CN104212753A - Arginine deiminase genetically engineered bacteria construction and purpose thereof - Google Patents

Abstract

In the invention, two arginine deiminase genes which are shown in a SEQ ID: NO.1 and a SEQ ID: NO.3 are cloned in environmental microorganism, escherichia coli BL21 genetically engineered bacteria can be constructed, the preservation numbers are respectively CGMCCNo.7491 and CGMCCNo.7492, the purpose of the genetically engineered bacteria for producing L-citrulline is verified, and L-arginine conversion rate is 99%.

Description

Structure of arginine deiminase genetic engineering bacterium and uses thereof

Technical field

The invention belongs to industrial biotechnology and field of medicaments, relate to a kind of recombinase based on arginine deiminase, engineering bacteria and the purposes in citrulline is produced thereof.

Technical background

Arginine deiminase (Arginine desiminase, ADI) is that one can decompose arginic protein.First this enzyme is applied to industrial production one step conversion arginine and prepares citrulline (Applied Microbiology, 1971:992-999), is also be employed one of citrulline production method the most widely at present.Japanese scholars in 1978 is from pseudomonas putida purifying and have studied the character of this enzyme, and finding that this enzyme has higher take arginine as the activity (FEBS Letters, 1978,96 (2): 389-391) of substrate synthesis citrulline.Subsequently, this enzyme is confirmed as the key enzyme (Gene, 1990,87:37-43) of microorganism arginine metabolism process ADI pathways metabolism.

At medical field, scholar finds that ADI can cell growth inhibiting (Biochemical and Biophysical Research Communications in by the epidermic cell of mycoplasma contamination, 1999,261:10-14), be a kind of inhibitor of Growth of Cells.Further research finds that ADI is to the tumour cell of people; propagation as hepatocellular carcinoma cells, melanoma cell, breast cancer cell also has obvious restraining effect (International Journal of Cancer; 1992,51 (2): 244-249; Leukemia, 2000,14 (5): 826-829).Abroad ADI and modifier thereof are studied as a kind of new antitumor drug, be used for the treatment of the tumour such as Hepatic Sarcoma, melanoma (Expert Opinion on Investigational Drugs, 2006,15 (7): 815-822; British Journal of Cancer, 2003,89 (5): 907-914).

This patent is under the mode without microorganism pure culture, by to the genomic research of environmental microorganism, clone and have expressed two arginine deiminase genes of SEQ ID No.1 and SEQ ID No.3, related microorganisms bacterial classification is preserved in China Committee for Culture Collection of Microorganisms (address: No. 1, Beichen Lu, Chaoyang District, Beijing City No. 3 Institute of Microorganism, Academia Sinica of institute) on April 19th, 2013, preserving number is respectively CGMCC No.7491 and CGMCC No.7492, bacterium classification be colon bacillus ( escherichia coli), for Cit suitability for industrialized production and medicine drug development provide new approach and genetic resources.

Summary of the invention

The object of the present invention is to provide, a kind of arginine deiminase or engineering bacteria containing this enzyme of utilizing carries out the new way that bio-transformation obtains citrulline.

technical scheme of the present invention is as follows:

the basic fundamental route realizing above-mentioned purpose is:

Overall technological scheme is the encoding gene that clone comprises ADI, utilize suitable carrier and restriction enzyme enzyme fragment, build conversion carrier, by ADI gene clone to suitable host cell, include but not limited to intestinal bacteria, yeast, Chinese hamster ovary celI, insect cell etc.

1. the extraction of gene templateflora ambient temperature overnight enrichment culture on Arginine-rich substratum of cloning arginine deiminase will be waited, to suitable cell concentration, the step be familiar with according to persons skilled in the art, reagent and method extract the template that group's thallus DNA obtains as gene.

2. the clone of arginine deiminase geneaccording to pseudomonas putidaaTCC 4359 (Genbank accession number: U07185) conserved sequence, designs the primer with restriction enzyme site, and step, reagent and the method be familiar with according to persons skilled in the art, by pcr amplification arginine deiminase gene.

3. the screening of the structure of conversion carrier, conversion and positive colonythe step be familiar with according to persons skilled in the art, reagent, the carrier that can obtain from commercial channels completely, restriction enzyme and method, be cloned into carrier by goal gene, transformed host cell, and realize positive colony screening.

invention effect

Utilize the method that the technical program relates to, construct and realize with arginine being the microorganism cells model that substrate produces citrulline based on arginine deiminase, carried out positive-selecting, and carried out bio-transformation.

Accompanying drawing explanation

fig. 10.8% agarose gel electrophoresis analysis of PCR primer.1,2,3: the ADI gene PCR amplified production being template with environment DNA sample, M: molecular weight marker (DL2000).

fig. 2expression vector ndei and hindiII double digestion analyzes (0.8% agarose gel electrophoresis).1:pET-KSRD3-1(SEQ ID No.1), 2:pET-KSRD3-2(SEQ ID No.3), M: molecular weight marker (DL10000).

fig. 3sDS-PAGE analyzes.1: supernatant liquor after the Host Strains cytoclasis of not inducing, 2:IPTG induction after Host Strains ( e.coli/ pET-KSRD3-1) supernatant liquor after cytoclasis, Host Strains after 3:IPTG induction ( e.coli/ pET-KSRD3-2) supernatant liquor after cytoclasis, supernatant liquor after Host Strains (Genbank:U01785) cytoclasis after 4:IPTG induction, M: molecular weight marker (Premixed Protein Marker (Low)).

fig. 4transformation experiment is analyzed.1: citrulline and arginine mixed solution object of reference; 2: recombinant bacterium ( e.coli/ pET-KSRD3-1) transform rear supernatant samples; 3: recombinant bacterium ( e.coli/ pET-KSRD3-2) transform rear supernatant samples.

attached sequencethe environmental microorganism arginine deiminase gene order be cloned into

Specific embodiments

reagent and material

Experimental strain and carrier

Intestinal bacteria ( escherichia coli) DH5 α, BL21, pMD18-T simple vector, pET28a carrier, by this Laboratories Accession, also can obtain from commercial sources.

Experiment reagent: archaeal dna polymerase (Taq DNA Polymerase, PrimeSTAR ^?hS DNA Polymerase), restriction enzyme ( ndei and hindiII), T4DNA Ligase, dNTP, DNA marker DL10,000, Premixed Protein Marker (Low) is all purchased from Takara company; Bacterial genomes extracts test kit, plasmid Mini Kit, DNA purification kit purchased from Beijing Ding Guo Bioisystech Co., Ltd; Sulfuric acid card receives mycin, penbritin, IPTG purchased from Genview company; All the other chemical reagent are analytical pure.

  

1.2 experimental technique

Bacterial genomes extraction, plasmid extraction and the recovery of DNA purifying etc. are all according to test kit operation instruction, preparation, the enzyme of E. coli competent are cut, enzyme connects, the conversion of carrier, the operation of the conventional molecular biological such as agarose gel electrophoresis and SDS-PAGE is with reference to " Molecular Cloning: A Laboratory guide " third edition (Science Press, 2002).

1.2.1 carry the genomic extraction of enrichment culture and group of arginine deiminase bacterium

In the plantation field such as watermelon or muskmelon, collecting part pedotheque, after suspension dilution, sampling in containing 2% arginic LB substratum 500ml shaking flask, 150rpm incubated at room temperature 20 hours.

Collected by centrifugation thalline, and utilize bacterial genomes extraction test kit to obtain its genomic dna.

1.2.2 the amplification of target gene arginine deiminase encoding sequence

Log according to Genbank pseudomonas putidaencoding sequence design pair of primers (synthesis of Shanghai Ying Jun Bioisystech Co., Ltd), primer sequence and restriction enzyme site as follows:

ADIPF:5 '-GGAATTC cATATG(underscore is TCCGCTGAAAAACAGAAGTACG-3 ' ndei restriction enzyme site)

ADIPR:5 '-CCC aAGCTT(underscore is AGTAATCGATCGGGTCAC-3 ' hindiII digestion site).

1.2.3 reaction system and condition:

PCR reaction system (50 μ l): each 1 μ l of 5 × PCR buffer 10 μ l, dNTP 4 μ l, primer ADIPF and ADIPR (10 μMs), template 0.5 μ l, PrimeSTAR ^?hS DNA Polymerase 0.5 μ l, water to 50 μ l.

PCR reaction conditions (Touch down PCR): 95 DEG C of 5min; 98 DEG C of 10 sec, 60-51 DEG C 10 sec, 10 circulations, 72 DEG C extend 1 min; 98 DEG C of 10 sec, 55 DEG C of 10 sec, 20 circulations, 72 DEG C extend 1 min; 72 DEG C of 10 min.0.8 % agarose gel electrophoresis detects and purified pcr product.

PCR primer adds A reaction system (20 μ l): get 14.5 μ l PCR primer, adds 2 μ l 10 × Taq Buffer, 3 μ l dNTP, 2 U Taq DNA Polymerase, and after 72 DEG C of reaction 20 ~ 30 min, direct purification reclaims.

1.2.4 the Construction and identification of target gene clone library

According to pMD18-T simple vector test kit operation instructions, linking objective gene and carrier T, transform e. colidH5 α, utilizes blue hickie to screen positive transformant, and selects part transformant to deliver to Sangon Biotech's order-checking.

1.2.5 target gene clone and qualification

In the sequencing result of clone library, select the transformant carrying interest genes, 37 DEG C of shake-flask culture that spend the night, extract recombinant plasmid, utilize ndei and hindiII double digestion, reclaims interest genes through agarose gel electrophoresis purifying.

Utilize simultaneously ndei and hindiII double digestion pET28a carrier, and sepharose reclaims support products.

Then, T4 ligase enzyme 16 DEG C of enzymes that spend the night are utilized to connect pET28a carrier and interest genes, product conversion e. colidH5 α (CaCl ₂method), select part transformant to check order through Sangon Biotech (Shanghai) Co., Ltd..

Select the transformant that sequencing result is correct, extract recombinant plasmid (pET-ADI) and transform e. colibL21 (CaCl ₂method).

1.2.6 the expression of recombinant protein

Picking recombinant bacterium e. colibL21 is inoculated in and receives in the LB substratum of mycin containing sulfuric acid card, and 37 DEG C of shaken overnight are cultivated.Culture being inoculated in the fresh sulfuric acid card that contains receives in the LB substratum of mycin, and inoculum size 2%, 500 mL shaking flask 37 DEG C shaking culture are to OD ₆₀₀=0.6.30 DEG C of IPTG induction (final concentration 0.6mM), centrifugal collecting cell after 3h; 20 mM Tris-Cl (pH8.0) re-suspended cells, ultrasonication, the upper cleer and peaceful precipitation of centrifugal collection respectively.Cleer and peaceful precipitation in SDS-PAGE analysis.With zero load e. colibL21 is as negative control.

1.2.7 enzymatic conversion method produces citrulline

Under aseptic condition, picking one ring thalline, is linked into and is equipped with in the 250ml triangular flask of 50m1 fermented liquid, 37 DEG C, 200r/min, cultivates 3h.To cell OD ₆₀₀grow to 0.6, add IPTG to final concentration 0.2mM, 25-37 DEG C, 200 r/min cultivate 3h.Ferment complete, 5000r/min, centrifugal 10min collects thalline.Get 0.2g wet thallus, through brine twice, proceed to the acetate buffer (pH 6.0) that 20ml arginine concentration of substrate is 10%, 150r/min, 37 DEG C start enzyme reaction.

1.2.8 citrulline detection method

Adopt the method for thin-layer chromatography (TLC) qualitative analysis and ultraviolet spectrophotometry binding analysis.TLC analyzes: moving phase: V (methyl alcohol): V (ammoniacal liquor) is 6:1, and developer is the triketohydrindene hydrate of 0.3%.Mensuration (the reference: Chinese Journal of Pharmaceuticals of Cit content, 2007,38 (7), 519-521): according to Cit in strongly acidic solution with the single-minded color reaction of Diacetylmonoxime and react mixture in 490nm place absorbancy and the linear feature of Cit mass concentration, utilize the quantitative citrulline of development process.The mensuration (reference: Chinese food journal, 2007,7 (4), 126-131) of L-arginine content, with the sodium hypobromite solution of thymol for developer, measures with spectrophotometric colo..

2 experimental results

2.1 take community DNA as the pcr amplification of the arginine deiminase gene of template

With ADIPF and ADIPR for primer, with group's bacterial genomes for template, pcr amplification is to about 1260 bp DNA fragmentations (Fig. 1), and result conforms to theoretical (1265bp).

2.2 the structure of target gene clone library

PCR primer and pMD18-T simple carrier enzyme are connected, transforms e. colidH5 α, and utilize blue hickie screening positive clone, gather in the crops the transformant of about about 500.

2.3 the structure of the Cloning and Expression carrier of target gene

Select the interest genes transformant through order-checking, extract cloning vector, and double digestion results target gene, enzyme connects the pET28a carrier of two enzymic digestion, transforms e. colidH5 α.Selection positive colony, extract plasmid, and carry out double digestion checking, result shows that enzyme is linked to be merit as shown in Figure 2.

2.4 the expression of recombinant protein

Recombination bacillus coli BL21 (pET-ADI), after IPTG induction, through SDS-PAGE gel electrophoresis analysis, there is protein band (Fig. 3) clearly, be arginine deiminase in expression product at about 46kDa place; After induction, thalline samples supernatant SDS-PAGE electrophoresis after ultrasonication, and this albumen exists in supernatant as seen, has good solubility.

2.5 enzymatic conversion method produces citrulline

According to method for transformation described in 1.2.7, collected by centrifugation thalline, carries out transformation experiment.Transformed through 4 hours, 12000rpm is centrifugal, and kapillary gets supernatant, utilize thin layer chromatography Qualitative Identification (Fig. 4), detection display arginine final concentration 0.3g/L, citrulline final concentration is 99.4g/L, and it is active that result shows that recombinant bacterium has good enzymatic conversion.

Claims (7)

1. one kind contains construction process of the Recombinant organism of arginine deiminase and uses thereof, it is characterized in that, comprise and arginine deiminase gene is connected, build restructuring pET28a plasmid, transformation of E. coli BL21 competent cell, and utilize recombinase or Recombinant organism for the production of the method for Cit.

2. method according to claim 1, is characterized in that, described Recombinant organism be intestinal bacteria ( escherichia coli) BL21 CGMCC No.7491 and intestinal bacteria ( escherichia coli) BL21 CGMCC No.7492.

3. method according to claim 1, is characterized in that, described arginine deiminase gene contains nucleotide sequence SEQ ID:No. 1 or SEQ ID:No.3, containing aminoacid sequence SEQ ID:No. 2 or SEQ ID:No.4.

4. construction process according to claim 1, is characterized in that, described arginine deiminase gene is obtained by amplification, with the environmental microorganism group genome containing arginine deiminase gene order for template, designs at its upstream ndei restriction enzyme site, designs in its downstream hindiII digestion site, its primer is:

ADIPF：5’-?GGAATTC CATATG TCCGCTGAAAAACAGAAGTACG-3’

ADIPR:?5’-?CCC AAGCTT AGTAATCGATCGGGTCAC-3’?。

5. construction process according to claim 1, is characterized in that, in described structure restructuring pET28a plasmid, connects, import e. coli bl21 competent cell with the arginine deiminase gene of described amplification after adding A reaction with T4 ligase enzyme.

6. according to construction process according to claim 1, it is characterized in that, described abduction delivering: culturing gene engineering bacteria cell, to logarithmic phase, adds inductor induction 3-8 hour, culture temperature 25-37 DEG C.

7. method according to claim 1, is characterized in that, the method for described production Cit, wet thallus consumption 1%, arginine concentration of substrate about 10%, conversion of Arginine rate 99%.