CN111394332A - Optimized diguanylic acid cyclase gene, expression vector and fusion protein thereof - Google Patents

Abstract

The invention belongs to the field of bioengineering, and provides an optimized diguanylic acid cyclase gene, an expression vector and a fusion protein thereof.A diguanylic acid cyclase gene in Rhodococcus ruber is subjected to codon optimization, then the gene is synthesized by a chemical method, a recombinant expression plasmid pPGH-dgc is constructed, and then the recombinant expression plasmid is transformed into E.coli B L21 (DE3) for expression.

Description

Optimized diguanylic acid cyclase gene, expression vector and fusion protein thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a codon-optimized Rhodococcus ruber dinucleotide cyclase gene, an expression vector and a fusion protein thereof.

Background

Generally, diguanylate cyclase contains the typical GGDEF (Gly-Asp-Glu-Phe) or GGEEF (Gly-Glu-Phe) catalytic domain, also called active site or "a site", which can bind to two triphosphates (GTP) and catalyze the synthesis of c-di-GMP, "GGDEF" domain, which was first identified by GREGORY et al, a conserved region in Caulobacter crescentus that is involved in cellular differentiation in response to regulator PleD, diguanylate cyclase generally also contains an inhibitory site, i.e., a site, with the typical RXXD motif (X representing any amino acid) binding of the product c-di-GMP to the I site of the diguanylate c, which is a human motif with the typical RXXD motif (X representing any amino acid), which binds to the latter site of the latter, which is a transmembrane-inducible promoter, which is expressed in the human transmembrane domain, which is considered to be present in the transmembrane synthesis of the transmembrane targeting gene system of the intracellular targeting gene, the protein synthesis of the protein, which is expressed in many proteins expressed by the wild-mediated enzyme system, including the intracellular targeting gene, the protein kinase, the protein, the intracellular targeting gene, the protein synthesis of the protein, the protein synthesis of the protein, the protein synthesis of the protein, the protein synthesis of the protein, which is not expressed in the protein, the protein synthesis of the protein, the protein synthesis of the protein, which is expressed in the protein, which is not expressed in the protein, which is expressed in the protein, which is expressed in the protein, which is not expressed in the protein.

Diguanylate cyclase, a key enzyme ubiquitous in microorganisms, has up to 41 regulatory proteins comprising a GGDEF domain in Vibrio cholerae. It was found by NCBI database search that diguanylate cyclase genes were present in all reported classes of Rhodococcus, such as Rhodococcus ruber SD3, Rhodococcus sp.HS-D2, Rhodococcus sp.strain IcdP1, and the like. However, there are no reports on recombinant expression, purification and characterization of Rhodococcus ruber diguanylate cyclase.

Reference documents: enzymatic synthesis of c-di-GMP using a therophthallic di-guanylate cyclase [ J ]. Analytical Biochemistry,2009,389(2):138-142.

Disclosure of Invention

The invention provides an optimized diguanylic acid cyclase gene, which comprises a base sequence shown as a sequence table SEQ ID NO: 1.

Preferably, the optimized diguanylate cyclase gene is extracted from Rhodococcus ruber.

In one embodiment, the Rhodococcus ruber is Rhodococcus ruber SD3(Rhodococcus ruber SD3) described in patent publication No. CN102604875B entitled "Rhodococcus ruber and its use in degrading phenol contaminants".

The preparation method of the optimized diguanylic acid cyclase gene comprises the following steps:

(1) designing and synthesizing primers covering the diguanylic acid cyclase gene double chains, wherein the length of each primer is between 30 and 40bp, and at least 10 to 15bp of overlap is required between the primers;

(2) and mixing the primers together to perform PCR reaction, wherein the longest product in the PCR products is the diguanylic acid cyclase gene.

In another aspect, the present invention provides a recombinant vector comprising the optimized diguanylate cyclase gene.

In one embodiment, the recombinant vector is a pPGH plasmid comprising the optimized diguanylate cyclase gene.

The invention also provides a fusion protein, wherein the amino acid sequence of the fusion protein is shown in a sequence table SEQ ID NO. 2, and the sequence comprises a GST label.

The preparation method of the fusion protein comprises the following steps:

(1) transferring the recombinant vector into a host cell to obtain a recombinant host cell, and performing induced expression;

(2) after the recombinant host cell is crushed, the recombinant host cell is purified by affinity chromatography to obtain the diguanylate cyclase fusion protein.

In one embodiment, the host cell is e.coli B L21 (DE 3).

The fusion protein is an enzyme catalyst, can be used for catalyzing the generation of c-di-GMP, and has high activity and stability.

Drawings

FIG. 1 is a graph of the relative activity and thermostability of diguanylate cyclase fusion proteins at different temperatures.

FIG. 2 is a graph of the relative activity and pH stability of diguanylate cyclase fusion proteins at different pH values.

FIG. 3 is a DNA agarose gel identification result of DGC gene amplification in R.ruber SD3, wherein M is D L2000 DNA Marker, and 1-3 is DGC amplification product.

In the sequence table, SEQ ID NO 1 is a codon-optimized Rhodococcus ruber SD3 diguanylic acid cyclase gene sequence, SEQ ID NO 2 is an amino acid sequence of diguanylic acid cyclase fusion protein, and SEQ ID NO 3 is a Rhodococcus ruber SD3 diguanylic acid cyclase gene sequence; SEQ ID NO. 4 is the amino acid sequence of the diguanylate cyclase translated according to SEQ ID NO. 3.

Detailed Description

The following examples are provided to illustrate the advantageous effects of the present invention in order to help the reader to better understand the essence of the present invention, but should not be construed as limiting the scope of the present invention in any way.

Dgc gene of Rhodococcus ruber SD3(Rhodococcus ruber SD3 or R.ruber SD3) is optimized by codon to obtain optimized diguanylate cyclase gene shown in sequence table SEQ ID NO:1, the gene is connected with pPGH plasmid to obtain recombinant vector, the recombinant vector is introduced into E.coli B L21 (DE3) host for induction expression to obtain diguanylate cyclase fusion protein, the protein contains glutathione mercaptotransferase (GST) label, the amino acid sequence of the protein is shown in sequence table SEQ ID NO:2, and the recombinant expression comprises the following steps:

(a) connecting the codon-optimized Rhodococcus ruber diguanylic acid cyclase gene with a vector pPGH plasmid to obtain a recombinant plasmid pPGH-dgc;

(b) transforming E.coli Top10 with recombinant plasmid pPGH-dgc, and screening positive clones;

(c) extracting recombinant plasmid pPGH-dgc, and transferring the recombinant plasmid pPGH-dgc into E.coli B L21 (DE3) to obtain a recombinant bacterium for preparing guanylate cyclase fusion protein;

(d) e.coli B L21 (DE3) containing recombinant plasmid is inoculated into L B liquid medium for culture until bacterial liquid OD₆₀₀When the nm reaches about 0.4, IPTG is added to the mixture to be vibrated and induced by a low-temperature shaking table, and the Rhodococcus ruber diguanylate cyclase fusion protein is optimally expressed.

Wherein the optimal recombination reaction temperature of the diguanylate cyclase fusion protein is 47 ℃, the high activity is still maintained after incubation for 60min at 37-87 ℃, and the activity is still maintained at 94% after treatment for 1h at 87 ℃. The optimal pH value of the diguanylate cyclase fusion protein is 8.0, and the diguanylate cyclase fusion protein keeps good stability within the pH range of 4-9. The Km, Vmax and Kcat values of the diguanylate cyclase fusion protein were 9.8. mu.M, 0.7. mu.M/min and 1.3S, respectively^-1One unit of diguanylate cyclase fusion protein catalyzes the production of 75.9 mg/L c-di-GMP at pH 8.0 and 47 ℃.

Example 1 recombinant expression and purification of Rhodococcus ruber Diguanylate cyclase

(1) Strain breeding and genome extraction

Rhodococcus ruber SD3 bacterial solution at 3m L log phase was centrifuged to remove supernatant, and 500. mu. L of sterilized ddH was added₂O precipitation with resuspended cells, centrifugation at 8000r/min for 1min, repeated washing 3 times, re-introduction of 500. mu. L of sterilized ddH₂And (4) resuspending the precipitate, placing the precipitate in a 35 ℃ water bath kettle, continuously heating until the precipitate is boiled, boiling for 10min, and then freezing for 10min at the temperature of minus 20 ℃. Centrifuging at 8000r/min for 1min after thawing, and collecting supernatant for use.

(2) Cloning of dgc Gene in Rhodococcus ruber SD3

The total DNA of Rhodococcus ruber SD3 extracted above was used as a template for PCR amplification, and the DGC gene was obtained by PCR amplification according to the reaction system shown in Table 1. The upstream primers used were: 5' -GGAATTCCATATGATGGGCGATCGCCAGGTGCT-3' (NdeI), and the downstream primer is: 5' -CCGGAATTCCCGGGACACGTAACTGGGCTGTT-3' (EcoRI). the PCR amplification program for the DGC gene was set up as follows, pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 90s, for 30 cycles in total, sufficient extension at 72 ℃ for 5min, and incubation at 4 ℃ wherein, after subtracting 5 ℃ from Tm, gradient PCR was performed to determine the optimum annealing temperature, the PCR product was subjected to preliminary detection in 1% agarose gel electrophoresis, purification of the DGC gene amplification product was performed according to the procedure of DNA agarose gel recovery kit of Tiangen Biochemical technology (Beijing) Inc. (Tiangen Inc.), and the purified product was enzymatically linked to pGM-T cloning vector (kit available from Tiangen Inc.), transformed into E.coli Top10 competent cells by heat shock method, after blue spot screening, white colonies were picked up to L B liquid medium containing 100. mu.g/m L penicillin (available from Tiangen Inc.), and cultured in BBQ.6 min, and the growth of the strain was determined by PCR amplification system and then cultured in the Blackground-white spot culture, and the PCR was performed on the bacterial liquid culture system for identification under the conditions of the growth of the DGC gene amplification system and the growth of the strain.

TABLE 1 PCR amplification System for DGC genes

TABLE 2 bacteria liquid PCR identification reaction system

(3) Cloning of the DGC Gene

The result of DNA agarose gel identification of dgc gene amplification in ruber SD3 is shown in FIG. 3. The target fragment recovered by agarose gel electrophoresis is connected to a pGM-T vector, and the positive clone sequencing result shows that the target gene is 1332bp and conforms to the expected size. The dgc gene sequence has now been submitted to the GenBank database (accession number MH 482549).

(4) Codon optimization and gene synthesis of dgc gene in Rhodococcus ruber SD3 and construction of recombinant expression plasmid pPGH-dgc

According to the codon preference of Escherichia coli, using codon optimization software (such as OPTIMIZER software, Codonoptimization By general biol software, etc.), codon optimization of dgc gene in Rhodococcus ruber SD3, then design and synthesis of two-strand primers covering the two guanylate cyclase gene, each primer between 30-40bp, the primers require at least 30-40bp overlap, the PCR reaction, after 25 reactions, the system will have different size of PCR products, the longest product is the two guanylate cyclase gene₂And supplementing O to 50 mu L, then adopting a rapid enzyme connection method to perform enzyme connection on the enzyme digestion products at room temperature for 25min, wherein the connection system is that DNA is 4 mu L, plasmid is 4 mu L, 10 × T4 DNA ligase buffer solution is 1 mu L, and T4 DNA ligase is 1 mu L, taking 3 mu l of the enzyme connection products to be transformed to E.coli Top10 competent cells, identifying positive clone according to the bacterial liquid PCR method, and transforming the recombinant plasmid to E.coli B L21 (DE3) for subsequent expression after determining no errors through sequencing.

(5) Recombinant expression of Rhodococcus ruber diguanylate cyclase

E.coli B L21 (DE3) containing pPGH-dgc recombinant plasmid was activated on L B plates, and the activated monoclonals on L B plates were inoculated into 50m L L B liquid medium (containing 100. mu.g/m L ampicillin) and shake-cultured overnight at 37 ℃ on a shaker at 200 r/min.The next day, 2m L inoculum was added to 100m L fresh L B liquid medium (containing 100. mu.g/m L ampicillin), and shake-cultured at 37 ℃ in a constant temperature shaker at 180r/min to obtain bacterial liquid OD₆₀₀When the nm reaches about 0.4, adding 0.1mM IPTG, shaking and inducing at the low temperature of 16 ℃ for 10 hours by 180r/min, centrifuging for 10 minutes at the temperature of 4 ℃ and 5000 × g after the induction is finished, and collecting thalli precipitates.

(6) Purification of diguanylate cyclase fusion proteins

The thalli precipitation induced and expressed by every 100m L is resuspended by 1 × PBS solution (containing 5mM DTT and 1mM PMSF with final concentration, pH 7.4) with the concentration of 6m L, the thalli precipitation is placed in a 10m L centrifuge tube to be crushed by ice bath and ultrasonic wave, the crushing power is 60w, the mixture is crushed for 3s and 5s in the gap, the mixture becomes transparent after about 15min of crushing, the crushed bacterial liquid is frozen and centrifuged for 30min at 10000 × g at 4 ℃, the supernatant is separated, 8m L is taken and uniformly mixed with Beyogold^TMGST-tag Purification resin gel (wherein the gel content is 50%, the remainder is the stock solution), left to stand on ice for 15min to discard the stock solution, adding an equal volume of 1 × PBS solution (pH 7.4) to the gel to resuspend the gel, left to stand on ice for 15min to discard the solution, repeatedly equilibrating 3 times, adding about 30m L of bacterial lysis supernatant to a 50m L centrifuge tube containing the gel, gently shaking the bacterial lysis supernatant and the gel, gently shaking the mixture on a horizontal shaking bed under ice bath conditions for 2h, loading the mixture of bacterial lysis supernatant and gel into an affinity chromatography column empty tube, opening a dropper at the bottom of the Purification column, controlling the flow rate of the effluent to be about 6s per drop, collecting the flow-through solution with a sterilized clean centrifuge tube, and re-loading the column to ensure that the GST tag is sufficiently bound to the gel, washing the column 5 times with 1 × PBS solution (pH 7.4), adding 2 times of the volume of the solution of the gel, controlling the flow rate of the effluent to be about 6s per drop, ensuring that the GST tag is sufficiently bound to the gel, and eluting protein is sufficiently controlled with a 1-10 mM GST-tag elution buffer solution, and eluting the volume of the corresponding to be about 10. the corresponding to provide a stable protein eluting column, and eluting protein eluting gel (0.5 times of the corresponding to be about 10. the same time, and adding the corresponding to be about 10. the same time, and eluting proteinProtein sequence of cleavage site + amino acid corresponding to restriction site + DGC protein sequence + amino acid corresponding to restriction site) are:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYY IDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCF KKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPGIPGSTRAAASGPH MMGDRQVLHGATANLRVQRRRLLAWYLAISTGLAAVGIVAALLDPAVARSGSFAGMASAL ALGIAGLVILRWTPRRLGRTPIVAATACAVAAMPAVIAFHVDPSRILISVMGSMFLSMYLAAF WPPRQAVLWIGTLTAATLGAAALAPTRLLWVGYVVVAAALLGSGAIFGAVGRLLVNAATR DPLTGLRNRAGLDLELRSRPRDRGAWVCLGLLDVDGFKAFNDEHGHPAGDALLRDAAEA WAQQVPRGGILARLGGDEFLVVLPDTDEEEGRAVLEGLARANPVPVSFGVAAGVVSDPMD LYHDADMDLYRAKAAAWAQLPDRLAPLPLPDVLDLLPVPILAVRDGGTVAYVNPCFATLFG VDARNVENRPLHEVLTGGTWPDRSVTVRSLHDRTGQLCEFGRPDGTVVQATVGAARIRWENTLATLVVLGDVTEQPSYVSRLE。

example 2: characterization of the diguanylate cyclase fusion protein

The diguanylate cyclase fusion protein prepared in example 1 was used for characterization.

(1) Determination of optimum temperature and thermal stability of Diguanylate cyclase fusion proteins

10 mu L diguanylate cyclase fusion protein is taken to be evenly mixed with 50mM Tris-HCl (pH 8.0), the mixture is preheated for 10min in water bath at 27 ℃, 37 ℃, 47 ℃, 57 ℃ and 67 ℃, GTP substrate with the final concentration of 100 mu M is added, the mixture is immediately taken out and is heated for 10min at 95 ℃ after being reacted for 15min in water bath at 27 ℃, 37 ℃, 47 ℃, 57 ℃ and 67 ℃ respectively, so as to terminate the reaction, and then the enzyme activity is measured, as shown in figure 1, the optimal reaction temperature of the diguanylate cyclase fusion protein is 47 ℃.

10 mu L diguanylate cyclase recombinant protein is taken to be mixed with 50mM Tris-HCl (pH 8.0) evenly, preheated for 60min in water bath at 37 ℃, 47 ℃, 57 ℃, 67 ℃, 77 ℃ and 87 ℃ respectively, and then the enzyme activity is measured, as shown in figure 1, the diguanylate cyclase fusion protein still keeps high activity after being incubated for 60min at 37-87 ℃, and still keeps 94% of activity after being treated for 1h at 87 ℃.

(2) Determination of optimum pH value and pH stability of recombinant protein of diguanylate cyclase

10 mu L diguanylate cyclase fusion protein was mixed well with 50mM Tris-HCl (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) and preheated in 37 ℃ water bath for 10min, GTP substrate was added to a final concentration of 100. mu.M, reacted in 37 ℃ water bath for 15min, immediately taken out and heated at 95 ℃ for 10min to terminate the reaction, and then the enzyme activity was measured as shown in FIG. 2, the optimal pH of diguanylate cyclase fusion protein was 8.0.

The diguanylate cyclase fusion protein 10 mu L is uniformly mixed with 50mM Tris-HCl (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0), preheated in water bath at 37 ℃ for 60min, and then the enzyme activity is measured, as shown in figure 2, the diguanylate cyclase fusion protein keeps good stability in the pH range of 4-9.

(3) 10 mu L diguanylate cyclase fusion protein is mixed with 50mM Tris-HCl (pH 8.0) and preheated at 47 ℃ for 10min, after which 2, 4, 6, 8, 10 and 12 mu L1 mM GTP are added, respectively, and reacted at 47 ℃ for 15min, and then the enzyme activity is determined, the calculated Km, Vmax and Kcat values are 9.8 mu M, 0.7 mu M/min and 1.3S^-1。

(4) One unit of diguanylate cyclase fusion protein catalyzes the formation of 75.9 mg/L of c-di-GMP at pH 8.0 and 47 ℃.

Example 3: effect of different expression vectors and Induction conditions on protein Activity

We also constructed pET-28a (+) -Dgc recombinant plasmid by linking the optimized Dgc gene with pET-28a (+), compared to pPGH-Dgc recombinant plasmid, Dgc gene optimized sequence for pET-28a (+) -Dgc recombinant plasmid construction changed restriction enzyme sites at 5 'and 3' ends to NheI and HindIII, respectively, B L21 (DE3) thallus containing pET-28a (+) -Dgc recombinant plasmid was activated on L B plate, single colony was selected for expression, optimization of expression conditions was performed from three angles of temperature (16 ℃, 25 ℃ and 37 ℃), induction expression time (3h, 6h, 9h, 12h and 16h) and IPTG concentration (0.1mM, 0.5mM, 0.8mM and 1mM), respectively, optimization of expression conditions of pPGH-Dgc recombinant plasmid was performed similarly, expression conditions of soluble protein was optimized in corresponding table 3, and it was found that the soluble protein had higher activity than that of pGST, thus it was possible to express the soluble protein, which was higher than that of pGST-28 a PGH-DG vector.

TABLE 3 heterologous expression profiles of DGC membrane proteins under different methods

Comparative example 1

Feng Rao et al (enzymic synthesis of C-di-GMP using a therophilic differential cyclase, Analytical Biochemistry 389(2009) 138-142) determined the sequence of the gene encoding the C-terminus (82 nd to 241 nd amino acid residues) of diguanylate cyclase TM1788, which was then chemically synthesized by GenScript, PCR amplified, cloned into pET28B (+), and then expressed in E.coli B L21 (DE 3). in addition, they site-directed mutagenesis of the C-terminus of diguanylate cyclase TM1788 using a site-directed mutagenesis kit (Stratagene), followed by recombinant expression, resulted in an optimized protein mutant 158RA. the mutant has a kcat of 2.6Min at 55 ℃^-1。

Sequence listing

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ctcgtcgtcc tcccggacac ggacgaggag gaagggcgcg cggtgctcga gggcctggcc 840

cgcgcgaatc ccgtgccggt cagcttcggg gtcgcagccg gcgtcgtcag cgaccccatg 900

gacctgtacc acgacgccga catggacctg tatcgggcga aggccgcggc ctgggctcaa 960

ctgccggacc ggctggcccc gttgcccctg ccggatgtcc tcgatctgct gcccgtcccc 1020

atcctcgccg tgcgcgacgg cgggaccgtc gcctacgtca atccgtgctt cgccacgctg 1080

ttcggcgtcg acgcccggaa cgtggagaac cggccgctgc acgaggtcct caccggcggc 1140

acctggcccg acaggtctgt caccgtccgc tcgctgcacg accggaccgg gcagctgtgc 1200

gagttcggcc ggccggacgg gaccgtggtg caggcgacgg tcggcgccgc ccggatccgg 1260

tgggagaaca ccctggcgac gctggtggtg ctcggcgacg tcaccgaaca gcccagttac 1320

gtgtcccggt ga 1332

<210>4

<211>443

<212>PRT

<213> Artificial sequence (Artificial sequence)

<400>4

Met Gly Asp Arg Gln Val Leu His Gly Ala Thr Ala Asn Leu Arg Val

1 5 10 15

Gln Arg Arg Arg Leu Leu Ala Trp Tyr Leu Ala Ile Ser Thr Gly Leu

20 25 30

Ala Ala Val Gly Ile Val Ala Ala Leu Leu Asp Pro Ala Val Ala Arg

35 40 45

Ser Gly Ser Phe Ala Gly Met Ala Ser Ala Leu Ala Leu Gly Ile Ala

50 55 60

Gly Leu Val Ile Leu Arg Trp Thr Pro Arg Arg Leu Gly Arg Thr Pro

65 70 75 80

Ile Val Ala Ala Thr Ala Cys Ala Val Ala Ala Met Pro Ala Val Ile

85 90 95

Ala Phe His Val Asp Pro Ser Arg Ile Leu Ile Ser Val Met Gly Ser

100 105 110

Met Phe Leu Ser Met Tyr Leu Ala Ala Phe Trp Pro Pro Arg Gln Ala

115 120 125

Val Leu Trp Ile Gly Thr Leu Thr Ala Ala Thr Leu Gly Ala Ala Ala

130 135 140

Leu Ala Pro Thr Arg Leu Leu Trp Val Gly Tyr Val Val Val Ala Ala

145 150 155 160

Ala Leu Leu Gly Ser Gly Ala Ile Phe Gly Ala Val Gly Arg Leu Leu

165 170 175

Val Asn Ala Ala Thr Arg Asp Pro Leu Thr Gly Leu Arg Asn Arg Ala

180 185 190

Gly Leu Asp Leu Glu Leu Arg Ser Arg Pro Arg Asp Arg Gly Ala Trp

195 200 205

Val Cys Leu Gly Leu Leu Asp Val Asp Gly Phe Lys Ala Phe Asn Asp

210 215 220

Glu His Gly His Pro Ala Gly Asp Ala Leu Leu Arg Asp Ala Ala Glu

225 230 235 240

Ala Trp Ala Gln Gln Val Pro Arg Gly Gly Ile Leu Ala Arg Leu Gly

245 250 255

Gly Asp Glu Phe Leu Val Val Leu Pro Asp Thr Asp Glu Glu Glu Gly

260 265 270

Arg Ala Val Leu Glu Gly Leu Ala Arg Ala Asn Pro Val Pro Val Ser

275 280 285

Phe Gly Val Ala Ala Gly Val Val Ser Asp Pro Met Asp Leu Tyr His

290 295 300

Asp Ala Asp Met Asp Leu Tyr Arg Ala Lys Ala Ala Ala Trp Ala Gln

305 310 315 320

Leu Pro Asp Arg Leu Ala Pro Leu Pro Leu Pro Asp Val Leu Asp Leu

325 330 335

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

340 345 350

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

355 360 365

Glu Asn Arg Pro Leu His Glu Val Leu Thr Gly Gly Thr Trp Pro Asp

370 375 380

Arg Ser Val Thr Val Arg Ser Leu His Asp Arg Thr Gly Gln Leu Cys

385 390 395 400

Glu Phe Gly Arg Pro Asp Gly Thr Val Val Gln Ala Thr Val Gly Ala

405 410 415

Ala Arg Ile Arg Trp Glu Asn Thr Leu Ala Thr Leu Val Val Leu Gly

420 425 430

Asp Val Thr Glu Gln Pro Ser Tyr Val Ser Arg

435 440

Claims (8)

1. An optimized diguanylate cyclase gene comprising: comprises a base sequence shown as a sequence table SEQ ID NO. 1.

2. A recombinant vector characterized by: comprising the optimized diguanylate cyclase gene of claim 1.

3. The recombinant vector according to claim 2, wherein: the recombinant vector is pPGH plasmid containing the optimized diguanylate cyclase gene of claim 1.

4. A fusion protein, characterized in that: the amino acid sequence of the fusion protein is shown in a sequence table SEQ ID NO. 2.

5. The method for preparing the protein according to claim 4, comprising the steps of:

(1) transferring the recombinant vector of claim 3 into a host cell to obtain a recombinant host cell, and performing induced expression;

(2) after the recombinant host cell is crushed, the recombinant host cell is purified by affinity chromatography to obtain the diguanylate cyclase fusion protein.

6. The method of claim 5, wherein the host cell is E.coli B L21 (DE 3).

7. Use of a fusion protein according to claim 4.

8. Use according to claim 7, characterized in that: the fusion protein is used for catalyzing the generation of c-di-GMP.

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