CN107400635A

CN107400635A - Import rhodotorula glutinis recombinant bacterial strain of α MSH polypeptides and its preparation method and application

Info

Publication number: CN107400635A
Application number: CN201710370603.4A
Authority: CN
Inventors: 孙晗笑; 利时雨; 黄俊丽; 贺凯
Original assignee: Guangzhou Hong Kong Biotechnology Co Ltd
Current assignee: Guangzhou Hong Kong Biotechnology Co Ltd
Priority date: 2017-04-27
Filing date: 2017-05-23
Publication date: 2017-11-28

Abstract

The present invention relates to a kind of rhodotorula glutinis recombinant bacterial strain of importing α MSH polypeptides, and α MSH polypeptides are imported in the rhodotorula glutinis through the restructuring of the delta 8 desaturase genes of Δ 6.Invention additionally discloses a kind of preparation method and application of the rhodotorula glutinis recombinant bacterial strain of importing α MSH polypeptides.The present invention has the advantages that：Identified through double digestion, the purpose fragment direction of insertion on recombinant plasmid is correct；Realize D6D stabilization, permanent expression；Real-time fluorescence quantitative PCR shows that D6D expression improves 2 times or so；Through gas chromatography analysis, the GLA converted in bacterial strain is improved more than 3.3 times compared with wild strain；It was found that α MSH content increases in mice serum and vitals (liver, lung, spleen, kidney), particularly liver improves 3.24 times.

Description

Import rhodotorula glutinis recombinant bacterial strain of α-MSH polypeptides and its preparation method and application

Technical field

The present invention relates to gene engineering technology field, more particularly to a kind of rhodotorula glutinis recombinant bacterium of importing α-MSH polypeptides Strain and its preparation method and application.

Background technology

Many unrighted acids are got over because of its extensive physiologically active and its important function in terms of human health, nutrition Paid close attention to get over by people.

The building-up process of microbial grease is almost similar to animals and plants, and most important part is exactly the synthesis of aliphatic acid, main There are two kinds of kytoplasm enzyme system catalysis, acetyl CoA carboxylase and fatty acid synthase complex, be that carbon source is passed through repeatedly using acetyl-CoA Carbochain extends synthesizing saturated fatty acid, or again by desaturation synthesis unrighted acid.It is main during desaturation It is responsible for catalysis by various desaturases, is the key for synthesizing long-chain unsaturated fatty acid, C=C can be introduced on fatty acid carbon chain Double bond, the degree of unsaturation of aliphatic acid is improved, fatty acid desaturase is widely present in yeast.

And the enzyme for synthesizing unrighted acid is divided into two kinds, the fatty acid desaturase that methyl orientation and carboxyl orient.First The fatty acid desaturase of base orientation introduces carbon-carbon double bond at the carbon atom that methyl end is fixed, and is referred to as ω-desaturase；Carboxylic The fatty acid desaturase of base orientation introduces carbon-carbon double bond at the carbon atom that c-terminus is fixed, and is referred to as Δ (delta)-go to satisfy And enzyme, it is also referred to as " front-end " desaturase.First carbon carbon double bond is introduced into saturated fatty acid by Δ 9- desaturases, Second carbon-carbon double bond is responsible for introducing by Δ 12 and Δ 6- desaturases, and the desaturase of Δ 15 is then responsible for introducing the 3rd double bond.

After cell synthesizes 18C saturated fatty acids, stearic acid obtains oleic acid through stearoyl-CoA desaturase desaturations；Oil Acid is catalyzed generation linoleic acid through Δ 12- desaturases, and alpha-linolenic acid (ALA) is synthesized through Δ 15- desaturases；Linoleic acid is through Δ 6- Desaturase catalysis generation gamma-Linolenic acid GLA.Wherein, Δ 6- desaturases are the key enzymes in GLA route of synthesis, for the first time It is to separate clone from blue-green algae within 1993 to obtain to clone Δ 6- desaturases, and obtaining GLA by biotechnology for the first time is The Δ 6- delta 8 desaturase genes production GLA of blue-green algae was expressed in potato in 1996.GLA further dehydrogenations in human body extend shape Into physiological activators such as arachidonic acid (AA), prostanoid and leukotrieneses, there is important physiological significance to human body, thus In recent years people are higher to the research temperature of Δ 6- fatty acid dehydrogenase genes, also achieve greater advance.

Although having there is more reports to show, in yeast GLA contents can increase expression external source Δ 6- fatty acid dehydrogenases Add, but so far, not yet occurring can be with the yeast strain of high yield gamma-Linolenic acid (GLA) using genetic engineering structure Report.

Alpha-melanocyte stimulating hormone (α-melanocyte-stimulating hormone, α-MSH) is to belong to casting skin matter Family, it is a kind of endogenous neural peptide derived from by POMC (POMC), by the small peptide of 13 Amino acid profiles, Nature exists in the form of cation, is the precursor protein of all casting skin matter Hormone Peptides, is responsible for secreting, expressing by pituitary.To the greatest extent Pipe α-MSH are as caused by pituitary, but have its figure in defense cell, peripheral tissues such as skin, illustrate that it is immune Control characteristic.α-MSH physiological property illustrates its importance in immunity of organism particularly inherent immunity and in antibacterial Aspect can also play a significant role.It is more widely at present its anti-inflammatory effect to α-MSH applications, because it can adjust many inflammation Inflammation factor, illustrate that there are powerful anti-inflammatory properties.α-MSH have powerful inhibitory action to almost all kinds of inflammation, bag The anti-inflammatory of acute inflammation, chronic inflammation, system inflammation, local inflammation, allergic inflammation and central nervous system is included, effect can Played a role by maincenter, periphery in a variety of endocrines, immunological regulation system.But because α-MSH itself are there is also shortcoming, its Half-life period is shorter, in vivo half-life period there was only more than 20 minutes, so existing administering mode is injection, and need to constantly inject to tie up Drug effect is held, is made troubles to patient and painful, if directly oral polypeptide, internal gastral enzyme can be degraded, so needing A kind of new Oral administration is developed to realize the application of this peptide.

The content of the invention

For insufficient existing for prior art described above, it is more that the first object of the present invention is to provide a kind of importing α-MSH The rhodotorula glutinis recombinant bacterial strain of peptide.

The second object of the present invention is to provide the preparation method of the rhodotorula glutinis recombinant bacterial strain of the importing α-MSH polypeptides.

The third object of the present invention is to provide the application of the rhodotorula glutinis recombinant bacterial strain of the importing α-MSH polypeptides.

To solve above-mentioned technical problem, the present invention adopts the following technical scheme that：Import the rhodotorula glutinis weight of α-MSH polypeptides Group bacterial strain, α-MSH polypeptides are imported in the rhodotorula glutinis GM4-D6D through the restructuring of Δ 6- delta 8 desaturase genes.

Δ 6- desaturases (D6D) are incorporated into rhodotorula glutinis GM4-D6D genome as expression vector.

The Δ 6- desaturases (D6D) are obtained by the D6D Gene Isolations of cunninghamella echinulata with amplification.

The rhodotorula glutinis construction method of Δ 6- delta 8 desaturase genes restructuring, the step of it includes, are as follows：

(1) expression vector pPGK1Z-rD-ME extracting；

(2) PGK1 genes and D6D genes are connected；

(3) PGK1-D6D fragments are reacted with the double digestion of expression vector carrier, are connected.

The step of method of expression vector pPGK1Z-rD-ME extracting, is as follows：

(1) bacillus coli DH 5 alpha containing pPGK1Z-rD plasmids is inoculated into equipped with 5mL LB-amp culture mediums, in 37 250rpm shaken cultivations are stayed overnight at DEG C；

(2) drawing 1.5mL, in centrifuge tube, 12000rpm is centrifuged 1 minute bacterium solution at 4 DEG C, abandons supernatant overnight；

(3) fluid nutrient medium in centrifuge tube is blotted with filter paper, bacterial sediment is suspended in 200 μ L STET buffer solutions, Fully mix；

(4) lysozyme soln that 4mL is newly prepared is added, is mixed after standing 5 minutes at room temperature；

(5) centrifuge tube is placed in boiling water bath 45 seconds, 12000rpm is centrifuged 5 minutes at once after taking-up；

(6) sediment in picking centrifuge tube discards, and adds 8mL, 5%CTAB in the supernatant of centrifuge tube, uses blender After mixing, 13000rpm is centrifuged 5 minutes, abandons supernatant, the liquid in centrifuge tube is blotted with filter paper；

(7) 1.2M NaCl 300mL are added, sediment is fully dissolved, adds 750mL pre-cooled ethanol, are fully mixed Afterwards, 13000rpm is centrifuged 15 minutes, abandons supernatant；

(8) 70% cold ethanol of 1mL is taken, slowly elution centrifugation inside pipe wall, 13000rpm centrifugation 5min, supernatant is abandoned, with filter Paper blots the liquid on tube wall, and putting makes nucleic acid precipitation spontaneously dry 5-10min in room temperature；

(9) sediment is dissolved in 50mL TE buffer solutions, and blender mixes, and is saved backup in -20 DEG C.

Connection PGK1 genes and the primers of D6D genes are：

PGK1-Bam HI primer1：

5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)

PGK1-XhoIprimer 2：

5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′(XhoI)

D6D-XhoIprimer1：

5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)

D6D-XhoIprimer2：

5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)

Rhodotorula glutinis and the mol ratio of D6D genetic fragments will be controlled 1:3-10.

A kind of preparation method of the rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides, including the step of it is as follows：

(1) preparation engineering bacterial strain GM4-D6D bacterial strain competent cells.

(2) totally 100 μ L after being mixed after FITC- α MSH that 10 μ g have been modified dissolving with competent cell, are added to 0.2cm In the electricity conversion cup of precooling；

(3) electric shockization；

(4) after electric shock terminates, the 1M sorbitol solutions of 900 μ L precoolings are added into conversion cup immediately, is slightly inhaled and beaten with rifle Mix, then gone in sterilized centrifuge tube, 30 DEG C of standing l h, the fresh YPD culture medium weights of 1mL are added after centrifugation Outstanding thalline, 30 DEG C, 200rpm shakes 1 hour；

(5) bacterial strain finally obtained is respectively designated as GM4-D6D-F α MSH；

The electric shockization step is as follows：

(1) DNA to be transformed that the competent cell and 20 μ L (about 10 μ g) that oneself prepares by 80 μ L have linearized Mixing, it is added in the electricity conversion cup of 0.2cm precoolings；

(2) by the electricity conversion cup ice bath 5min equipped with mixed liquor；

(3) parameter of electroporation is adjusted:Voltage 1.5kV, electric capacity 25uF, the Ω of resistance 200, electric shock the duration be about 5ms or so；

(4) after electric shock terminates, the 1M sorbitol solutions of 1mL precoolings are added into conversion cup immediately, slightly inhales to beat with rifle and mixes It is even, then gone in sterilized centrifuge tube, 30 DEG C of standing l h, then YPD culture mediums fresh addition 1mL, 30 DEG C, 200rpm shakes 1 hour；

(5) 3000rpm at room temperature, 4min is centrifuged, thalline is resuspended with 200 μ L ddH2O；

(6) liquid that will disappear is coated on YPD flat boards (containing 50 μ L Zeocin), is placed in 30 DEG C of incubators and cultivates 2-3d, Zhi Daochang Untill going out single bacterium colony.

The rhodotorula glutinis recombinant bacterial strain of the importing α-MSH polypeptides is used to treat inflammatory bowel disease medicine and feature food Application in product preparation, can make biological capsule.Particularly be used to treat application in colitis medicine, can be aqua or Person's pulvis.

The rhodotorula glutinis recombinant bacterial strain of the importing α-MSH polypeptides can be also used for the food or medicine related to MSH effects Application in product preparation.

The present invention has the advantages that：Foreign gene D6D expression vector pPGK1Z-rD-D6D are successfully constructed, are passed through Double digestion identifies that the purpose fragment direction of insertion on recombinant plasmid is correct；Recombinant plasmid is imported in rhodotorula glutinis, inverted bacterium Strain PCR identifications, recombinant plasmid are successfully imported in rhodotorula glutinis and are inserted into the genome of rhodotorula glutinis, realize the steady of D6D Fixed, permanent expression；Real-time fluorescence quantitative PCR shows that D6D expression improves 2 times or so；Through gas chromatography analysis, turn The GLA changed in bacterial strain is improved more than 3.3 times compared with wild strain；The serum of the mouse of GM4-D6D- α MSH groups and liver Central Asia oil Acid, gamma-Linolenic acid, arachidonic relative amount dramatically increase compared with Normal groups, GM4-D6D- α MSH wherein in blood The GLA contents of group improve about 7.26 times compared with Normal groups, and arachidonic acid improves about 1.55 times；GM4-D6D- α in liver The GLA contents of MSH groups improve about 4.33 times compared with Normal groups, and arachidonic acid improves about 1.28 times；Because GM4- itself D6D- α MSH bacterial strains can produce more gamma-Linolenic acid, so Mouse oral can increase the content of the GLA in serum, simultaneously It is probably because part gamma-Linolenic acid is to arachidonic that arachidonic acid, which increases, compared with control group, in GM4-D6D- α MSH groups Acid conversion.

Brief description of the drawings

Fig. 1 is the Technology Roadmap of rhodotorula glutinis integrating expression vector pPGK1Z-rD-D6D structure；

Fig. 2 is colonial morphology figure observation photo；

Fig. 3 is the PCR amplified band figures of PGK1 (A) and D6D (B) gene；

Fig. 4 is that (wherein 1,2,3 attach most importance to for rhodotorula glutinis conversion bacterial strain GM4-D6D Insert Fragment D6D PCR detections figure Group bacterial strain, 4,5,6 be empty carrier bacterial strain)；

Fig. 5 is that recombinant plasmid pPGK1Z-rD-D6D double digestions qualification figure (is compareed, wherein 1 is with pPGK1Z-rD PPGK1Z-rD-D6D, 2 be pPGK1Z-rD)；

Fig. 6 is rhodotorula glutinis D6D gene expression dose figures；

Fig. 7 is that the polypeptide of Laser Scanning Confocal Microscope picture analyzing FITC marks is contaminated into the position oil droplet after intracellular with Nile red Chromatic graph, wherein (A) red fluorescence channel, (B) green fluorescence channel, (C) A and B two open the stacking chart of figure；

Fig. 8 is concentration map of the electricity conversion into the polypeptide of intracellular；

Fig. 9 be the concentration of α-MSH in serum such as；

Figure 10 is α-MSH Tissue distribution figure；

Figure 11 is for the concentration curve of α-MSH in colon mucus；

Figure 12 is the activity of MPO in colon；

Figure 13 is cytokine-expressing figure in colon；

Figure 14 is colon's pathological section figure (100X).

Specific embodiment

In order to preferably illustrate present invention, illustrated below with some preferably specific embodiments.But these have Body embodiment is simply to illustrate that present disclosure, rather than present invention is limited.2.1 experiment materials and instrument Device explanation

2.1.1 strain

Rhodotorula glutinis (Rhodotorula glutinis) GM4 is through screening and preserving (CCTCC M 2012203)；Wine brewing Yeast (Saccharomyces cerevisiae) 2.1445 is purchased from China General Microbiological culture presevation administrative center (CGMMCC)；Cunninghamella echinulata (Cunninghamella echinulata) MIAN6；Escherichia coli (Escherichia coli)DH5α。

2.1.2 plasmid

Expression vector pPICZ-rD, build and preserve.

2.1.3 culture medium and main agents

(1) YPD culture mediums (1L), YPD-zeocin culture mediums (1L), Luria-Bertani (LB) culture medium (1L), LB- Ampicillin culture mediums (g/L), less salt LB-zeocin culture mediums, Potato dextrose agar (PDA) culture mediums (g/ L)；

(2) STET buffer solutions, TE buffer solutions, 10%SDS；

(3) plasmid extraction liquid；

(4) 10xDNA buffer link buffer solution；

(5) Tricine SDS-PAGE electrophoresis reagents；

Negative pole cathode buffer liquid (10x), gel buffer liquid (3x), fixer, dyeing liquor, destainer, sample buffer, AB-3 storing liquids (49.5%T, 3%C), AB-6 storing liquids (49.5%T, 3%C), 16% separation gel, 10% squeegee, 4% is dense Contracting glue.

Above solution is by known existing fixing means configuration.

2.2.1 the separation and amplification of target gene

2.2.1.1 the separation and amplification of saccharomyces cerevisiae PGK1 genes

1st, separate：

Saccharomyces cerevisiae is inoculated in 50mL/250mL YPD fluid nutrient mediums, 30 DEG C, and 180rpm is cultivated to OD₆₆₀When=3 from The heart (12000r/min, 5min, 4 DEG C), supernatant is abandoned, collect thalline.Aseptic water washing thalline centrifuges afterwards twice, afterwards with cold sterilized water Thalline is resuspended, bacteria suspension, which is gone in the mortar containing liquid nitrogen and 1g alumina, grinds broken somatic cells, then goes to 5mL DNA extractions (50mM TRIS, 10mM MgCl in buffer solution₂, 50mM NaCl, 1% (wt/vol) SDS, pH 7.4) centrifuge tube in.By Phenol/chloroform extracting and methanol precipitation step, saccharomyces cerevisiae DNA repeatedly is separated.

2nd, PCR is expanded：

According to PGK1 gene orders, primer is designed：

- the CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3 ' of forward primer 5 ' (Bam HI)

- the TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAG-3 ' of reverse primer 5 ' (XhoI)

PCR reactions are anti-by Takara PCR reaction kits operation (being purchased from precious bioengineering (Dalian) Co., Ltd) PCR Answer condition：94 DEG C of 5min, 94 DEG C of 0.5min, 55 DEG C of 1min, 72 DEG C of 1min, 72 DEG C of 10min, 30 circulations, 5 μ L PCR are taken to expand Product is treated to analyze through 2% agarose gel electrophoresis.

2.2.1.2 the separation and amplification of cunninghamella echinulata D6D genes

1st, separate：

The extraction operating process of cunninghamella echinulata total serum IgE uses Oligotex with reference to Wan and Zhang et al. method MRNA Mini Kit (Qiagen) extract mRNA from total serum IgE.

2nd, PCR is expanded：

According to cunninghamella echinulata D6D gene orders (the GenBank accession number announced： DQ177498), D6D primers are designed.

- the CCG of forward primer 5 'CTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)

- the CATG of reverse primer 5 'CCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)

3rd, reverse transcription (RT) is reacted：

(1) reverse transcription is by Takara reverse transcription reagent box operation (being purchased from precious bioengineering (Dalian) Co., Ltd)；

(2) 37 DEG C are reacted 1 hour；

15 minutes terminating reactions of (3) 75 DEG C of incubations, it is stored in -20 DEG C or is directly used in downstream experiment.

4th, PCR reacts：

2.2.1.3 gel electrophoresis is reclaimed with PCR primer

1st, gel electrophoresis：

Operated according to gel electrophoresis Conventional procedures.

2nd, PCR primer reclaims：

Go out target DNA band using ultraviolet photodevelopment, cut corresponding agar band with slicer, insert in centrifuge tube；Tool The operation of body is：Binding Buffer II (7~4 μ l/1mg agar gels) are added, are shaken up in 60 DEG C of water-baths, until completely Dissolving；Put the agar gel of dissolving into UNIQ-10 pillars 5min, filtrate, the centrifugation of 8000rpm normal temperature are collected with collecting pipe 15min；The waste liquid of collection is discarded, removes UNIQ-10 posts, enters 600 μ l Wash Solution, 8000rpm centrifugations 2min；Repeat The waste liquid of collection is discarded, removes UNIQ-10 posts, enters 600 μ l Wash Solution, 8000rpm centrifugations 2min；Take out UNIQ- 10 posts are put in new centrifuge tube, add 40 μ l Elution Buffer in pillar film center, 5min is placed in 50 DEG C；Room temperature Lower 12000rpm centrifuges 1min, and liquid is to reclaim DNA, -20 DEG C freeze it is standby.

2.2.2 external source D6D integration vectors pPGK1Z-rD-D6D structure is expressed

2.2.2.1 integration vector pPGK1Z-rD-D6D structure route map

D6D gene integration expression vectors are designed using homologous recombination principle, constructing technology route is shown in Fig. 1：

2.2.2.2 expression vector pPGK1Z-rD-ME extracting

PPGK1Z-rD plasmids are what this laboratory built and preserved, are extracted according to conventional plasmid extraction step.

2.2.2.3 over-lap PCR connects PGK1 and D6D

Overlap PCR primers for PGK1 and D6D are as follows：

PGK1-Bam HI primer1：

5′-CGCGGATCCTATTTAGATTCCTGACTTCAACTC-3′(Bam HI)

PGK1-XhoIprimer2：

5′-TATCCGCTCGAGTGTTTTATATTTGTTGAAAAAGTAGATGTCGCCTATTATT-3′(XhoI)

D6D-XhoIprimer1：

5′-TCTGCTTTCTTCGCTCCGCTCGAGATGTCAGGGCAAACTCGAG-3′(XhoI)

D6D-XhoIprimer2：

5′-CATGCCATGGATCATCTAAAACATCTTTTGAGAG-3′(NcoI)

PGK1 and D6D fragments, using PGK1 the and D6D fragments of recovery as co-template, PGK1-Bam are reclaimed after 5 circulations HI primer1 and D6D-XhoIprimer2 are that upstream and downstream primer carries out Overlap PCR, obtain PGK1-D6D fragments.

Erlap PCR reactions (are purchased from precious bioengineering (Dalian) limited public affairs by the operation of Takara PCR reaction kits Department) .PCR reaction conditions：94 DEG C of 5min, 94 DEG C of 0.5min, 55 DEG C of 1min, 72 DEG C of 1min, 72 DEG C of 10min, 30 circulations.

2.2.2.4 the reaction of the double digestion of PGK1-D6D fragments and carrier, connection

The 1st, Overlap PCR primers and pPGK1Z-rD are carried out to NcoI and Bam HI double digestions respectively

Plasmid pPGK1Z-rD NcoI/Bam HI double digestion reaction systems：

Plasmid pPGK1Z-rD-ME：15 μ l, NcoI restriction endonucleases：2 μ l, Bam HI restriction endonucleases：2 μ l,

10×buffer：5 μ l, 1%BSA：5 μ l, ddH₂O：26μl.

Fragment PGK1-D6D NcoI/Bam HI double digestion reaction systems：

Fragment PGK1-D6D：10 μ l, NcoI restriction endonucleases：2 μ l, Bam HI restriction endonucleases：2 μ l,

10×buffer：5 μ l, 1%BSA：5 μ l, ddH₂O：26μl.

Endonuclease reaction was in 37 DEG C of water-baths 3 hours.

2nd, coupled reaction

After endonuclease reaction, construction recombination plasmid pPGK1Z-rD-D6D.

Coupled reaction reaction system is as follows:

Fragment PGK1-D6D digestion purified products：6 μ l, carrier pPGK1Z-rD digestion purified products：2 μ l, 10 × T4 DNALigase Buffer：1 μ l, T4 DNA ligases：1μl.

The mol ratio of 16 DEG C of water-bath reaction overnights, carrier and exogenous dna fragment will be controlled 1:3-10.

2.2.3 recombinant plasmid pPGK1Z-rD-D6D electricity conversion rhodotorula glutinis competent cell

2.2.3.1 the preparation of rhodotorula glutinis competence

Picking yeast single bacterium colony is seeded in 5mL YPD fluid nutrient mediums, and in 30 DEG C, 250rpm shaken cultivations are stayed overnight；With 1% Inoculum concentration is forwarded to 100mL YPD fluid nutrient mediums, in 30 DEG C, 250rpm shaken cultivations to cell OD600 ≈ 1.4；Bacterium solution is in 4 DEG C, 3000g centrifugation 5min sedimentation cells, supernatant is abandoned, is resuspended with 100mL sterilized waters；It is repeated once；4 DEG C, 3000g centrifugations 2min Sedimentation cell, supernatant is abandoned, cell is resuspended with the 1M sorbierites of 20mL ice precoolings；4 DEG C, 3000g centrifugation 2min sedimentation cells, abandon Clearly, cell is resuspended with 200 μ L 1M sorbierites, for converting.

2.2.3.2 plasmid linearization

About 10 μ g recombinant plasmids pPGK1Z/rD/D6D are subjected to single endonuclease digestion with Sac I.

Digestion system is as follows:

Sac I：1 μ l, 10 × buffer：5 μ l, plasmid pPGK1Z-rD-D6D：10 μ l, ddH₂O：7μl.

2.2.3.3 electricity conversion

By the recombinant plasmid linearized electricity conversion to rhodotorula glutinis GM4, electric step of converting is according to conventional electric method for transformation Operation.

2.2.3.4 recombinate the PCR detections of rhodotorula glutinis

(1) the preferable single bacterium colony of growing way is transferred in YPD fluid nutrient mediums in picking YPD resistant panels, 30 DEG C, 250rpm Shaken cultivation is to OD600 more than 2；

(2) take 1mL bacterium solutions 12000rpm to centrifuge 2min, abandon supernatant, add 100 μ L TE buffer solutions and thalline is resuspended, water-bath is boiled 5-10min is boiled, -20 DEG C of refrigerators is immediately placed on and freezes 15min, stand dissolves bacteria suspension at room temperature, 12000rpm centrifugation 5min, The μ L of supernatant 5 are taken to enter performing PCR detection for template.PCR reactions (are purchased from precious bioengineering by the operation of Takara PCR reaction kits (Dalian) Co., Ltd) PCR reaction conditions：94 DEG C of 5min, 94 DEG C of 0.5min, 55 DEG C of 0.5min, 72 DEG C of 1.5min, 72 DEG C 5min, 30 circulations.

2.2.3.5 the digestion verification of plasmid

After bacterium solution PCR preliminary identifications, the plasmid in correct positive colony is extracted, distinguishes counterweight with Nco I and Bam HII Group plasmid carries out double digestion identification, and reaction temperature is 37 DEG C.

Endonuclease reaction system is as follows：

Plasmid：2 μ l, 10 × buffer：4 μ l, NcoI：1 μ l, BamHII：1 μ l, ddH2O：41μl.

2.2.3.6 transformant Detection of Stability

Rhodotorula glutinis transformant is inoculated in 40mL YPD fluid nutrient mediums, 30 DEG C, 250rpm shaken cultivations 24h- 36h.1mL bacterium solutions are taken to be diluted to 10 respectively with sterilized water^-2,10^-3,10^-4, respectively take the dilution of 200 μ L difference dilution factors to apply respectively Common YPD flat boards and YPD-Zeocin resistant panels are distributed in, clump count is calculated, is designated as total bacteria count respectively and with integrated plasmid Clump count.YPD fluid nutrient mediums are forwarded to 1% inoculum concentration, 30 DEG C, 250rpm shaken cultivations, using 24h as 10 generations, trained altogether Support 50 generations.A plate count is carried out from generation to generation every 10.

Calculate plasmid stability：Stability (%)=clump count ÷ total bacteria count × 100% with integrated plasmid.

2.2.4 transformant D6D expressions determine

Real-time fluorescence quantitative PCR detects expressions of the D6D in bacterial strain is converted.Wild strain GM4 and conversion bacterial strain GM4-D6D is collected in YPD culture mediums after fermentation 24h, 48h, 72h respectively, extraction wild strain and the total serum IgE for converting bacterial strain, Method is with reference to above 2.2.1.3；CDNA is obtained through reverse transcription, method is with reference to 2.2.1.3.CDNA after synthesis is diluted 30 times, Instruct to carry out according to fluorescence quantitative kit SYBR PrimeScript TM RT-PCR Kit.

D6D real-time fluorescence quantitative PCR primer is：

F:5′-ATGTCAGGGCAAACTCGAG-3′；R:5′-ATCATCTAAAACATCTTTTGAGAG-3′

Quantitative fluorescent PCR is examined (limited purchased from the rich day science and technology in Hangzhou according to BioRT real-time fluorescence RT-PCRs kit method Company)

PCR amplification conditions are：95 DEG C of 3s, 95 DEG C of 5s, 60 DEG C of 31s, 40 circulations.Simultaneously to Real-time PCR primers SYBR melting curve analysis is carried out, each sample is parallel to do 4 secondary responses, repeats experiment 2 times, and with reference to Pfaffl (2001) side Method carries out data analysis.

2.2.5 bacterial strain GM4-D6D fatty acid compositional analysis are converted

1st, the extraction of bacterial strain GM4-D6D total fatty acids

The GM4-D6D bacterial strains of stationary phase are collected, are cleaned twice with distilled water after centrifugation, 5000g centrifuges 5min in 4 DEG C and collected Constant weight is dried to after thalline, grind into powder, bacterium powder is wrapped with filter paper and is dried to constant weight again；The bacterium powder that filter paper wraps up is put in In extracting barrel, injection absolute ether submergence sample, backflow extracting 8h or so in 70 degree or so of water bath with thermostatic control, after removing solvent Grease it is stand-by；Add 1mL 2% (wt/vol) sulfuric acid-methanol, 60 DEG C of esterification 2h；1mL hexanes are added after cooling, are vortexed 10min；The μ L of step ethane 800 are taken to be added to progress GC analyses in vial.

2nd, GC is analyzed

Match somebody with somebody hydrogen flameionization (FID) detector and capillary chromatographic column HP-INNOWAX with Bruker 450-GC instruments (30m×0.25mm).Qualitative and quantitative analysis is carried out to different aliphatic acid according to GC routine operations.

As a result prove

2.3.1 the colonial morphology of rhodotorula glutinis

After rhodotorula glutinis GM4 grows 3 days on Bangladesh's flat board, form such as Fig. 2.Bacterium colony 3~5mm of average diameter, color For red or Chinese red, gloss, surface is smooth and neat in edge, rounded projection.

2.3.1 PCR augmentation detection purpose fragments

Design specific primer enters performing PCR amplification to know the real situation gene PGK1 and D6D, is analyzed by agarose gel electrophoresis, Fig. 3 In (A) be shown in 800bp or so and amplify purpose band, (B) is shown in 13000bp or so and amplifies purpose band in Fig. 3.

Recombinant plasmid is imported into rhodotorula glutinis competent cell by electrotransformation, the cell pyrolysis liquid of rhodotorula glutinis is entered Performing PCR detects, and enters performing PCR amplification with target gene D6D specific primer, is analyzed by agarose gel electrophoresis, Fig. 5 is shown 1st, 2,3 passages (recombinant bacterial strain) amplify purpose band in 13000bp or so, and negative control (empty carrier bacterial strain, i.e., it is wild viscous red Yeast strain GM4, without recombinant plasmid transformed) then without band.Illustrate that recombinant plasmid pGK1Z-rD-D6D is successfully transferred to viscous red ferment Mother is simultaneously inserted into rhodotorula glutinis genome.

Recombinant bacterium GM4-D6D extracts plasmid after culture, is imaged by EcoRI and HindIII double digestions rear electrophoresis, As a result such as Fig. 4, it can be seen that have band (passage 1) in 2100bp and 3100bp or so, the clip size after digestion and inserted Target gene clip size is consistent, and illustrates that the direction of insertion of target gene fragment on recombinant plasmid is correct.

2.3.2 recombinant plasmid stability detects

In order to detect recombinant plasmid pPGK1Z-rD-D6D genetic stability, we are to conversion bacterial strain in non-selection pressure Lower Shaking culture 60 from generation to generation, picks bacterium solution in being coated with, cultivating in Zeocine resistant panels, calculates clump count.As a result such as table 2-1 It is shown, the rhodotorula glutinis bacterial strain of conversion is shown after continuously 60 generations of culture, and stability still can reach 99.31%, show Under non-selection pressure, the plasmid in rhodotorula glutinis has good genetic stability.

The Detection of Stability of table 2-1 recombinant plasmids

2.3.3 rhodotorula glutinis bacterial strain D6D expression analysis is converted

The result of real-time fluorescence quantitative PCR analysis D6D gene transcription levels is shown in Fig. 6, and the D6D expressions for converting bacterial strain are 2 times or so of wild strain, wild strain D6D transcriptional levels drop to reduced levels after cultivating 72 hours, and convert bacterial strain and exist It grows and gamma-Linolenic acid expression phase, its D6D transcriptional level are all higher than wild strain always.D6D high transcriptional level can With can for gamma-Linolenic acid and synthesis provide needed for enough enzymes so that the synthetic quantity of gamma-Linolenic acid maintain it is higher Level.

2.3.4 the accumulation research of gamma-Linolenic acid

The GLA of higher level whether can be produced in order to be overexpressed the rhodotorula glutinis of D6D genes conversion bacterial strain, in experimentation Its fatty acid composition is determined and analyzed, it is found that the GLA contents of conversion bacterial strain significantly increase, is carried by original 3.12% It is high that to 10.36%, while the content of the substrate linoleic acid of GLA synthesis substantially reduces, it can thus be seen that with wild strain phase Than conversion bacterial strain can accumulate more GLA.

Table 2-2 converts the aliphatic acid composition of bacterial strain and wild strain

In the present invention, Δ 6- fatty acid desaturases are the rate-limiting enzymes of synthesis of long-chain polyunsaturated fatty acids, respectively in n-3 Oleic acid (LA) dehydrogenation generation GLA being catalyzed in approach in ALA dehydrogenations generation SDA and n-6 approach.Closed in the biology of gamma-Linolenic acid During, LA the 6th carbon atom dehydrogenation is changed into GLA by △ 6- fatty acid desaturases.Constantly exerted by researchers Power, the encoding gene for having had many GLA key enzymes are cloned and realize functional verification, and some genes are also utilized In the modification of fatty acid metabolism approach, and obtain ideal effect.Such as Laoteng is successfully separated the Δ 6- fat in Mucor rouxii Fat acid desaturase, it is found that it has the similitude of height in the Δ 6- fatty acid desaturases of plant, and make it in saccharomyces cerevisiae It is middle successfully to be expressed；He Lu et al. have found that substantial amounts of GLA is had in the fermentation process of fermented soya bean to be produced, therefore isolate height GLA rhizopus is produced, and therefrom clone obtains Δ 6- desaturase genes, being expressed in saccharomyces cerevisiae can cause GLA's Accumulation；Δ 6- fatty acid desaturases in cunninghamella echinulata bacterium are expressed in tangerine woods saccharomyces oleaginosus by Wang Ping et al., are made Its GLA content accounts for the 1.2% of total fatty acids.And in this experiment, by fatty acid analysis, it is found that the LA in conversion bacterial strain is obvious Less than wild strain, this can also reflect LA from side and further synthesize GLA through D6D catalysis, so that in conversion bacterial strain LA content reduces, and GLA content increases.

Rhodotorula glutinis is the blast resistance of a plant height Lipid-producing, standing to be used for producing microbial grease and unsaturated fat Acid, carotenoid etc..This laboratory screens one plant of production fat ability stronger rhodotorula glutinis early stage, and its fat content can be up to 22.54%.In this experiment, we make use of the rhodotorula glutinis expression plasmid construction work of this laboratory early stage, successfully build Integrating expression vector pPGK1Z-rD-D6D, two rDNA fragments containing rhodotorula glutinis and saccharomyces cerevisiae is strong on the carrier Promoter PGK1, the stable high copy expression of target gene is realized using rDNA as integration site, so as to which external source is pierced into small gram of silver of spore The D6D gene integrations of Chinese mould can be stablized, high efficient expression on the chromosome of rhodotorula glutinis, as a result show and pass on 60 Dai Hou, plasmid remain to be stable in the presence of in transformant, and the D6D expressions of rhodotorula glutinis conversion bacterial strain are apparently higher than wild Bacterial strain, its GLA yield also significantly improve.

Generally speaking, the present invention successfully constructs foreign gene D6D expression vector pPGK1Z-rD-D6D, is reflected through double digestion Fixed, the purpose fragment direction of insertion on recombinant plasmid is correct；Recombinant plasmid is imported in rhodotorula glutinis, inverted bacterial strain PCR mirror Fixed, recombinant plasmid is successfully imported in rhodotorula glutinis and is inserted into the genome of rhodotorula glutinis, realizes D6D stabilization, permanent Expression；Real-time fluorescence quantitative PCR shows that D6D expression improves 2 times or so；Through gas chromatography analysis, bacterial strain is converted In GLA improved compared with wild strain more than 3.3 times.

The rhodotorula glutinis engineering bacteria and allogenic polypeptide for introducing allogenic polypeptide prepare material in the positioning of intracellular

3.1.1 test strain

Rhodotorula glutinis recombinant bacterial strain GM4-D6D

3.1.2 culture medium

Same 2.1.2

3.1.3 main agents

0.5mg/mL Nile red coloring agent；α-the MSH and H22LP of FITC and Plam modifications

The method for introducing allogenic polypeptide

3.2.1 electroporation method mediation allogenic polypeptide enters GM4-D6D intracellulars

1st, preparation engineering bacterial strain GM4-D6D bacterial strain competent cells, the same 2.2.3.1 of method.

2nd, totally 100 μ L after being mixed after FITC- α MSH and FITC-H22LP that 10 μ g have been modified dissolving with competent cell, It is added in the electricity conversion cup of 0.2cm precoolings；

3rd, electric shock process routinely operates；

4th, the bacterial strain finally obtained is respectively designated as GM4-D6D-F α MSH and GM4-D6D-FH22LP；

5th, α-MSH are transferred to GM4-D6D intracellulars by electroporation, obtain bacterial strain GM4-D6D- α MSH.

3.2.2 laser co-focusing detects polypeptide in the position of intracellular

0.1mL GM4-D6D-F α MSH and GM4-D6D-FH22LP bacterium solutions are taken respectively in 1.5mL EP pipes, 3000rpm from Heart 5min, it is resuspended with 0.1mL distilled water, adds a few drop Nile red dyes, 2-3min is kept in dark place, it is then burnt micro- by being copolymerized Mirror (TCS SP2) is observed.Green fluorescence 488nm lasing light emitter is observed, observes red fluorescence with 514 nanometers of lasing light emitter.

3.2.3 sepectrophotofluorometer quantitative analysis

(1) FITC- α MSH and FITC-H22LP concentration gradient titer are prepared；And 1mL titers are taken to 1 milliliter of colorimetric Ware, fluorescence intensity Fs to be determined；1 milliliter of GM4-D6D-F α MSH and GM4-D6D-FH22LP bacterium solution is pipetted to 1 milliliter of cuvette, Fluorescence intensity Fx to be measured；Blank solution is prepared in 1 milliliter of cuvette, fluorescence intensity F0 to be measured；

(2) standard curve between (Fs-F0) and testing sample standard concentration C is done；According to (Fx-F0) from standard curve On try to achieve the content of sample.

(3) data analysis

Data analysis uses GraphPad Prism 5.0, and statistics is represented with Mean ± SD, the otherness between group point Analysis uses single factor test variance (one-way ANOVA).

3.3 experimental result

3.3.1 the method validation polypeptide of Laser Scanning Confocal Microscope (CLSM) enters the position of intracellular

In order to detect polypeptide whether enter intracellular and they in the position of intracellular, contaminated with hydrophobic fluorescence material Nile red Material dyeing fat drips are as shown in fig. 7, respectively using red and green fluorescence channel then respectively it can be seen that the fat drips that Nile red dyes With aobvious green polypeptide FITC-F α MSH and FITC-FH22LP, and two figures are superimposed, it is found that red and green phosphor dot is complete Full weight closes (green fluorescence point is smaller than the phosphor dot of red), is in yellow after superposition, it is same to show that two kinds of materials are present in In individual nano-particle.This can be explained two kinds of polypeptides and is incorporated into intracellular, and because the lipotropism of itself is entered in fat drips.

3.3.2 fluorescence spectrophotometry intracellular polypeptide FITC- α MSH and FITC-H22LP content

After electricity conversion, we centrifuge away the polypeptide in solution, bacterium solution are collected, with fluorescence spectrophotometry intracellular Polypeptide FITC- α MSH and FITC-H22LP content, from figure 8, it is seen that the concentration difference of two kinds of polypeptides is little, it is 3mg/ ML or so, and initially electricity conversion when polypeptide concentration be 10mg/mL, illustrate electricity conversion after intracellular polypeptide concentration reach initially The 1/3 of concentration, and two kinds of polypeptides do not have significant difference in the concentration of intracellular.

Allogenic polypeptide is made it into rhodotorula glutinis GM4-D6D intracellulars by us by the method for electroporation, obtains bacterial strain GM4-D6D-F α MSH and GM4-D6D-FH22LP because we to have polypeptide fat-soluble, from the figure of laser co-focusing It can be seen that they are entered in the oil droplet of rhodotorula glutinis, while pass through fluorescence spectrophotometry intracellular polypeptide FITC- α MSH and FITC-H22LP content, find more 1/3 allogenic polypeptide can be transferred into rhodotorula glutinis by the method for electroporation Intracellular, and the maximum that Mark et al. is rotated into bovine serum albumin(BSA) in red blood cell using the method for electroporation is 1/4；And two The content difference that kind of polypeptide is entered by electroporation after intracellular is little, is 3 μ g/mL, illustrates that the method is also applied for polypeptide Conversion enters rhodotorula glutinis intracellular.

Once there is article to report that as the carrier of vaccine, micro-capsule is fabricated to as natural lipid with by yeast for oral viable yeast Body packaging medicine, or the yeast of oral killed transmit polyunsaturated fatty acid, and yeast is acting as natural biological glue herein The role of capsule, the transport of medicine and unrighted acid etc. can be realized by oral whole yeast.

Generally speaking, the present invention makes FITC- α MSH and FITC-H22LP enter rhodotorula glutinis by the method for electroporation GN4-D6D intracellulars；Laser co-focusing experiment proves that two kinds of polypeptides are entered in oil droplet；Fluorescence spectrophotometry intracellular polypeptide FITC- α MSH and FITC-H22LP content, finding the content of two kinds of peptides does not have significant difference, and is the 1/3 of primary quantity.

Healthy mice In vivo study

4.1 experiment materials and instrument

4.1.1 experimental strain

Bacterial strain GM4-D6D- α MSH and GM4-D6D

4.1.4 experiment mice

15 SPF level BALB/c mouses, buy in Zhongshan University's Experimental Animal Center.

4.2 experimental method

4.2.1 GM4-D6D- α MSH mouse In vivo study

4.2.1.1 concentration of the α-MSH in serum

1st, mice group

(1) GM4-D6D- α MSH groups, 6；

(2) GM4-D6D groups, 6 as control；

2nd, eyeball of mouse takes blood

The 1st, 3,5,7 day after feeding, eyeball rear vein beard blood taking method took blood (once take a blood sample 0.2mL).

3rd, α-MSH concentration in serum is surveyed

2500rpm centrifuges 15min, and the standard items for taking debita spissitudo to be 300pg/mL are diluted to a series of the dilute of concentration gradients Release liquid；α-MSH concentration in serum is surveyed with ELISA kit, operating method follows ELISA kit explanation.

4.2.1.2 α-MSH Tissue distributions

Above-mentioned mouse by 7 days administration and after taking blood, mouse cervical dislocation is put to death at the 7th day, take respectively liver, Spleen, lung, kidney, related adipose tissue and connective tissue above it are removed, normal saline flushing is clean, will organize to inhale with filter paper It is dry；Clip is organized in right amount, 1mL physiological saline is added after being precisely weighed homogenate is made；The sample of homogenate is made in 200rpm, 25 DEG C Lower concussion 20min, then centrifuges 15min under 25000rpm；Supernatant is crossed into 0.22 μm of miillpore filter, used after removing magazine ELISA kit surveys α-MSH concentration, and method is same as above.

4.2.2 serum and liver fat acid constituents measure

4.2.2.1 mice group

GM4-D6D- α MSH groups, take 6 normal mouses, daily except normal feed is fed, also feed GM4-D6D- α MSH Bacterial strain, every daily scale of feeding is 1 × 1010CFU；Normal groups, take 6 normal mouses, only feed daily normal feed and The physiological saline of equal volume, as control.

4.2.2.1 determination of fatty acid in serum

In feeding 7 days, mouse took blood by eyeball, by blood serum sample formicester after centrifugation, took supernatant to carry out GC- after processing MS is analyzed, the same 2.2.5 of method.

4.2.2.2 determination of fatty acid in liver

Mouse equally is put to death in the 7th day cervical dislocation, takes 80mg livers, adds chloroform:Methanol=2:1 extractant, Moved into after homogenate in centrifuge tube, 25000rpm centrifugation 15min, precipitation is repeated once extraction process, and merging centrifuges supernatant twice.Add Enter the 1.45mM of 0.2 times of volume NaCl solution, remove a layer nitrogen drying, esterification handles same blood serum sample.

4.2.3 data processing

Data processing and mapping use GraphPad Prism 5.0, and statistics is represented with average ± standard deviation, group Between contrast use one-way analysis of variance (one-way ANOVA), the data analysis between two groups uses Student ' s Test, p<0.05 is considered significant difference be present between group.

4.3 experimental result

4.3.1 detections of the α-MSH in serum and tissue

In order to detect GM4-D6D- α MSH deliveries α-MSH effect, after we make mouse take GM4-D6D- α MSH, the 1st, blood was taken by eyeball in 3,5,7 days, the concentration of α-MSH in serum is detected with ELISA kit, the mouse for taking GM4-D6D makees Control.As illustrated, in GM4-D6D- α MSH groups, contents of the α-MSH in serum increases with the increase for taking number of days, 1 to 5 day, α-MSH content linearly increased, and after 5 days, hardly increases and is intended to steadily, and in GM4-D6D Group, α-MSH content are always maintained at poised state, and contents of the GM4-D6D- α MSH groups α-MSH in serum in finite concentration It is significantly higher than GM4-D6D groups (p<0.05).

From mouse tissue distribution map (Figure 10), GM4-D6D- α MSH deliveries α-MSH make it in liver, lung, spleen, kidney Distribution dramatically increases, and is especially improved in the distributed density of liver by 5.35pg/g to 17.36pg/g, improves 3.24 times, and almost Do not enter cardiac muscular tissue.This is probably that we guess that α-MSH are likely to fat with lipid mainly the reason for the liver metabolism Matter forms chylomicron and is carried, and enters lymphatic system together, finally enters blood, is transported to each tissue of body, especially It is the major organs liver of lipid-metabolism.

4.3.2 Analysis of Fatty Acids Composition

Can be seen that from the data in table 4-1 the mouse of GM4-D6D- α MSH groups serum and liver Linoleic acid, γ- Leukotrienes, arachidonic relative amount dramatically increase compared with Normal groups, the GLA of GM4-D6D- α MSH groups wherein in blood Content improves about 7.26 times compared with Normal groups, and arachidonic acid improves about 1.55 times；GM4-D6D- α MSH groups in liver GLA contents improve about 4.33 times compared with Normal groups, and arachidonic acid improves about 1.28 times.Because GM4-D6D- α MSH itself Bacterial strain can produce more gamma-Linolenic acid, so Mouse oral can increase the content of the GLA in serum, while and control group Compare, it is probably because part gamma-Linolenic acid converts to arachidonic acid that arachidonic acid, which increases, in GM4-D6D- α MSH groups.

Table 4-1 serum fatty acid constituent analysis tables (%)

Table 4-2 liver fat acid constituent analysis tables (%)

Anti-inflammatory activity evaluation experimental proves

5.1 experiment material

5.1.1 culture medium and preparation of reagents

(1) LPS (lipopolysaccharides) solution, RMPI-1640 nutrient solutions；

(2) protease inhibitors, electricity turn buffer solution, lysate, transferring film liquid, glycine running buffer, PBST；

Above reagent, is prepared according to a conventional method.

5.2 experimental method

5.2.1 PBMC (PMNC) separation

The blood 36ml of healthy volunteer is extracted into centrifuge tube, adds 4ml ACD anti-coagulants；It is slowly added to close to tube wall 72ml lymphocyte separation mediums, 2000rpm centrifuges 15min at 25 DEG C；The white single core of the second layer is drawn with syringe Cell；The mononuclearcell of absorption is placed in new test tube, measures volume, the lymphocyte separation medium of equivalent is added, 25 1500rpm centrifuges 10min at DEG C, collects mononuclearcell, repeats 5 times；The mononuclearcell being collected into is used and contains 10% The RMPI-1640 nutrient solutions of hyclone are resuspended, and take and are dyed on a small quantity with trypan blue dye liquor, (dead in micro- Microscopic observation cell Cell is blue, and living cells is colourless)；Calculate cell viability (cell viability=viable count/TCS x100%)；By cell Auto-regulating System of Density of Heavy Medium is 1x106/ml, and constant incubator culture is standby.

3.2.2 the PBMC that recombinant bacterium cell culture supernatant processing LPS is stimulated

(a) DMEM buffer culture medium cultures 10h bacterial culture fluid is taken, 3000rpm centrifugations 10min, discards bacterium under normal temperature Body, supernatant is collected, it is degerming with 0.22 μm of membrane filtration partly with after 3 μ g/ml furin digestions, collect filtrate；

(b) with the α-MSH concentration in α-MSH ELISA kits detection recombinant bacterium culture supernatant, α-MSH concentration is used DMEM buffer culture mediums are adjusted to 80pg/ml, and the supernatant of other non-recombinant bacterium also dilutes identical multiple；

(c) PBMC cells are inoculated in Tissue Culture Plate by every hole 1ml, remove culture medium before experiment, added after starting It is above-mentioned that the bifid bacterium culture supernatant after one times is diluted with the RMPI-1640 nutrient solutions for concentrating one times.Test and be divided into six ancestrals, every group If 5 multiple holes.Normal group (N groups), only replacing PBS solution dilutes one times of RMPI-1640 nutrient solution of concentration to the group in an experiment； LPS groups, replacing PBS solution dilute 1h after one times of RMPI-1640 nutrient solution of concentration, and 8 are stimulated with final concentration of 5 μ g/ml LPS Hour；PDG7 groups (empty carrier group), after discarding culture medium, add after diluting one times with the RMPI-1640 nutrient solutions for concentrating one times Empty carrier bifid bacterium culture supernatant be incubated 1h, afterwards with final concentration of 5 μ g/ml LPS stimulation 8 hours；pDG7+Furin Group, identical with pDG7 group processing, simply bifid bacterium culture supernatant digests by furin；PBDMSH groups, with pDG7 groups It is the rhodotorula glutinis recombinant bacterial strain for the importing α-MSH polypeptides for secreting α-MSH unlike processing；PBDMSH+Furin groups, 1h, Zhi Houyong are incubated with the rhodotorula glutinis recombinant bacterial strain culture supernatant of the importing α-MSH polypeptides digested by furin Final concentration of 5 μ g/ml LPS is stimulated 8 hours；α-MSH groups are used as control for the α-MSH of synthesis.

5.2.3 Western Blot detect the expression of NF- kB proteins

A. total protein extraction

Cell after collection processing, is moved in centrifuge tube, and 4000rpm centrifuges 8min at 4 DEG C, removes supernatant, is added Ice-cold PBS liquid washs 3 times, and 4000rpm centrifuges 8min again at 4 DEG C, removes supernatant, is placed at once cold on ice Freeze；The mixed liquor of cell pyrolysis liquid and protease inhibitors is added, is blown and beaten with strength, 12000rpm is in 4 DEG C after placing 0.5h on ice Lower centrifugation 10min, supernatant is transferred in another clean centrifuge tube, using BCA methods measure protein content, it is standby or- 80 DEG C of preservations；Sample total protein content is 100~150 μ g or so, take albumen sample-loading buffer and and supernatant, in 1/3 ratio Mix, boiling water water-bath 15min makes albuminous degeneration, in standing 10min on ice, cools down rear electrophoresis.

B. electrophoresis

Running gel is prepared according to a conventional method.

When starting electrophoresis, voltage maintains 100v, and (2h is taken around) when going to separation gel etc. bromophenol blue, and voltage is improved To 150v, when electrophoresis to bromophenol blue is reached at the top of separation gel, stop electrophoresis, prepare transferring film.

C. routinely operating method carries out transferring film to transferring film.

D. immune detection

Film is cleaned 3 times, each 10min with PBST, is placed in containing in confining liquid, vibrates closing on decolorization swinging table at room temperature 1h；Primary antibody dilutes is incubated a night at 1000 times, 4 DEG C on decolorization swinging table, cleans；Secondary antibody dilutes 1000 times, decolorization swinging table under normal temperature Upper incubation 1.5h；Film is soaked in eluent, 55 DEG C of baking boxs are incubated 30min, are cleaned 3 times with PBST, each 10min；Into As being imaged in system, gray scale is analyzed with image J.

3.2.4 detect culture supernatant TNF-α secretion

The post-stimulatory culture supernatants of LPS are collected, 3000rpm centrifugation 15min, collect supernatant, employment double-antibody sandwich The concentration of TNF-α, its step operate according to TNF-α ELISA kit in TNF-α ELISA kit detection supernatant.

5.2.5 data processing

Data processing and mapping use software GraphPad Prism 5.0, statistics average ± standard deviation table Show, the contrast between group uses one-way analysis of variance (one-way ANOVA), and p ＜ 0.05 are considered significant difference be present between group.

5.3 experimental result

5.3.1 PBMC vigor after separating

Active PBMC, because its cell membrane is intact, trypan blue can not enter cell, show colourless；Dead PBMC, Trypan blue can enter intracellular, aobvious blue；200 cells are observed, colourless cell there are 192, cell viability 96%, can be used for Follow-up α-MSH anti-inflammatory activities detection.

5.3.2 NF- κ B p65 protein expressions in PBMC

Using β-actin as reference, NF- κ B p65 expression is analyzed with image J, the results showed that：NF- κ after DSS inductions B p65 expression raises (the p ＜ 0.01 compared with normal group) significantly；Use the rhodotorula glutinis recombinant bacterial strain for importing α-MSH polypeptides After culture supernatant processing, NF- κ Bp65 expression effectively declines (pDG7 groups, pDG7+Furin groups the p ＜ 0.05 compared with LPS groups), The culture supernatant for converting empty carrier is discharged without influence (pDG groups and pDG7+Furin before and after furin enzymolysis on TNF-α Group contrast, p ＞ 0.05) and using after the Bacteria Culture supernatant processing for secreting α-MSH precursor proteins, this effect suppresses NF- κ The effect of B p65 expression is more notable (pBDMSH groups, pBDMSH+Furin groups are compared with LPS groups, pDG7 groups, p ＜ 0.01), such as It is shown in Table shown in 3-1.

Table 3-1 NF- κ B p65 express relative gray values

Table 3-1 Relative gray value of NF-κB p65 expression

Note：^aP ＜ 0.01 contrast with N groups,^bP ＜ 0.05 contrast with LPS groups,^cP ＜ 0.01 are compared with LPS groups, pDG7 groups, n =5.

5.3.3 recombinant bacterium culture supernatant, which suppresses LPS, stimulates PBMC TNF-α release

PBMC is added after LPS stimulates 8h, and the TNF-α concentration in supernatant largely raises (compared with normal group p<0.01)； The culture supernatant for converting empty carrier is discharged without influence (pDG groups and pDG7+Furin before and after furin enzymolysis on TNF-α Group compares, p ＞ 0.05)；The supernatant liquid energy for converting the bifid bacterium of empty carrier significantly inhibits the generation for the TNF-α that LPS is stimulated, i.e., PDG7 groups, pDG7+Furin the groups p compared with LPS groups<0.05；Bacterial supernatant and the conversion for secreting α-MSH precursor proteins are unloaded Body group, LPS groups are compared to pBDMSH groups compared with LPS groups, pDG7 groups), the secretion (p of TNF-α can be significantly inhibited<0.05)；But again Group bacterium supernatant after furin digests, its suppress TNF-α expression activity greatly improve (pBDMSH+Furin groups and LPS groups, pDG7 groups, p<0.01) complete α-MSH are obtained after, illustrating enzymolysis and the α-MSH have played its anti-inflammatory activity.

Import effect experiment of the rhodotorula glutinis recombinant bacterial strain of α-MSH polypeptides to experimental colitis

6.1 material

6.1.1 main agents：3.5%DSS solution

6.1.3 experiment mice

46 SPF level BALB/c mouses are bought is in Zhongshan University's Experimental Animal Center, certificate of competency number：SCXK Guangdong 2011--00296.2 method

6.2.1 the structure of animal model

40 mouse are taken, give 3.5%DSS solution as free drinking water, it is continuous to feed 7 days, the excrement of mouse is observed, Diarrhoea status, TNF-α content in mice serum is detected, modeling is determined, gives up for the unsuccessful mouse of modeling and do not have to.

6.2.2 packet transaction

N groups (normal group), 6 mouse not modeled；DSS groups, the mouse of 8 modelings；

PDG7 groups (empty carrier group), the mouse of 8 modelings is taken, daily except normal feed is fed, also feeding conversion is unloaded The rhodotorula glutinis recombinant bacterial strain of the importing α-MSH polypeptides of body, every daily scale of feeding is 1 × 1010CFU；PBDMSH groups, take 8 The mouse of modeling, daily except normal feed is fed, also feeding secretion α-MSH restructuring imports the rhodotorula glutinis of α-MSH polypeptides Recombinant bacterial strain BL-pBDMSH, every daily scale of feeding are 1 × 10 10CFU.The rhodotorula glutinis weight of α-MSH polypeptides is imported in feeding Group bacterial strain is after seven days, and be euthanized mouse, colon rear end tissue is cut, for detecting the MPO in tissue, TNF-α, IL-1 β, IL- 6th, IL-10 and pathology section examination；α-MSH in knot mucous gut membrane are detected, then are the 1st, 3,5,7 after bifid bacterium is fed Its detection.

6.2.3 colonic mucus α-MSH are detected

The 1st, 3,5,7 day after bifid bacterium is fed, colonic mucus is collected, 20min is centrifuged under 3000rpm normal temperature, it is heavy to remove Form sediment, the α-MSH employment α-MSH ELISA kits detection in supernatant, specific method is shown in 2.2.5.

6.2.4 MPO is detected

MPO detection uses Fabia [57] method, and 4.2.5 cytokine TNFs-α, IL-1 β, IL-6 are operated according to kit With IL-10 measure

Colon 0.1g is weighed, addition PBS to 0.5ml, is fully homogenized, 5000rpm centrifugations 20min at 4 DEG C, Precipitation is discarded, it is standby to collect supernatant.

A.TNF- α are detected

Using the kit of equation company, operating procedure illustrates B.IL-1 β, IL-6 and IL-10 measure according to kit

Kit of the Bo Ling sections for company is used, step illustrates that 4.2.6 tissue pathological slices are observed according to kit

Colon is taken, is cleaned 3 times with physiological saline, cuts irregular part, clean tissue is placed in containing 10% first 5h is soaked in the solution of aldehyde；Dehydration, transparent, waxdip, embedding, section, dewaxing, dye, seal rear micro- Microscopic observation up for safekeeping and take pictures.

6.2.7 data processing

Data processing and mapping use software GraphPad Prism 5.0, statistics average ± standard deviation table Show, the contrast between group uses one-way analysis of variance (one-way ANOVA), and the data analysis between two groups uses Student' S t test, p<0.05 is considered significant difference be present between group.

6.3 result

6.3.1 model construction

Mouse drank the aqueous solution containing 3.5%DSS after seven days, symptom of having loose bowels occurred, excrement scattered paste shape, companion in excrement With bloodstain, the TNF-α in serum rises to 19.22 ± 1.07pg/ml (p by 9.20 ± 0.41pg/ml<0.05) modeling, is illustrated Success.

6.3.2 α-MSH are detected

After colitis mice takes bifid bacterium, the 1st, 3,5,7 day after taking, colonic mucus α-MSH concentration is detected. PBDMSH groups, it will be seen that with the number of days taken, α-MSH amount sustainable growth：At 1-5 days, α-MSH increased sharply, At 5-7 days, the increased amounts of α-MSH were relatively few, tended to a stable state, its reason be probably at latter several days, colon The rhodotorula glutinis recombinant bacterial strain arrival peak for importing α-MSH polypeptides is not further added by；Expression quantity compared with pDG7 (empty carrier group) group, p<0.001.And in pDG7 groups, expression quantity is always maintained at poised state (being shown in Table 4-1 and Figure 11) within degree of detection.

α-MSH concentration (pg/ml) in table 4-1 colon mucus

Table 4-1 Concentration of α-MSH in colon mucinous

Note：* * p ＜ 0.001 and pDG7 is passed through compared with n=5.

6.3.3 MPO activity in colon

After being handled seven days with the rhodotorula glutinis recombinant bacterial strain for importing α-MSH polypeptides, the MPO in tissue is detected with ELISA Activity, as a result find compared with DSS groups, the MPO of pDG7 groups and pBDMSH groups activity all effectively arrive decline, exist notable Difference (p ＜ 0.05), but pBDMSH groups are significantly (p ＜ 0.01), are shown in Table 4-2 and Figure 12.

MPO activity in table 4-2 colons

Table 4-2 MPO activity in the colon tissue

Note：* p ＜ 0.05 are compared with DSS groups, and * * p ＜ 0.01 are compared with pDG7, DSS group, n=5.

6.3.4 colon's cytokine TNF-α, IL-1 β, IL-6 and IL-10 expressions

After the long rhodotorula glutinis recombinant bacterial strain for importing α-MSH polypeptides is taken seven days, cell factor IL-1 β, TNF-α, IL- There is great variety in 6 and IL-10 expression.In pDG7 groups, compared to DSS groups, anticusp inflammation factor TNF-α and IL-6 can have Effect declines (p ＜ 0.05), and anti-inflammatory cytokines IL-10 substantially rises (p ＜ 0.05).With respect to DSS groups and pDG7 groups, pBDMSH groups In anticusp inflammation factor TNF-α, IL-1 β, IL-6 are significantly reduced (p ＜ 0.05), and IL-6 is more significantly (p ＜ 0.01), resists Inflammatory cytokines IL-10 also dramatically increases (p ＜ 0.05), is shown in Table 4-3 and Figure 13.

Cell factor IL-1 β, TNF-α, IL-6 and IL-10 expression in table 4-3 colons

Table 4-3 Expression of cytokines IL-1 β, TNF-α, IL-6 and IL-10 in the colon tissue

Note：* p ＜ 0.05 are compared with pDG7, DSS group, and * * p ＜ 0.01 are compared with pDG7, DSS group, #p ＜ 0.05 and DSS groups Compare, n=5.

6.3.5 mouse Colon histotomy is observed

Shown in Figure 14, after hematoxylin-eosin dye dyeing, in normal group (N groups), it may be seen that complete colon Mucous layer, there is clearly brush border, the arrangement of top layer columnar cell is closely orderly, without any broken sign；In DSS groups, It can be seen that rotten to the corn mucous membrane, brush border receive damage, it can not be identified and, can't see the columnar cell on top layer, column is thin Obvious layering is not seen under born of the same parents, shows that serious inflammatory cell invades profit and typical ulcer yet；In empty carrier group (pDG7 Group), it was observed that broken brush border, moreover it is possible to see fuzzy top layer columnar cell, can also see picture between columnar cell's cell The same white space between cells of bubble；In express alpha-MSH recombinant bacterium group (pBDMSH groups), it can be seen that clearly brush relatively Edge, surface are serrated, and the columnar cell on top layer also can be also than more visible, but arranges in a jumble, is layered below columnar cell bright In vain.

Ulcerative colitis (UC), it is really sick although experienced decades of research as a kind of complicated inflammatory bowel disease Because failing to understand so far.Current treatment method mainly using anti-inflammatory agent glucocorticoid, minosalicylates, drugs and is immunized Inhibitor etc., can't solve problem at all.In our experiment, we are tested to import the rhodotorula glutinis of α-MSH polypeptides Anti-inflammatory polypeptide α-MSH are delivered to colon, research carries α-MSH and imports the viscous of α-MSH polypeptides by recombinant bacterial strain as transport vehicle Antiphlogistic effects of the rhodotorula recombinant bacterial strain to colitis.A series of UC unconventionality expression for primarily showing as inflammatory cytokines, Body is damaged, therefore suppresses inflammation just as the treatment sick top priority.α-MSH are a kind of human endogenous's nerves Property polypeptide, it is good to human body affinity, there is very strong antiinflammatory action, the various inflammation medium factors can be adjusted, suitable for this Disease of the kind with complicated inflammation background, has been reported that confirmation, the polypeptide is expelled in inflammatory bowel disease models Mice Body, Ke Yiqi To good therapeutic effect, but there is very big problem in this experiment.First, α-MSH half-life period only 20 minutes, injection are only capable of tieing up The effect of holding in the short time, it is impossible to applied to reality；On the other hand, α-MSH, which have, suppresses pathogenic bacteria such as staphylococcus aureus With the effect of Candida albicans, cause inflammatory bowel disease for such bacterium, it is harmful thin that injection obviously can not act on α-MSH Bacterium, therefore be a kind of very reasonless mode, furthermore, α-MSH are polypeptide, orally just also unrealistic.

In order to change this awkward situation, we attempt to import the viscous red ferment of α-MSH polypeptides by oral express alpha-MSH Female recombinant bacterial strain so form utilizes α-MSH.The rhodotorula glutinis recombinant bacterial strain for importing α-MSH polypeptides is mainly grown in knot Intestines, the degraded of stomach and enteral protease to α-MSH can be avoided, by the rhodotorula glutinis recombinant bacterium for importing α-MSH polypeptides Strain, to realize lasting administration, to maintain valid density, reaches therapeutic effect constantly in the secretion α-MSH of colon；Meanwhile can So that α-MSH directly act on some pathogenic bacteria, this is extremely important to inflammatory bowel disease, because most anti-inflammatory agents only have anti-inflammatory effect Fruit, the function without suppressing germ.For drug delivery, the rhodotorula glutinis recombinant bacterial strain field planting for importing α-MSH polypeptides exists Colon, it is also the position of colitis breaking-out herein, is delivered by the rhodotorula glutinis recombinant bacterial strain for importing α-MSH polypeptides, α-MSH are just Inflammatory tissue can be directly targeted, improves the utilization rate of medicine.The result of mouse colitis model shows, takes express alpha-MSH Precursor protein bacterial strain, α-MSH precursor proteins can effectively be secreted in colon, be converted in the presence of colon's furin For α-MSH.5-7 days after taking, α-MSH reached a plateau in the amount of colonic mucus, and valid density it It is interior, show the effect of a lasting administration.Our data showed that an inflammatory conditions and the mark of tissue damage because Sub- MPO, after express alpha-MSH bacterial strain is taken, its activity substantially reduces；Anticusp inflammation factor IL-1 β, TNF-α and IL-6 are then Significantly reduce, also anti-inflammatory cytokines IL-10, be considered to have the albumen of potential treatment inflammatory bowel disease, also significantly increased Add, illustrate a good anti-inflammatory effect, colon's pathology section examination below has also confirmed our result.We Experiment change this injection occupation mode of α-MSH polypeptides, realize oral delivery α-MSH, solve asking for its half-life short Topic.

On the other hand, the treatment of IBD only focuses on that to control inflammation be far from being enough, because this disease is also Relevant with enteric flora disturbance, rational therapy should be also including regulation gut flora.Studies have reported that some bacteriums are for example big The pathogenic bacteria such as enterobacteria and avain tuberculosis mycobacteria dramatically increase in inflammatory bowel disease patient's enteron aisle, mycobacteria, monocyte Increasing property listeria spp, Chlamydia and enterobacteriaceae lactobacteriaceae can cause inflammatory bowel disease.Therefore, control pathogenic bacteria are being treated Inflammatory bowel disease just seems natural.The rhodotorula glutinis recombinant bacterial strain of α-MSH polypeptides is imported as probiotics, except some health cares Outside function, moreover it is possible to play barrier action to alimentary canal mucous membrane, suppress the bacteria growing that causes a disease, maintain gut flora balance, also have some to grind Study carefully the rhodotorula glutinis recombinant bacterial strain for showing to import α-MSH polypeptides to can be used for controlling inflammatory bowel disease, it may be possible to its antibacterial functions have Close, therefore be applied to inflammatory bowel disease.More there are some researches show the quantity for importing the rhodotorula glutinis recombinant bacterial strain of α-MSH polypeptides is being burst It is to reduce in the colon of the patient of ulcer colitis, the rhodotorula glutinis recombinant bacterial strain that supplement imports α-MSH polypeptides just seems non- Often it is necessary.In summary, the rhodotorula glutinis recombinant bacterial strain bacterial strain of oral express alpha-MSH importing α-MSH polypeptides can be used as one Effective means of the kind with reference to anti-inflammatory and gut flora adjustment for the treatment of inflammatory bowel disease

α-MSH are a kind of human endogenous's nerve polypeptides, and our trials are carrying out mouth with yeast as its carrier Clothes, by strain construction above, α-MSH is entered intracellular and shown that it is located in oil droplet, passed through this chapter reality Issue after examination and approval now, after Mouse oral carries α-MSH bacterial strain GM4-D6D- α MSH, α-MSH content is in serum and organs in adult Middle content significantly increases, and changes α-MSH medication, solves the problems, such as half-life short.It is and real by Tissue distribution It is more notable to issue after examination and approval content increases of the existing α-MSH in liver, therefore is beneficial to for treating liver diseases.

GLA inherently has the function that antibacterial, anti-inflammatory as polyunsaturated fatty acid, and has many health cares to make With.We realize GLA overexpression by being overexpressed D6D in rhodotorula glutinis, and unrighted acid is and glycerine mostly It is stored in reference to rear generation triacylglycerol in oil droplet, then our this experiment is exactly to realize both to be rich in GLA in oil droplet, is contained again There are α-MSH, by the recombination yeast of oral killed, realize transmission of the yeast for two kinds of anti-inflammatory substances, while red ferment is glued in the strain Female its fatty acid composition is similar to vegetable oil [10] through this laboratory checking early stage, and containing the carrotene enriched, its into Dividing has good health-care effect.We have found that after oral whole GM4-D6D- α MSH bacterial strains, not only GLA content increases through experiment Add, and GLA further product arachidonic acid is that ARA content also increases, and realizes a variety of insatiable hungers in Mice Body With the increase of content of fatty acid.In summary, the oral whole yeast rich in GLA and α-MSH can simply, effectively realize two Kind material oral delivery, then this method is also applied for the oral delivery as other polypeptide drugs or dewatering medicament.

After Mouse oral GM4-D6D- α MSH bacterial strains, α-MSH concentration increase in serum and liver, lung, kidney, spleen, In vital tissue organ, the concentration increase of the α-MSH in liver is more notable；After Mouse oral GM4-D6D- α MSH bacterial strains, blood GLA, ARA of cleer and peaceful liver concentration increase.

The present invention, as carrier, improves bacterial strain GLA content, and pass through electroporation with rhodotorula glutinis by being overexpressed D6D Realize that allogenic polypeptide α-MSH and H22LP enter in intracellular oil droplet, and through the oral whole yeast strain GM4-D6D- α of healthy mice MSH evaluates this method oral delivery α-MSH and GLA effect, and concrete outcome is summarized as follows：

(1) by building foreign gene D6D expression vector pPGK1-rD-D6D, realize Δ 6- delta 8 desaturase genes viscous Stable heredity in rhodotorula, and D6D overexpression is realized, the GLA contents in recombinant bacterial strain is improved 3.3 compared with wild strain It is more again.

(2) by electroporation method, realize that allogenic polypeptide enters intracellular, found by laser co-focusing into the more of intracellular Peptide is located in oil droplet；And the method is applied to two kinds of different polypeptide α-MSH and H22LP, two kinds of polypeptides enter containing for intracellular Measure no notable difference.

(3) the oral whole bacterial strain GM4-D6D- α MSH for carrying GLA and α-MSH, have found mice serum and vitals α-MSH content increase in (liver, lung, spleen, kidney), particularly liver improves 3.24 times；GLA's and GLA in serum and liver Next step product ARA content is also improved, and GLA contents improve about 7.26 times wherein in blood, and arachidonic acid carries It is high about 1.55 times；GLA contents relatively improve about 4.33 times in liver, and arachidonic acid improves about 1.28 times.

The characteristics of yeast existing unicellular microorganism, such as yeast smaller only several microns, easily culture, cell growth in itself It is fast etc., also there is feature possessed by eukaryotic, can such as carry out translation after the transcription of eukaryotic, without endotoxin, and And up to the present yeast studied on science of heredity and structure the characteristics of it is more clear, beneficial to carrying out genetic modification, Engineering bacteria is built, while yeast can realize industrialization large-scale production, be related to the launch of yeast at present.Yeast serves as Carrier, the transmission of unrighted acid is realized it has been reported that the unique distinction that we test is can by oral whole yeast Simultaneously deliver unrighted acid and polypeptide drugs, the special cell wall structure of yeast so that its have to gastral hydrochloric acid in gastric juice compared with Strong resistant function, good protection can be provided for large biological molecule as a kind of natural biological capsule, protect unsaturated lipid Fat is not oxidized, protects polypeptide drugs not to be degraded by enzymes.

Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent replacement mode is should be, is included within protection scope of the present invention.

Claims

1. a kind of rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides, it is characterised in that α-MSH polypeptides are imported and gone through Δ 6- In the rhodotorula glutinis of saturation enzyme gene restructuring.

A kind of 2. rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 1, it is characterised in that：Δ6- Desaturase is obtained by the separation of the D6D genes of cunninghamella echinulata with amplification.

A kind of 3. rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 2, it is characterised in that：Δ6- The rhodotorula glutinis construction method of delta 8 desaturase genes restructuring, the step of it includes, are as follows：

(1) expression vector pPGK1Z-rD-ME extracting；

(2) PGK1 genes and D6D genes are connected；

A kind of 4. rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 5, it is characterised in that：It is viscous red Yeast and the mol ratio of D6D genetic fragments will be controlled 1:3-10.

5. a kind of preparation method of the rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 1, its feature It is：Including the step of it is as follows：

(1) preparation engineering bacterial strain GM4-D6D bacterial strain competent cells；

(2) totally 100 μ L after mix after the dissolving of α-MSH polypeptides that 10 μ g have been modified with competent cell, are added to electricity and convert cup In；

(3) electric shockization；

(4) after electric shock terminates, the 1M sorbitol solutions of 900 μ L precoolings are added into conversion cup immediately, is slightly inhaled with rifle and plays mixing, Then gone in sterilized centrifuge tube, 30 DEG C of standing 1h, the fresh YPD culture mediums of 1mL added after centrifugation thalline is resuspended, 30 DEG C of temperature, 200rpm shake 1 hour；

(5) bacterial strain finally obtained.

6. a kind of preparation method of the rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 5, its feature It is：The electric shockization step is as follows：

(1) competent cell that oneself prepares by 80 μ L mixes with the 20 μ L DNAs to be transformed linearized, is added to In the electricity conversion cup of 0.2cm precoolings；

(3) parameter of electroporation is adjusted:Voltage 1.5kV, electric capacity 25uF, the Ω of resistance 200, electric shock duration are 5ms；

(4) after electric shock terminates, the 1M sorbitol solutions of 1mL precoolings are added into conversion cup immediately, is slightly inhaled with rifle and plays mixing, so Gone to afterwards in sterilized centrifuge tube, 30 DEG C of standing 1h, then add the fresh YPD culture mediums of 1mL, 30 DEG C of temperature, turn Fast 200rpm shakes 1 hour；

(6) liquid that will disappear is coated on YPD flat boards, is placed in 30 DEG C of incubators and cultivates 2-3d, untill growing single bacterium colony.

A kind of 7. rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 1, it is characterised in that：For Treat the application in inflammatory bowel disease medicine and functional food preparation.

A kind of 8. rhodotorula glutinis recombinant bacterial strain of importing α-MSH polypeptides according to claim 1, it is characterised in that：For Application in the food related to MSH effects or medicine preparation.