fragment lacking most of the C-terminal amino acids is called an Enzyme Donor (ED). The phenomenon that the two exist independently without enzyme activity and can be polymerized to form the whole enzyme with enzyme activity when the two exist simultaneously is defined as alpha-complementation.

This property of E.coli beta galactosidase has been applied in molecular biology and Cloned Enzyme Donor Immunoassay (CEDIA). The CEDIA technology allows the detection of small molecule compounds in clinical biochemical diagnostics.

CEDIA is an immunoassay technique that combines the principles of β galactosidase α complementation with the specific binding properties of antigen antibodies. Small molecule antigen (Ag) is conjugated to ED to form an ED-Ag conjugate, which binds spontaneously to EA to form active beta galactosidase. However, if an antibody (Ab) against the small molecule antigen is added to the reaction system, the specific binding of the antigen antibody will form an ED-Ag-Ab complex. At this point, the ED-Ag-Ab complex can no longer bind to EA to form active β -galactosidase due to the steric hindrance of the antibody. If the sample contains the small molecule antigen to be detected, the reaction system contains two forms of antigen: one is free Ag in the sample; one is ED-Ag in the kit. Both antigens have the ability to specifically bind to antibodies. Obviously, the two forms of antigen competitively bind to the Ab in the reaction system, the more free Ag in the sample, the more Ab bound, and thus the more the remaining ED-Ag conjugate (the portion not bound to the antibody) can spontaneously bind to EA to form active β -galactosidase, and the level of the enzyme activity is proportional to the amount of free antigen in the sample.

The Escherichia coli beta galactosidase EA can be applied to the development of clinical biochemical diagnostic reagents of CEDIA technology, is an important raw material enzyme and has a great application value.

There are many genetically engineered EA or EA mutants reported so far, for example, in the papers published by Langley KE and Zabin I (biochemistry.1976Nov 2; 15(22):4866-75) a beta galactosidase is mentioned, which lacks the amino acid residues at positions 11 to 41 (called M15) as compared with wild-type EA.

As another example, M112 deleted for amino acids 23 to 31; EA5 deleted for amino acids 35 to 52; EA14 deleted for amino acids 30 to 37; EA11 deleted for amino acids 35 to 54; EA17 deleted for amino acids 21 to 53; EA18 deleted for amino acids 13 to 45; EA20 deleted for amino acids 26 to 45; EA22 deleted for amino acids 13 to 40; EA23 deleted for amino acids 16 to 35; EA24 which lacks amino acids 22 to 35 (Daniel R.Henderson, Clinical Chemistry, Vol.32, No.9,1986, 1637-1641). Yet scholars report beta-galactosidase EA lacking amino acids 1 to 5 and 14 to 40 simultaneously (expression of fusion protein of Escherichia coli beta-galactosidase EA and ED; J. Zhonghua microbiology and immunology 2003,23, No 2: 113-114).

TABLE 1 summary of the EA mutants reported

However, the enzyme receptors in the prior art still have the phenomenon of high detection background. In view of this, there is a need in the art for a novel beta galactosidase enzyme that has higher yield, lower background, and stronger complementary activity between the enzyme acceptor and the enzyme donor.

Disclosure of Invention

According to some embodiments, there is provided a beta galactosidase receptor whose amino acid sequence is SEQ ID No. 2. According to the beta galactosidase receptor of the present application, the amino acid residues at positions 13 to 33 are deleted compared to the wild type.

According to the biological structure and function of the beta galactosidase of Escherichia coli and the complementation principle of EA and ED, the amino acids from 13 th to 33 th positions of the beta galactosidase are deleted in a fixed point manner to obtain the beta galactosidase receptor of the application. The inventors have unexpectedly found that recombinant beta galactosidase receptors engineered in this way have a very low detection background, while facilitating improved expression and complementation activities.

According to some embodiments, there is provided a polynucleotide encoding a beta galactosidase receptor of the present application. In some embodiments, the polynucleotide is DNA; in other embodiments, the polynucleotide is RNA. In a specific embodiment, the polynucleotide is DNA. In a specific embodiment, the nucleotide sequence of the polynucleotide is SEQ ID No.1, the complement of SEQ ID No. 1.

According to some embodiments, an expression vector is provided that expresses the beta galactosidase receptor of the present application. In some embodiments, the expression vector of the present application comprises the polynucleotide described above. In some embodiments, the expression vector of the present application comprises the polynucleotide set forth in SEQ ID No. 1. In a specific embodiment, the polynucleotide shown in SEQ ID No.1 is operably linked to an expression vector. In some embodiments, the expression vector is a prokaryotic expression vector. In some embodiments, the expression vector is selected from the group consisting of pET41a, pET28a, pET20a, and combinations thereof. In a specific embodiment, the expression vector is pET41 a.

According to some embodiments, there is provided a host cell comprising an expression vector of the present application. In some embodiments, the host cell of the present application is a prokaryotic host cell. In a specific embodiment, the prokaryotic host cell is e. In a specific embodiment, the E.coli is selected from the group consisting of Rosetta-gami2(DE3) pLysS, BL21, Rosetta and combinations thereof.

The skilled person will appreciate that, in addition to using a host cell to express the product of interest, a cell-free expression system may be used to express the beta galactosidase receptor of the present application.

According to some embodiments, there is provided an agent comprising a beta galactosidase receptor of the present application. In some embodiments, the reagent is a clonase donor immunoassay reagent. In some embodiments, the reagent comprises one, or two, or three, or more separate containers. In some embodiments, the beta galactosidase receptor of the present application is contained in one container; in another container a beta galactosidase donor is contained. In other embodiments, the beta galactosidase donor can be free; or the beta galactosidase donor may be coupled to an antigen, such as a smaller molecular weight antigen, or a hapten. In some embodiments, the antigen is selected from: peptides, polysaccharides, lipids, nucleic acids, drugs. In some embodiments, the beta galactosidase receptor and the beta galactosidase donor of the present application are in different containers. In some embodiments, the beta galactosidase donor can be any beta galactosidase donor known in the art or of the future.

According to some embodiments, there is provided the use of any one or a combination of the following in the preparation of an agent: beta galactosidase receptor of the present application, polynucleotide of the present application, expression vector of the present application, host cell of the present application. In some embodiments, the reagent is a clonase donor immunoassay reagent.

According to some embodiments, there is provided a beta galactosidase comprising a beta galactosidase donor, and a beta galactosidase receptor of the present application. In some embodiments, the beta galactosidase donor and the beta galactosidase receptor of the present application interact to form an active beta galactosidase. In some embodiments, the beta galactosidase donor and the beta galactosidase receptor of the present application are non-covalently bound. In some embodiments, the beta galactosidase donor can also be coupled to an antigen, such as a smaller molecular weight antigen, or a hapten. In some embodiments, the antigen is selected from: peptides, polysaccharides, lipids, nucleic acids, drugs. In some embodiments, the beta galactosidase donor can be any beta galactosidase donor known in the art or of the future.

According to some embodiments, there is provided a method of preparing a beta galactosidase receptor, comprising the steps of:

1) culturing a host cell of the present application under conditions that allow expression of the beta galactosidase receptor; and

2) purifying and collecting the beta galactosidase receptor.

In some embodiments, the culturing is by fermentation. In some embodiments, the fermentation is in a manner selected from the group consisting of: batch fermentation, continuous fermentation, fed-batch fermentation, and combinations thereof. In some embodiments, the culturing is performed in a vessel. In particular embodiments, the container is selected from the group consisting of: shake flasks, petri dishes and fermentors.

In some embodiments, the culturing is performed at a temperature of 28 to 40 ℃, 30 to 40 ℃, 35 to 38 ℃, preferably 37 ℃.

In some embodiments, the culturing is performed under aerobic conditions. In a specific embodiment, the process is carried out with dissolved oxygen of greater than 40%.

In some embodiments, the culturing is under conditions of pH between 6.8 and 7.2.

In some embodiments, the expression is constitutive expression or inducible expression; depending on the type of expression vector.

In some embodiments, an induction step is further included between step 1) and step 2). In a specific embodiment, an inducer is added to the culture of step 1) to induce the expression of the beta galactosidase receptor. In a specific embodiment, the inducer is IPTG.

In some embodiments, the purification is affinity purification. The method of purification depends on the type of expression vector. For example, but not limited to, when the expression vector contains a His tag, Ni-NTA affinity chromatography is used.

In a specific embodiment, there is provided a method of preparing a beta galactosidase receptor comprising the steps of:

1) by 1.5X 10⁸(ii) density of/L, inoculating the host cells of the present application in a fermenter in a primary medium containing an antibiotic;

2) culturing the host cell at 37 ℃ until the OD of the host cell₆₀₀Up to 3 to 5;

3) the temperature of the culture was lowered to 25 ℃ and a first feed was added followed by IPTG to a final concentration of 1 mM;

4) adding a second feeding material at the speed of 10ml/h/L for culturing for 8 to 12 hours;

5) collecting the culture;

6) purifying and collecting beta galactosidase receptor by Ni-NTA affinity chromatography;

7) optionally, desalting is performed on the beta galactosidase receptor.

In a specific embodiment, the antibiotic-containing initial medium comprises: 10g/L tryptone, 5g/L yeast extract, 0.5 to 2g/L magnesium sulfate, 7 to 10g/L dipotassium hydrogen phosphate, 3 to 6g/L sodium dihydrogen phosphate, 10ml/L microelement mother liquor, 50mg/L kanamycin, and 20% to 25% glucose.

In a specific embodiment, the first feed comprises 100. + -.20 g/L tryptone, 250. + -.50 g/L yeast extract, 10. + -.2 g/L lactose.

In a specific embodiment, the second feed comprises 400 ± 50g/L glycerol.

Drawings

FIG. 1: expression of beta galactosidase receptor. And (3) left lane: protein molecular weight Marker; and (3) right path: band of E.coli beta galactosidase receptor.

Detailed Description

Example 1 construction of expression vector for E.coli beta-galactosidase EA

Determining a wild type EA gene sequence according to an escherichia coli beta galactosidase nucleotide sequence published by an NCBI website; deleting amino acid residues from 13 th to 33 th to obtain a sequence shown as SEQ ID No. 2; entrusts Beijing Sanbo Polygala biological technology company to carry out whole gene synthesis, and adds two enzyme cutting sites (such as Nco I and Xho I) at two ends of the sequence respectively, inserts the synthesized gene sequence on the plasmid;

carrying out double enzyme digestion on the plasmid, and recovering an escherichia coli beta galactosidase EA gene fragment through electrophoresis; carrying out double enzyme digestion treatment on the vector plasmid pET41a, and carrying out electrophoretic purification on the plasmid subjected to enzyme digestion;

connecting the digested pET41a plasmid with an escherichia coli beta galactosidase EA gene at 16 ℃ to obtain an expression plasmid, transforming the expression plasmid into escherichia coli DH5 alpha, and coating a Kan resistant culture medium plate for screening; screening the obtained positive clones, extracting expression plasmids, and sequencing for confirmation.

Example 2 obtaining of expression host cells

The expression plasmid obtained in example 1 was transformed into host cells E.coli Rosetta-gami2(DE3) pLysS to give expression host cells.

Example 3 preparation of beta galactosidase EA

The expression host cells obtained in example 2 were inoculated in a seed medium (10g of tryptone, 5g of yeast extract, 10g of NaCl were weighed and dissolved in 900ml of deionized water, pH was adjusted to 7.0 with 10M NaOH, deionized water was added to the solution to a constant volume of 1L, autoclaving was carried out at 121 ℃ for 20min, cooling and storage at room temperature were carried out, kanamycin was added to the solution until the final concentration was 50mg/L during use), and culture was carried out at 37 ℃ until OD was reached₆₀₀To 3 to 5;

by 1.5X 10⁸(density of L) host cells were inoculated into fermentation initiation medium (tryptone 10g, yeast extract 5g, magnesium sulfate 0.5 to 2g, dipotassium hydrogenphosphate 7 to 10g, sodium dihydrogenphosphate 3 to 6g, 10ml of microelement mother liquor (10g FeSO)₄·7H₂O、0.1g NH₄6Mo₇O₂₄、0.5g MnSO₄·5H₂O、2.25g ZnSO₄·7H₂O、0.23g Na₂B₄O₇·10H₂O、1g CuSO₄·7H₂O、2g CaCl₂Dissolved in 5M hydrochloric acid), adding 900ml of deionized water to dissolve, adjusting the pH value to 6.8 to 7.2, adding deionized water to constant volume to 1L, sterilizing at high pressure for 20min, and cooling for use. Adding kanamycin to a final concentration of 50mg/L, adding sterilized glucose to a final concentration of 20% to 25%), adjusting aeration, allowing growth under dissolved oxygen of more than 40%, and adjusting the pH value of the culture medium to 6.8 to 7.2 by dropwise adding acid or alkali;

when the components in the initial culture medium are gradually exhausted and the oxygen solubility value of the culture medium is reduced to 40%, the culture temperature is reduced to 25 ℃, and meanwhile, the supplementary material 1(100 plus or minus 20g/L tryptone, 250 plus or minus 50g/L yeast extract, 10 plus or minus 2g/L lactose, high-temperature sterilization at 121 ℃ for 20min and then room-temperature storage) is supplemented according to the proportion of 100 ml/L. After the supplement 1 is completed, IPTG is added until the final concentration is 1mM, and induction is started;

adding supplementary material 2(400 + -50 g/L glycerol, high temperature sterilization at 121 deg.C for 20min, and storing at room temperature) at a speed of 10ml/h/L, and adjusting pH of the culture medium to 6.8-7.2 with ammonia water or sulfuric acid; after the induction is carried out for about 10 hours at the low temperature of 25 ℃, the fermentation culture is finished;

centrifuging and collecting the obtained cells by using a tubular centrifuge, and precooling the cells to a temperature below 4 ℃ by using a high-pressure homogenizer;

resuspending the cells, homogenizing for 3 times at 850bar high pressure by a high pressure homogenizer to break the cells and release the Escherichia coli beta galactosidase EA;

centrifuging the cell lysate at 18000rpm at 4 deg.C for 30min, and filtering the supernatant with 0.22 μm filter membrane;

loading the filtered supernatant to a Ni affinity column; removing impurities which are not combined with the column or have low combination force; eluting target protein escherichia coli beta-galactosidase EA, and collecting a protein peak at the same time;

loading to desalting column for desalting, and collecting protein peak.

Test example

Test example 1 determination of protein purity and yield

The protein obtained in example 3 was subjected to SDS-PAGE gel electrophoresis to determine the purity of the protein (FIG. 1), and the concentration of the protein was measured by BCA method to calculate the EA production. The purity of the EA produced herein is 90%.

TABLE 2 yields

Test example 2 Activity measurement

The activity of E.coli beta-galactosidase EA obtained in example 3 was examined.

Coli beta galactosidase EA is able to complement ED to form beta galactosidase. Beta galactosidase is capable of hydrolyzing o-nitrophenyl beta-D-galactopyranoside (ONPG) to produce galactose and yellow o-nitrophenol (ONP). The enzymatic activity of galactosidase, and thus the complementary activity of EA, can be determined by measuring the amount of ONP.

(1) EA was diluted to 0.2mg/ml with PB buffer (100mM PB, 150mM NaCl, 0.1% NaN3, pH7.3) and ED was diluted to 1. mu.g/ml with PB buffer. Complementary activities were measured with siemens biochemical analyzer 1800:

100 μ l EA solution as R1;

as R2, 100. mu.l of an ONPG substrate solution (5mg/ml ONPG, 100mM PB, 150mM NaCl, 10mM MgCl2, 0.1% NaN3, pH 7.3);

mu.l of enzyme donor solution was used as sample S for determining the complementary activity of EA.

10 μ l PB buffer as blank control sample for determination of background activity of EA;

running an EA-ED program, determining the reaction rate,

(2) the above operations were performed in parallel, using M15 as a control.

(3) As a result:

the EA complementation activity of the present application was 11.2% higher than M15, whereas the background activity was 21% lower.

TABLE 3 complementary Activity

EA	OD₅₇₁	％
			M15	0.21874	100
EA of the present application	0.24332	111.2％

TABLE 4 background Activity

EA	OD₅₇₁	％
			M15	0.00167	100
EA of the present application	0.00132	79

Sequence listing

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Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser

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Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val

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Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val

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Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr

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Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala

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Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln

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Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp

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Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu

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Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu

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Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr

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Arg Ala Val Val Glu Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala

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Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu

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Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr

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Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser

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Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg Asn His Pro Ser Val

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Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His Gly Ala Asn His Asp

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Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln

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Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys

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Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu

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Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe

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Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn

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Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn

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Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro

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His Pro Ala Leu Thr Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe

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Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg

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His Ser Asp Asn Glu Leu Leu His Trp Met Val Ala Leu Asp Gly Lys

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Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys

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Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln

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Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser

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Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn

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Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala Ile Pro His Leu Thr

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Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln

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Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys

740 745 750

Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu

755 760 765

Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala

770 775 780

Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala

785 790 795 800

Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr

805 810 815

Thr Ala His Ala Trp Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg

820 825 830

Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp

835 840 845

Val Glu Val Ala Ser Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn

850 855 860

Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly

865 870 875 880

Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg

885 890 895

Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser

900 905 910

Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His

915 920 925

Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln

930 935 940

Gln Leu Met Glu Thr Ser His Arg His Leu Leu His Ala Glu Glu Gly

945 950 955 960

Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly Ile Gly Gly Asp Asp

965 970 975

Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln Leu Ser Ala Gly Arg

980 985 990

Tyr His Tyr Gln Leu Val Trp Cys Gln Lys

995 1000

Claims

1. A method of making a beta galactosidase receptor comprising the steps of:

-culturing the prokaryotic host cell under conditions that allow expression of the beta galactosidase receptor; and

-purifying and collecting said beta galactosidase receptor;

wherein the beta galactosidase receptor is shown as SEQ ID No. 2.

2. The method of claim 1, wherein:

the culturing is carried out at a temperature of 28 to 40 ℃;

the culturing is carried out under aerobic conditions;

the culturing is carried out at a pH of 6.8 to 7.2.

3. The method of claim 2, wherein:

the culturing is carried out at a temperature of 30 to 40 ℃.

4. The method of claim 3, wherein:

the culturing is carried out at a temperature of 35 to 38 ℃.

5. The method of claim 4, wherein:

the cultivation is carried out at a temperature of 37 ℃.

6. The method of any of claims 2 to 5, wherein:

the culturing is carried out under the condition that the dissolved oxygen is more than 40 percent.

7. The method of claim 1, wherein:

the prokaryotic host cell is E.coli.

8. The method of claim 7, wherein the E.coli is selected from the group consisting of: rosetta-gami2(DE3) pLysS, BL21, Rosetta.

9. The method of claim 1, wherein:

the prokaryotic host cell comprises a prokaryotic expression vector;

the prokaryotic expression vector comprises a polynucleotide shown by SEQ ID No.1 or a complementary sequence of SEQ ID No. 1.

10. The method of claim 9, wherein the prokaryotic expression vector is selected from the group consisting of: pET41a, pET28a, pET20 a.