CN106967157A - A kind of pBpp albumen and its function verification method - Google Patents

Abstract

The invention provides a kind of pBpp albumen and its function verification method, the technical scheme is using zebra fish faecal microbiota DNA as template, amplification obtains pBpp expressing genes, it is carrier to recycle pET28c plasmids, prior to expanding plasmid in Escherichia coli TOP10, then it is transformed into e. coli bl21 and carries out protein expression.On this basis, the present invention devises suitable protein expression and inductive condition for the characteristic of recombinant bacterial strain, for the pBpp albumen of intracellular expression, the present invention is carried out after ultrasonication to thalline, free albumen is combined first with nickel bead, albumen wash-out is carried out again, PBS is finally utilized respectively and glycerine is dialysed, it is achieved thereby that effective purifying of pBpp albumen.Experiment finds, pBpp albumen provided by the present invention can definitely in inducing pancreatic β cells propagation so that the diabetes caused by being damaged because of beta Cell of islet play therapeutic effect.

Description

A kind of pBpp albumen and its function verification method

Technical field

The present invention relates to genetic engineering and Protein purification techniques field, and in particular to a kind of pBpp albumen and its functional verification Method.

Background technology

Diabetes be by inherent cause, immunologic function disorder, microorganism infection and its toxin, free radical toxin, spirit because The various virulence factors of element etc. act on sugar, protein, the fat that body causes hypoinsulinism, insulin resistance etc. and triggered A series of metabolic disorder syndromes such as fat, water and electrolyte, clinically using hyperglycaemia as main feature.

At present, the illness rate of China's type II diabetes increases very fast, according to the investigation in the population of China 300,000 in 1979, sugar The sick illness rate of urine is 0.6%, is within 1989 2.02%, and average annual to increase by 0.1% or so, China generally investigates 200,000 populations within 1994, suffers from Sick rate is had gone up as 2.5%, and diabetes prevalence is about 3% or so in current 20~75 years old crowd.Patients with impaired glucose tolerance is not Less than 3%.In general, the diabetes prevalence in China areas of well-being is higher than poverty-stricken area, city is higher than rural area, fat high In normal type, advanced age is higher than low age.Current China diabetes morbidity is 1/1000 or so, the average illness rate of more than 50 years old For more than 7%, illness rate is raised with advancing age within less than 40 years old, and the patient peak age was at 50~70 years old.

China's illness rate was higher with Beijing, Liaoning, Ningxia, Gansu, Yunnan, Fujian, according to 1979~1997 years investigation results It is shown to be first of the whole nation, and Xinjiang, Guizhou, Shanxi are relatively low.The higher Beijing of the incidence of disease, Liaoning and minimum Guizhou, Xinjiang it Between differ up to 10 times, the city incidence of disease is higher than 1~4 times of rural area, and Guangxi province investigation proves that the regional diabetes prevalence of production sugar is high In 2 times of Fei Chantang areas.

Although multiple therapy methods can control blood sugar concentration, the radical cure of diabetes is still not implemented at present.Prior art In, pharmaceutical intervention is still the Main Means for the treatment of diabetes, wherein more wide with the application such as GLP-1 analogs, DPPIV inhibitor It is general.PBpp albumen (promoting β-Cell proliferation protein), refer to can induce pancreas in Beta cell proliferation, And then promote a class functional protein of insulin secretion, because it does not participate in human body glycometabolism mechanism directly, therefore hypoglycemic is made It is smaller to human body side effect with more gentle.However, the large-scale production of this albuminoid is restrict its clinical practice at present straight Connection technology problem, the acquisition of wherein people's source protein is more difficult, and its drug effect of pBpp albumen and security in other biological source It is difficult to be protected again.

The content of the invention

It is contemplated that for prior art technological deficiency there is provided a kind of pBpp albumen and its function verification method, with Solve the technical problem that pBpp albumen in the prior art is difficult to prepare with scale.

Another technical problem to be solved by the present invention is that obtained with genetic engineering means, the restructuring expressed by Institute of Micro-biology PBpp albumen, it purifies more difficult.

The invention solves the problems that another technical problem be that pBpp albumen is imitated to the regulation of mammal blood glucose in the prior art Fruit is difficult to obtain accurately to quantify to investigate.

To realize above technical purpose, the present invention uses following technical scheme：

A kind of pBpp albumen, the pBpp albumen is prepared by following methods：

1) using the STb gene of zebra fish faecal microbiota as template, with sequence as shown in SEQ ID No.1 and sequence such as SEQ ID Fragment shown in No.2 is respectively that upstream and downstream primer performs PCR amplifications, obtains DNA piece of the sequence as shown in SEQ ID No.3 Section, as pBpp protein-encoding genes；

2) step 1 is taken) the pBpp protein-encoding genes that are obtained, it is connected on pET28c carriers, that is, obtains recombinant plasmid pET28c-pBpp；

3) take step 2) obtained by recombinant plasmid pET28c-pBpp, be transferred in E. coli Top10, obtain weight Group bacterial strain；

4) incubation step 3) obtained by recombinant bacterial strain, therefrom extract recombinant plasmid pET28c-pBpp, be transformed into large intestine Bacillus E.coli BL21, that is, obtain recombinant strains pET28c-pBpp-BL21；

5) incubation step 4) obtained by recombinant strains pET28c-pBpp-BL21, collect thalline；

6) step 5 is resuspended using EQ Buffer) gained thalline, ultrasonication, then with 10000rpm rotating speed centrifugation 10min, takes supernatant；Nickel bead is taken, is suspended in EQ Buffer, 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, suspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected, is resuspended in EQ Buffer, obtains nickel bead Suspension, is added into the supernatant, and 2h is kept under the conditions of 4 DEG C, then centrifuges 15min with 4000rpm rotating speed, is collected Precipitation, is suspended in Wash Buffer, then the normal temperature rotation 10min on rotation blending instrument, then with 4000rpm rotating speed 15min is centrifuged, precipitation is collected, is resuspended in EQ Buffer, then 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, is resuspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected；

7) take step 6) gained sediment be suspended in elution Buffer in, rotation blending instrument on normal temperature rotation 15min, with 4000rpm rotating speed centrifugation 15min, collects supernatant, then collect supernatant with 4000rpm rotating speed centrifugation 15min；

8) take step 7) obtained by supernatant, 30~60min is centrifuged with 13000rpm rotating speed, takes supernatant to load dialysis Bag, first with 1 × PBS dialysis 3h, then with the glycerine water solution dialysis 3h of 25% volumetric concentration, that is, obtains the pBpp Albumen.

Preferably, step 5) in be used for cultivate recombinant strains pET28c-pBpp-BL21 culture medium be containing The LB culture mediums of 100 μ g/ml ampicillins.

Preferably, step 5) in, the recombinant strains pET28c-pBpp-BL21 bacterium initially accessed into culture medium Liquid measure is the 1% of culture volume.

Preferably, step 5) in culture to nutrient solution OD values for 0.6 when add final concentration of 1mmol/L's thereto IPTG, induces 6~8h under the conditions of 25 DEG C, terminates culture.

Preferably, step 2) in the connection of pBpp protein-encoding genes and pET28c carriers be first double through NcoI and XhoI Digestion, digestion products are connected on pET28c using T4 ligases after purification.

Preferably, step 6) described in ultrasonication, until solution clarify untill.

Preferably, step 6) described in 15min is centrifuged with 4000rpm rotating speed, by centrifugation thing be fill in Centrifuged in 15ml centrifuge tubes.

Preferably, step 6) suspension of the nickel bead in EQ Buffer, the volume ratio of the two is 1:15.

Preferably, step 8) using 1 × PBS dialysis 3h during, successively change PBS 3 times.

Meanwhile, present invention also offers a kind of function verification method of above-mentioned pBpp albumen, comprise the following steps：

1) mouse is taken, continuous 5d injects Streptozotocin with daily 50mg/kg dosage, obtains type ii diabetes model small Mouse；

2) the pBpp albumen is taken, in tail vein injection mode to step 1) obtained by type ii diabetes model mice give Medicine, the change of blood sugar of the type ii diabetes model mice was monitored every 7 days.

The invention provides a kind of pBpp albumen and its function verification method, the technical scheme is with zebra fish faecal microbiota DNA is template, and amplification obtains pBpp expressing genes, and it is carrier to recycle pET28c plasmids, prior to being expanded in Escherichia coli TOP10 Plasmid, is then transformed into e. coli bl21 and carries out protein expression.On this basis, the present invention is directed to the characteristic of recombinant bacterial strain Suitable protein expression and inductive condition is devised, for the pBpp albumen of intracellular expression, it is broken that the present invention carries out ultrasound to thalline After broken, free albumen is combined first with nickel bead, then carries out albumen wash-out, PBS is finally utilized respectively and glycerine is dialysed, from And realize effective purifying of pBpp albumen.

On this basis, it is the hypoglycemic effect of investigation pBpp albumen, the present invention establishes corresponding function verification method, should Method builds type ii diabetes model mice first with Streptozotocin, and pBpp eggs are then carried out by the way of tail vein injection Give medicine free of charge, analyzed with mouse blood sugar situation of change after administration come the hypoglycemic effect to pBpp albumen.Experiment is found, of the invention The pBpp albumen provided can definitely in inducing pancreatic β cells propagation so that the glycosuria caused by being damaged because of beta Cell of islet Disease plays therapeutic effect.

Embodiment

The embodiment to the present invention is described in detail below.In order to avoid excessive unnecessary details, It is will not be described in detail in following examples to belonging to known structure or function.In addition to being defined, institute in following examples Technology and scientific terminology have the identical meanings being commonly understood by with those skilled in the art of the invention.

Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following examples；The experiment Method, is conventional method unless otherwise specified；Quantitative test in following examples, is respectively provided with three repetition experiments, as a result Average；% in following examples, is weight/mass percentage composition unless otherwise instructed.

In following examples, used E.coli Top10, E.coli BL21, pET28c vector plasmid is purchased from raw work Bioengineering (Shanghai) limited company；Used restriction enzyme, T4DNA ligases, DNA ligase are purchased from NEB Company；Used LB solid mediums and fluid nutrient medium is voluntarily prepared by this laboratory.

In following examples, each buffer formulation is as follows：

4L EQ Buffer：NaH2PO4.H2O 1.46g；Na2HPO4.7H2O:50.76g；Finally add water to 4L, filtering Degerming, it is 8.0 to adjust PH, sterilizing.

Wash Buffer：Plus 0.34g imidazoles is in 500ml EQ Buffer, ph is adjusted to be sterilized for 8.0..(now with the current).

Elute Buffer：Plus 1.7g imidazoles, to EQ buffer, final volume is 100ml, it is 8.0 to adjust pH, and sterilizing is (now with existing With).

100mM IPTG：2.4g IPTG are dissolved in 100ml water, filtration sterilization.

Embodiment 1

1st, the structure of pBpp Protein reconstitutions expression system

Using zebra fish faecal microbiota DNA as template, using SEQ ID No.1 and SEQ ID No.2 sequences as primer, PCR expands Increase the pBpp fragments shown in acquisition SEQ ID No.3, be named as pBpp.

Acquired pBpp fragments and plasmid pET28c, through NcoI and XhoI double digestions, digestion products after purification, using T4 Ligase is connected in pET28c (SEQ ID No.4) plasmid, and recombinant plasmid is named as pET28c-pBpp, and converts entrance In E.coli BL21, the recombinant strains of acquisition are named as pET28c-pBpp-BL21.

2nd, the expression and purification of pBpp albumen

(1) pBpp protein expressions：200ml LB culture mediums are prepared, 200ul ammonia benzyl mother liquors are added after cooling, make it dense eventually Spend for 100ug/ml；PET28c-pBpp-BL21 single bacterium colonies are selected to carry out staying overnight Shaking culture；Contain by the access of 1% inoculum concentration is fresh In the LB culture mediums for having ammonia benzyl antibiotic；Final concentration of 1mM IPTG are added when its OD is 0.6 to be induced, 25 DEG C continuously lure Thalline is centrifuged after leading culture 6-8h, -80 DEG C save backup.

(2) pBpp protein purifications：

A associated proteins：The thalline 35ml EQ Buffer of harvest in step (1) are suspended, it is saturating that clarification is presented in ultrasonication After bright solution, 10000rpm/min centrifugation 10min collect supernatant standby；1ml nickel beads are taken, nickel bead is resuspended with 15ml centrifuge tubes (filling 15mL), 4000rpm is centrifuged 15 minutes, is repeated twice；Nickel bead is resuspended with 1mL EQ Buffer；By the nickel bead washed Add in albumen supernatant, be placed in 4 degree and make nickel bead and protein binding 2h；4000rpm is centrifuged 15 minutes, abandons supernatant.Use Wash Nickel bead is resuspended in Buffer, is placed in 15ml centrifuge tubes, plus Wash Buffer are to 15ml, are placed on rotary apparatus in normal temperature Rotation 10 minutes, 4000rpm is centrifuged 15 minutes.Repeat the operation 3 times.

B elutes albumen：The precipitation harvested in step A is suspended with 1ml elutions Buffer, and at normal temperatures on gyroscope Rotation 15 minutes, 4000 leave the heart 15 minutes, and this step is repeated twice, and recovery cumulative volume is 2mL protein solutions, while reclaiming nickel Pearl.

C is washed：The supernatant of elution is transferred in 1.5ml centrifuge tubes, maximum (top) speed 13000rpm centrifuges 30-60min, small The heart takes supernatant, loaded in bag filter, using 1 × PBS 3h, changes PBS solution and is repeated 3 times；Finally use 25% glycerine Dialyse 3h, careful to draw albumen supernatant, determines after concentration, packing, -80 freeze it is standby, and using SDS-PAGE glue checking albumen Size and purity；

3rd, the functional authorization of pBpp albumen

Reagent is configured：0.1M citrate buffers (PH4.5)：(0.21014 citric acid is dissolved in 0.1M citron acid solutions 4.5mL In 10mL distilled water)+0.1M sodium citrate liquid 5.5mL (0.29412 sodium citrate is dissolved in 10mL distilled water)；7.5mg/mL nothing Bacterium STZ solution (takes 37.5mgSTZ to be dissolved in 5mL citrate buffers, filtered with super-clean bench 0.22um filters, packing is stored in -20 DEG C ) (attention lucifuge)

Experiment flow：

(1) dosage and administering mode：Positive group：50mg/kg/ days STZ solution；Negative group：With positive group Isodose Chinese holly Rafter acid buffer；

Administering mode：Intraperitoneal injection；

(2) 12 healthy male wister rats are taken, 24h is observed, it is without exception to be tested afterwards；Mouse is randomly divided into 2 groups, weighs； Water is can't help in Nature enemy 24h before being administered for the first time, fasting；After weighing, after positive group dose lonvestion, with abdominal cavity in super-clean bench STZ medicines are injected, after having beaten, feed are normally given, sterilized water is fed, continuous 5 days same time injection STZ medicines；And in beating The blood glucose after mouse empty stomach 8h and the then blood glucose after feeding 2h are determined with test paper within complete 3rd, 7,10,14,21,28 day, if positive Group is more than 11.1mmol/L, then modeling success；

(3) after modeling success, daily tail vein injection pBpp albumen, frequency 2 days once, normal diet；Negative group, tail is quiet Arteries and veins injects sterile PBS and is used as control；

(4) at the 7th day, the 14th day, blood glucose after the 21st day detection mouse empty stomach 8h and the then blood glucose after feeding 2h are seen Examine blood glucose whether there is decline；

Embodiment 2

A kind of pBpp albumen, the pBpp albumen is prepared by following methods：

1) using the STb gene of zebra fish faecal microbiota as template, with sequence as shown in SEQ ID No.1 and sequence such as SEQ ID Fragment shown in No.2 is respectively that upstream and downstream primer performs PCR amplifications, obtains DNA piece of the sequence as shown in SEQ ID No.3 Section, as pBpp protein-encoding genes；

2) step 1 is taken) the pBpp protein-encoding genes that are obtained, it is connected on pET28c carriers, that is, obtains recombinant plasmid pET28c-pBpp；

3) take step 2) obtained by recombinant plasmid pET28c-pBpp, be transferred in E. coli Top10, obtain weight Group bacterial strain；

4) incubation step 3) obtained by recombinant bacterial strain, therefrom extract recombinant plasmid pET28c-pBpp, be transformed into large intestine Bacillus E.coli BL21, that is, obtain recombinant strains pET28c-pBpp-BL21；

5) incubation step 4) obtained by recombinant strains pET28c-pBpp-BL21, collect thalline；

6) step 5 is resuspended using EQ Buffer) gained thalline, ultrasonication, then with 10000rpm rotating speed centrifugation 10min, takes supernatant；Nickel bead is taken, is suspended in EQ Buffer, 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, suspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected, is resuspended in EQ Buffer, obtains nickel bead Suspension, is added into the supernatant, and 2h is kept under the conditions of 4 DEG C, then centrifuges 15min with 4000rpm rotating speed, is collected Precipitation, is suspended in Wash Buffer, then the normal temperature rotation 10min on rotation blending instrument, then with 4000rpm rotating speed 15min is centrifuged, precipitation is collected, is resuspended in EQ Buffer, then 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, is resuspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected；

7) take step 6) gained sediment be suspended in elution Buffer in, rotation blending instrument on normal temperature rotation 15min, with 4000rpm rotating speed centrifugation 15min, collects supernatant, then collect supernatant with 4000rpm rotating speed centrifugation 15min；

8) take step 7) obtained by supernatant, 30~60min is centrifuged with 13000rpm rotating speed, takes supernatant to load dialysis Bag, first with 1 × PBS dialysis 3h, then with the glycerine water solution dialysis 3h of 25% volumetric concentration, that is, obtains the pBpp Albumen.

On the basis of above technical scheme, following condition is met：

Step 5) in be used to cultivate recombinant strains pET28c-pBpp-BL21 culture medium be containing 100 μ g/ml ammonia The LB culture mediums of parasiticin.

Step 5) in, the recombinant strains pET28c-pBpp-BL21 bacterium solutions amount initially accessed into culture medium is culture The 1% of matrix product.

Step 5) in culture to nutrient solution OD values for 0.6 when add final concentration of 1mmol/L IPTG thereto, in 25 DEG C Under the conditions of induce 6~8h, terminate culture.

Step 2) in the connection of pBpp protein-encoding genes and pET28c carriers be first through NcoI and XhoI double digestions, digestion It is connected to after product purification using T4 ligases on pET28c.

Step 6) described in ultrasonication, until solution clarify untill.

Step 6) described in 15min is centrifuged with 4000rpm rotating speed, by centrifugation thing filled in 15ml centrifuge tubes Centrifuged.

Step 6) suspension of the nickel bead in EQ Buffer, the volume ratio of the two is 1:15.

Step 8) using 1 × PBS dialysis 3h during, successively change PBS 3 times.

A kind of above-mentioned function verification method for stating pBpp albumen, it is characterised in that comprise the following steps：

1) mouse is taken, continuous 5d injects Streptozotocin with daily 50mg/kg dosage, obtains type ii diabetes model small Mouse；

2) the pBpp albumen is taken, in tail vein injection mode to step 1) obtained by type ii diabetes model mice give Medicine, the change of blood sugar of the type ii diabetes model mice was monitored every 7 days.

Embodiment 3

A kind of pBpp albumen, the pBpp albumen is prepared by following methods：

1) using the STb gene of zebra fish faecal microbiota as template, with sequence as shown in SEQ ID No.1 and sequence such as SEQ ID Fragment shown in No.2 is respectively that upstream and downstream primer performs PCR amplifications, obtains DNA piece of the sequence as shown in SEQ ID No.3 Section, as pBpp protein-encoding genes；

2) step 1 is taken) the pBpp protein-encoding genes that are obtained, it is connected on pET28c carriers, that is, obtains recombinant plasmid pET28c-pBpp；

3) take step 2) obtained by recombinant plasmid pET28c-pBpp, be transferred in E. coli Top10, obtain weight Group bacterial strain；

4) incubation step 3) obtained by recombinant bacterial strain, therefrom extract recombinant plasmid pET28c-pBpp, be transformed into large intestine Bacillus E.coli BL21, that is, obtain recombinant strains pET28c-pBpp-BL21；

5) incubation step 4) obtained by recombinant strains pET28c-pBpp-BL21, collect thalline；

6) step 5 is resuspended using EQ Buffer) gained thalline, ultrasonication, then with 10000rpm rotating speed centrifugation 10min, takes supernatant；Nickel bead is taken, is suspended in EQ Buffer, 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, suspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected, is resuspended in EQ Buffer, obtains nickel bead Suspension, is added into the supernatant, and 2h is kept under the conditions of 4 DEG C, then centrifuges 15min with 4000rpm rotating speed, is collected Precipitation, is suspended in Wash Buffer, then the normal temperature rotation 10min on rotation blending instrument, then with 4000rpm rotating speed 15min is centrifuged, precipitation is collected, is resuspended in EQ Buffer, then 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, is resuspended 15min is centrifuged in EQ Buffer, then with 4000rpm rotating speed, precipitation is collected；

7) take step 6) gained sediment be suspended in elution Buffer in, rotation blending instrument on normal temperature rotation 15min, with 4000rpm rotating speed centrifugation 15min, collects supernatant, then collect supernatant with 4000rpm rotating speed centrifugation 15min；

8) take step 7) obtained by supernatant, 30~60min is centrifuged with 13000rpm rotating speed, takes supernatant to load dialysis Bag, first with 1 × PBS dialysis 3h, then with the glycerine water solution dialysis 3h of 25% volumetric concentration, that is, obtains the pBpp Albumen.

Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention, It is not intended to limit the invention.All any modifications, equivalent substitutions and improvements done in the application range of the present invention etc., all should Within protection scope of the present invention.

SEQUENCE LISTING

<110>University Of Nanchang

<120>A kind of pBpp albumen and its function verification method

<130> 2017

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 36

<212> DNA

<213>Artificial sequence

<400> 1

cgtgccatgg ggatgaacaa gcgtaactgg ttgctg 36

<210> 2

<211> 31

<212> DNA

<213>Artificial sequence

<400> 2

taagctcgag gcggctcgtt tcagtcaagt c 31

<210> 3

<211> 783

<212> DNA

<213>Native sequences（Zebra fish）

<400> 3

atgaacaagc gtaactggtt gctggccttg tcactgagtc tggccttctc accctgttat 60

gccgactggg ccaagctcaa ggccgcagcg tccgatctgg gagcggcagt gagcgagacc 120

agcaaggagg tgtggcagga tgtcagcgat ttctccaaga agagctgggc ttccatctct 180

gcatggggtg aagaggcgtt caataccgcc ggggtctgga ccgacaagag catcgcgacc 240

ggcaaggagt ggctgaaggc agccgacaag gagctcaacg agatgctcaa ccccaagacg 300

gcgaaagagg cgaggatcgc gatcaatacc atggccgaca ctgcgctgat ccggttgttc 360

aacgagcagc catcagccaa gttgttgttt gacaaggctt atggttatgc ggtgttcgat 420

tcgcgcaagt tctccctgat gctgcatacc aatcaggggg ccggggtggc agtcaatcgc 480

aaaaccggca agcacaccta tatgaagatg tttggtgcgg gtctggcggc cgggattggc 540

ggcaagttct atcagcaggt gatcctgttt gaagacaagg ccagattcga tgccttcgtg 600

acccaggggt gggaagctac ctccgaagtg ggtgtggtgg ccggcaagga gagcgctgag 660

ctgaccgcca aatataatgg cggcatggcc atctaccaga ttggcgagaa gggtttgctg 720

ctcgatgcca atatctccgg ctctaaatac tggattgaca aggacttgac tgaaacgagc 780

cgc 783

<210> 4

<211> 5367

<212> DNA

<213>Artificial sequence

<400> 4

atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60

ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120

tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt 180

cgacggagct cgaattcgga tccgacccat ttgctgtcca ccagtcatgc tagccatatg 240

gctgccgcgc ggcaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatggt 300

atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca 360

attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg 420

acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga 480

catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt 540

gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc 600

attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca 660

ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc 720

gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag 780

taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg 840

tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg 900

agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga 960

ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta 1020

aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg 1080

tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca 1140

ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc 1200

cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg 1260

aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc 1320

tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat 1380

atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt 1440

ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg 1500

ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg 1560

ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc 1620

cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct 1680

tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg 1740

tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 1800

attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 1860

gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga 1920

gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 1980

atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt 2040

ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc 2100

ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc 2160

gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg 2220

tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg 2280

atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca 2340

tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc 2400

accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca 2460

tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc 2520

cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta 2580

acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat 2640

cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc 2700

tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa 2760

caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc 2820

gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 2880

gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 2940

ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 3000

ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc ggtgtgaaat 3060

accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac 3120

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3180

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3240

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3300

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 3360

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 3420

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 3480

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 3540

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 3600

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 3660

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 3720

aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 3780

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 3840

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 3900

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt 3960

ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt 4020

gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 4080

gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc 4140

cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 4200

tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 4260

ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat 4320

tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 4380

ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 4440

ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 4500

gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac 4560

cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 4620

aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 4680

ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 4740

aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 4800

ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa 4860

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt 4920

ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 4980

atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca 5040

gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 5100

gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg 5160

aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 5220

ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 5280

gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg 5340

ccgctacagg gcgcgtccca ttcgcca 5367

Claims (10)

1. a kind of pBpp albumen, it is characterised in that the pBpp albumen is prepared by following methods：

1) using the STb gene of zebra fish faecal microbiota as template, with sequence as shown in SEQ ID No.1 and sequence such as SEQ ID Fragment shown in No.2 is respectively that upstream and downstream primer performs PCR amplifications, obtains DNA piece of the sequence as shown in SEQ ID No.3 Section, as pBpp protein-encoding genes；

2) step 1 is taken) the pBpp protein-encoding genes that are obtained, it is connected on pET28c carriers, that is, obtains recombinant plasmid pET28c-pBpp；

3) take step 2) obtained by recombinant plasmid pET28c-pBpp, be transferred in E. coli Top10, obtain recombinant bacterium Strain；

4) incubation step 3) obtained by recombinant bacterial strain, therefrom extract recombinant plasmid pET28c-pBpp, be transformed into Escherichia coli E.coli BL21, that is, obtain recombinant strains pET28c-pBpp-BL21；

5) incubation step 4) obtained by recombinant strains pET28c-pBpp-BL21, collect thalline；

6) step 5 is resuspended using EQ Buffer) gained thalline, ultrasonication, then with 10000rpm rotating speed centrifugation 10min, Take supernatant；Nickel bead is taken, is suspended in EQ Buffer, 15min is centrifuged with 4000rpm rotating speed, precipitation is collected, is suspended in EQ In Buffer, then with 4000rpm rotating speed centrifugation 15min, precipitation is collected, is resuspended in EQ Buffer, obtains nickel bead suspension, It is added into the supernatant, 2h is kept under the conditions of 4 DEG C, 15min is then centrifuged with 4000rpm rotating speed, collects precipitation, It is suspended in Wash Buffer, then the normal temperature rotation 10min on rotation blending instrument, is then centrifuged with 4000rpm rotating speed 15min, collects precipitation, is resuspended in EQ Buffer, then centrifuges 15min with 4000rpm rotating speed, collects precipitation, is resuspended in EQ In Buffer, then with 4000rpm rotating speed centrifugation 15min, collect precipitation；

7) take step 6) gained sediment be suspended in elution Buffer in, rotation blending instrument on normal temperature rotation 15min, with 4000rpm rotating speed centrifugation 15min, collects supernatant, then collect supernatant with 4000rpm rotating speed centrifugation 15min；

8) take step 7) obtained by supernatant, 30~60min is centrifuged with 13000rpm rotating speed, takes supernatant to load bag filter, first Using 1 × PBS dialysis 3h, then with the glycerine water solution dialysis 3h of 25% volumetric concentration, that is, obtain the pBpp albumen.

2. a kind of pBpp albumen according to claim 1, it is characterised in that step 5) in be used to cultivate recombinant strains PET28c-pBpp-BL21 culture medium is the LB culture mediums containing 100 μ g/ml ampicillins.

3. a kind of pBpp albumen according to claim 2, it is characterised in that step 5) in, initially accessed into culture medium Recombinant strains pET28c-pBpp-BL21 bacterium solutions amount is the 1% of culture volume.

4. a kind of pBpp albumen according to claim 2, it is characterised in that step 5) in culture to nutrient solution OD values be 0.6 When add final concentration of 1mmol/L IPTG thereto, 6~8h is induced under the conditions of 25 DEG C, terminates culture.

5. a kind of pBpp albumen according to claim 1, it is characterised in that step 2) in pBpp protein-encoding genes with The connection of pET28c carriers is that, first through NcoI and XhoI double digestions, digestion products are connected to pET28c using T4 ligases after purification On.

6. a kind of pBpp albumen according to claim 1, it is characterised in that step 6) described in ultrasonication, until molten Untill liquid is clarified.

7. a kind of pBpp albumen according to claim 1, it is characterised in that step 6) described in the rotating speed with 4000rpm 15min is centrifuged, is filled and is centrifuged in 15ml centrifuge tubes by centrifugation thing.

8. a kind of pBpp albumen according to claim 7, it is characterised in that step 6) suspension of the nickel bead in EQBuffer, The volume ratio of the two is 1:15.

9. a kind of pBpp albumen according to claim 1, it is characterised in that step 8) utilize 1 × PBS dialysis 3h During, successively change PBS 3 times.

10. the function verification method of pBpp albumen described in a kind of claim 1, it is characterised in that comprise the following steps：

1) mouse is taken, continuous 5d injects Streptozotocin with daily 50mg/kg dosage, obtains type ii diabetes model mice；

2) the pBpp albumen is taken, in tail vein injection mode to step 1) obtained by type ii diabetes model mice administration, often The change of blood sugar of the type ii diabetes model mice was monitored every 7 days.