CN104818227B - A kind of food grade lactic acid galactococcus of surface anchoring people I type metallothioneins and preparation method and application - Google Patents
A kind of food grade lactic acid galactococcus of surface anchoring people I type metallothioneins and preparation method and application Download PDFInfo
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Abstract
Food grade lactic acid galactococcus and preparation method and application the present invention relates to a kind of surface anchoring people I type metallothioneins, step are as follows：(1) by over-lap PCR by cA genes and hMT gene splicings, linker is introduced between two genes, N-terminal is introduced and promotees expression label, obtains recombinating cA hMT genes；(2) restructuring cA hMT genes and expression vector are attached, the connection product for obtaining is converted to competent escherichia coli cell；(3) the engineering bacteria induced expression recombination fusion protein for obtaining step (2), is centrifuged after harvesting thalline ultrasonication, and supernatant is directly incubated with food grade lactic acid galactococcus, and fusion protein is then anchored into Lactococcus lactis surface without purifying.The food grade lactic acid bacterium of this surface anchoring someone I type metallothioneins prepared by the inventive method, can enriching heavy metal, preparation method have the advantages that efficiently, safety non-toxic, low cost, be not required to protein purification.
Description
Technical field
The invention belongs to the DNA recombinant expression technical field of genetic engineering field, and in particular to a kind of surface anchoring someone
The food grade lactic acid galactococcus preparation method and application of I type metallothioneins.
Background technology
Metallothionein (Metallothionein, MT) has very conservative primary structure and similar space structure,
Be distributed widely in animal, plant and microorganism, be a class rich in cysteine, low-molecular-weight (6000~7000 dalton),
Protein (Vasak M.Advances in metallothionein with stronger bind metal ion ability
structure and functions.J Trace Elem Med Biol,2005,l19:13–17.).MT has a combination huge sum of money
Important physiological function (the Yang F et such as category, removing free radical, radioresistance, anti-aging and regulating microelement balance
al.High-yield expression in Escherichia coli of soluble human MT2A with
native functions.Protein Expr Purif,2007,53:186–194.).At present, MT has turned into medical science, biology
The hot research topic of the scientific research fields such as chemistry, molecular biology and environmental protection.Simultaneously because the efficient heavy metal solutions of MT
Malicious function and security, have been widely used in the production fields such as medical treatment, cosmetics, health products and environmental protection.
Soluble M T can be obtained using heavy metal induced animal, but yielded poorly and purification step complexity is cumbersome, cause production
Cost is high, limits the application of MT.Because MT molecular weight is small, cysteine content is high, expresses easily being formed in protokaryon system
Inclusion body, there is not yet largely producing report (the Butt TR et al.Ubiquitin fusion of soluble M T with Escherichia coli
augments the yield of cloned gene products in Escherichia coli.Proc Natl Acad
Sci USA,1989,86:2540-2544).At present, using the dissolution labels such as GST, Sumo recombination expression MT albumen (Huang
Y et al.Expression and purification of glutathione transferase-small
ubiquitin-related modifier-metallothionein fusion protein and its neuronal
and hepatic protection against D-Galactose-induced oxidative damage in mouse
model,JPET,2009,329:469–478.)。
Lactic acid bacteria (Lactic acid bacteria) is the probiotics in a kind of human body, is widely used in dairy produce life
Produce, the aspect such as meat, vegetables, fermentation of bread, therefore be generally considered safe level microbe (Generally Recognized
As Safe,GRAS；Gilliland SE.Health and nutritional benefits from lactic acid
bacteria.FEMS MicrobiolRev.1990,7(1-2):175-188).Since the eighties in 20th century, people begin to
It is devoted to entering the biological characteristics and molecular mechanism of various lactic acid bacterias especially Lactococcus lactis (Lactococcuslactis)
Row research.Lactococcus lactis have its unique advantage as the desirable strain of surface display foreign protein：First, its own secretion
Protein it is few, extracellular protease it is active low, the expression of target protein will not be subject to Lactococcus lactis oneself protein matter
Influence；Secondly, antigenicity is weak, will not produce endotoxin, and strong immune response will not be caused into human body；Finally, lactic acid breast
Coccus does not colonize in intestines and stomach, can avoid because its colonize for a long time caused by immune tolerance (Feng Jin, Lactococcus lactis surface
Display technique, the chemistry of life, 2013,33 (1):91-95).Therefore Lactococcus lactis express place as a kind of food-grade
It is mainly used to expression and the various antigens of surface display, growth factor and functional protein (Ribeiro LA, et al.Production
and targeting of the Bmedhabortus antigen L7/L12in Lactococcuslactis:a first
step towards food-grade live vaccines against Brucellosis,Applied and
Environmental Microbiology 2002,68(2):910-916).But there is table using lactic acid bacteria expression alien gene
Up to measure it is low, recombinant lactic acid bacteria can introduce foreign gene, using antibiotic can be introduced as selection markers resistant gene etc. some not
Hold the problem ignored.
The content of the invention
It is an object of the invention to provide a kind of food grade lactic acid galactococcus system of surface anchoring someone I type metallothioneins
Preparation Method, can be anchored on feature people's I type metallothioneins on Lactococcus lactis surface in E. coli first
In vain (hMT), it is not required to that hMT is anchored into food grade lactic acid bacterium surface by by cumbersome steps such as purifying after bacteria breaking.
The present invention provides the Lactococcus lactis after a kind of grappling hMT simultaneously, and the Lactococcus lactis do not carry any resistance base
Cause and foreign gene, therefore can safely be applied to the industries such as food, health products and cosmetics.
There are the Lactococcus lactis of hMT simply and efficiently to obtain grappling, and can large-scale application, the technology of the present invention
Scheme is as follows：
A kind of food grade lactic acid galactococcus preparation method of surface anchoring people I type metallothioneins, comprises the following steps：
(1) the recombination, amalgamation and expression carrier of the people's I type metallothioneins containing anchor unit is built：
By over-lap PCR by cA genes and hMT gene splicings, linker is introduced between two genes, N-terminal is introduced and promotees expression mark
Sign, obtain recombinating cA-hMT genes；The nucleotide sequence of cA genes such as SEQ ID NO:Shown in 1, the nucleotide sequence of hMT genes
Such as SEQ ID NO:Shown in 2, the nucleotide sequence such as SEQ ID NO of linker:Shown in 3；
(2) will restructuring cA-hMT genes and expression vector pET21a connections, the connection product pET21a-cA-hMT for obtaining turns
Change to competent escherichia coli cell；
(3) the engineering bacteria induced expression recombination fusion protein cA-hMT for obtaining step (2), harvests ultrasonication after thalline
Cell, supernatant is directly incubated with Lactococcus lactis, and fusion protein cA-hMT is then anchored into phage surface without purifying.
To improve soluble protein expression quantity during step (2) expression of recombinant proteins, also need to add molecular chaperones, specific steps are such as
Under：
Connection product pET21a-cA-hMT described in step (2) and the expression vector containing molecular chaperones are successively converted
To the expression bacterial strain of Escherichia coli.
The molecular chaperones is dnaK, one or more in dnaJ, grpE, groES and groEL.
Except wet thallus can directly in addition to grappling, the wet thallus to the Lactococcus lactis add acetic acid (pH=1) to process and boil
Grappling is carried out with recombination fusion protein cA-hMT again after 30min, PBS washing, recombination fusion protein is remarkably improved and is anchored into breast
The efficiency of sour bacterium.
The step of step (3) induction engineering bacterium expression recombination fusion protein cA-hMT, is as follows：
A. engineering bacteria is inoculated in the LB liquid medium containing ampicillin and chloramphenicol, is shaken in 37 DEG C, 180rpm
Overnight, then by 1：During 100 ratio is inoculated in containing ampicillin, chloramphenicol, the LB liquid medium of L-arabinose,
The isopropylthiogalactoside (IPTG) of final concentration of 0.5~1.0mM is added when OD is up to 0.4~0.8, it is low in 200rpm
Temperature continues to collect bacterium solution after cultivating 12h, is centrifuged, and removes supernatant；
B. PBS washing step a gained thalline is used 2 times, resuspended with bacterial lysate, ultrasound is broken under being placed in ice bath
It is broken, the centrifugation of the homogenate after ultrasound is taken, supernatant and inclusion body are collected respectively.
The step of fusion protein cA-hMT is anchored to Lactococcus lactis surface by step (3) is as follows：
Supernatant is resuspended with Lactococcus lactis, 1h is stored at room temperature, 9000g centrifugations 2min abandons supernatant, precipitation PBS solution
Washing 3 times, resuspended to 4 DEG C preservations.
Step (3) described Lactococcus lactis are galactococcus MG1363, MG1614, LM0230, enterococcus faecalis FA2-2, OGIX
Or Lactobacillus plantarum.
The incubation step of the Lactococcus lactis is as follows：
Lactococcus lactis are cultivated in GM17 fluid nutrient mediums, 30 DEG C of anaerobism quiescent cultures to exponential phase late period or
Plateau, OD600nmUp to 3~4, room temperature 9000g centrifugation 5min, wet thallus are collected.
The food grade lactic acid galactococcus of surface anchoring people I type metallothioneins obtained in the above method can make an addition to food,
Cosmetics and field of environment protection, for human body heavy metal detoxification, remediating heavy metal oxidative damage, metal ion Metabolism regulation with
And pollutant treatment and environment remediation.
The lactic acid bacteria that grappling has hMT is carried out into Cd2+、Pb2+Adsorption experiment
After hMT is anchored into Lactococcus lactis by the anchored mode of step 3, the Cd of 1mL is added2+Or Pb2+(100 μM) weights
Outstanding, room temperature places 2h, and period fully mixes, and makes the abundant Adsorption of Heavy Metals of albumen.8000rpm is centrifuged 1min, abandons supernatant, and thalline is used
PBS is put into 65 DEG C of drying boxes and dries 12h after washing 3 times, after the dry mycelium for obtaining is weighed, overnight thoroughly digested with 60% nitric acid.
Plus ddH2O is diluted to 5mL for Atomic Absorption Spectrometry.
The present invention compared with prior art, has the following advantages that：
(1) present invention utilizes molecular chaperones (dnaK or dnaJ or grpE or groES or groEL) coexpression system in large intestine
Expression hMT recombinant proteins can be melted in bacillus, total expression quantity reaches 176mg/L, and wherein soluble recombinant protein reaches 134mg/L, solvable
Property expression quantity considerably beyond document report containing GST, SUMO fusion tag restructuring MT albumen.
(2) it is centrifuged after carrying out ultrasonication to restructuring thalline, supernatant directly can carry out grappling with lactic acid bacteria, eliminate
The tedious steps that recombinant protein is isolated and purified, reduce the loss of albumen.
(3) breaking the part recombinant protein inclusion body after bacterium can be dissolved with 8M urea, correctly be folded through dialysis renaturation, multiple
Property rate is up to 82%, and the recombinant protein after renaturation also can carry out grappling with lactic acid bacteria.
(4) anchorin that the present invention is used is the C-terminal of Lactococcus lactis N- acetyl muramidases, can be with Gram-positive
The cell membrane of bacterium carries out specific non-covalent combination, and its grappling Host Strains can be not limited to one kind.
(5) lactic acid bacteria of grappling hMT is food-grade microorganisms, both delivers the harm to environment without foreign gene,
Any resistant gene is not carried.
The preparation method of lactic acid bacteria surface display metallothionein of the present invention have efficiently, safety non-toxic, into
The advantages of this is low and is not required to purifying, so as to be the extensive preparation and its application of the foods and cosmetics of metallothionein lactic acid bacteria
Lay the foundation.
Brief description of the drawings
Fig. 1 pET21a-cA-hMT construction of recombinant plasmid figures；
Fig. 2 recombinant plasmid double digestion qualification figures；Wherein：Swimming lane M:DNA Marker, swimming lane 1：PET21a-cA-hMT is recombinated
Plasmid double digestion；
The SDS-PAGE identifications of Fig. 3 restructuring cA-hMT expression；Wherein：Swimming lane M：Protein Marker, swimming lane 1：Do not lure
Lead full bacterium, swimming lane 2：Supernatant, swimming lane 3 are not induced：Non- induced precipitation, swimming lane 4：Induce full bacterium, swimming lane 5：Induction supernatant, swimming lane 6：
Induced precipitation；
Fig. 4 restructuring cA-hMT is anchored into before and after lactic acid bacteria total protein SDS-PAGE electroresis appraisals in supernatant；Wherein：Swimming lane
M：Protein Marker, swimming lane 1：Non- grappling supernatant, swimming lane 2：Supernatant after grappling；
Fig. 5 restructuring cA-hMT is anchored into ELISA detections after lactic acid bacteria；Wherein：1：The lactic acid bacteria of surface anchoring hMT, 2：hMT
Recombinant protein, 3：Lactic acid bacteria 4：PBS solution；
Fig. 6 restructuring cA-hMT is anchored into the detection of lactic acid bacteria rear stability；
Fig. 7 restructuring cA-hMT is anchored into after lactic acid bacteria to Cd2+(a)、Pb2+(b) absorption detection；Wherein：1：Active lactic acid
Bacterium, 2：The biodiasmin of surface anchoring GFP, 3：The biodiasmin of surface anchoring hMT, 4：Lactic acid bacteria after acid treatment, 5：
The acid treatment lactic acid bacteria of surface anchoring GFP, 6：The acid treatment lactic acid bacteria of surface anchoring hMT.
Specific embodiment
The present invention is described in detail hereinafter with reference to the drawings and specific embodiments.
Embodiment 1
Recombinate the structure of cA-hMT protein engineering bacterium
CA genes are spliced with hMT genes by over-lap PCR, linker is introduced between two genes, N-terminal is introduced and promotees table
Label up to standard, are connected after NdeI and XhoI double digestions with expression vector pET21a, you can realize recombinant plasmid pET21a-cA-hMT
Structure, its specific building process is as shown in Figure 1.
1. genes of interest fragment, the design of primer and synthesis
According to Lactococcus lactis MG1363N- acetyl muramidase gene orders (GenBank ID:CAL96887.1), exist
Its C-terminal introduces linker (SEQ ID No.3), thus designs primer, and wherein forward primer is F1:
5’-AATTCCATATG(underscore is marked for GGATCCAATACTAATTCTGGTGGCTC-3 ', NdeI containing restriction enzyme site
Part), reverse primer is R1:
5 '-TGAACAATTAGGATCCATCTGCAGACCACCACCTTTTATTCGTAGATACT-3 ', contain linker sequences
Row (italicized item is linker complementary series).
According to people's I types metallothionein sequence (SEQ ID No.2), linker sequences are introduced in its N-terminal, thus design is drawn
Thing, wherein forward primer are F2:
5 '-CAGTATCTACGAATAAAAGGTGGTGGTCTGCAGATGGATCCTAATTGTTC-3 ', italicized item is
Linker sequences, reverse primer is R2:
5’-CCACTCGAGAGCACAACAGGAACACT-3 ', contains restriction enzyme site XhoI (underscore mark part).
2.PCR is expanded and construction of recombinant plasmid
With Lactococcus lactis MG1363 genomes as template, with the nucleotides sequence row shown in F1, R1 for upstream and downstream primer enters
Performing PCR expands cA, and its reaction system is as follows：
PCR amplification programs are as follows：
Gained PCR primer is stand-by in 4 DEG C of preservations, 1% agarose gel electrophoresis identification PCR primer, obtains size for 713bp
The genetic fragment comprising restriction enzyme site and linker sequences, to this fragment gel extraction.
With pET28a-hMT as template, expanded for upstream and downstream primer enters performing PCR with the nucleotides sequence row shown in F2, R2
HMT, its reaction system is as follows：
PCR amplification programs are as follows：
Gained PCR primer is stand-by in 4 DEG C of preservations, 1% agarose gel electrophoresis identification PCR primer, obtains size for 225bp
The genetic fragment comprising restriction enzyme site and linker sequences, to this fragment gel extraction.
With cA, hMT as template, with the nucleotides sequence row shown in F1, R2 for upstream and downstream primer carries out over-lap PCR amplification cA-
HMT, its reaction system is as follows：
PCR amplification programs are as follows：
Gained overlapping PCR products are stand-by in 4 DEG C of preservations, 1% agarose gel electrophoresis identification PCR primer, and obtaining size is
The genetic fragment comprising restriction enzyme site and linker sequences of 888bp, to this fragment gel extraction.
Above-mentioned gained overlapping PCR products with NdeI and XhoI in 37 DEG C of double digestions 5h, pET21a also with NdeI and XhoI in
37 DEG C of double digestion 3h.Double digestion system is as follows：
Gel extraction double digestion product, 10h is connected in 16 DEG C, and linked system is as follows：
Gained connection product conversion competent escherichia coli cell DH5 α.
3. recombinant plasmid identification
Method：Above-mentioned connection product conversion competent escherichia coli cell DH5 α, 9 monoclonal Amplification Cultures of picking, and
Carry out bacterium solution PCR identifications, PCR conditional synchronizations rapid 2 choose the clone for being accredited as the positive through bacterium solution PCR and extract plasmid, NdeI and
37 DEG C of double digestion 2h of XhoI, double digestion product enters row agarose gel electrophoresis identification.Select corresponding positive colony sample presentation to China
Big gene carries out sequencing identification.
As a result：As shown in Fig. 2 being obtained after recombinant plasmid double digestion and pET21a large fragments of the same size and and cA-hMT
The consistent small fragment of gene size, its double digestion collection of illustrative plates is as shown in Figure 2, it was demonstrated that this clone is positive colony.Sequencing result with it is pre-
Phase nucleotide sequence is completely the same.
Conclusion：PET21a-cA-hMT recombinant plasmids successfully build.
Embodiment 2
The expression and identification of cA-hMT recombinant proteins
Method：Positive colony in above-described embodiment 1 extract the plasmid that obtains with containing molecular chaperones (dnaK, dnaJ,
GrpE, groES and groEL) plasmid converted into E. coli expression strains competent cell BL21 (DE3), and picking is through bacterium colony
The positive colony of PCR identifications is seeded to 3mL and contains 100 μ g/mL Amp, 25 μ g/mL chloramphenicol, 1mg/mL L-arabinoses
37 DEG C of Amplification Cultures in LB fluid nutrient mediums, in OD600For 0.5 when add 0.5mM IPTG, be collected by centrifugation after low temperature induction 12h
Thalline, ultrasonic degradation, supernatant precipitation is separately added into sample-loading buffer boiling water boiling 5min.Glue 80V is concentrated, separation gel 120V is carried out
SDS-PAGE electrophoresis, electrophoresis is finished and dyeed with coomassie brilliant blue staining liquid, and destainer decolourizes.
As a result：As shown in figure 3, restructuring cA-hMT albumen successfully obtains induced expression.Restructuring cA-hMT is precipitated in supernatant
In be all distributed, wherein soluble restructuring cA-hMT albumen accounts for the 39.0% of supernatant total protein.
Conclusion：The success of cA-hMT expression of recombinant proteins.
Embodiment 3
Restructuring hMT fusion proteins are anchored into lactic acid bacteria and detection
1. the culture of Lactococcus lactis and the preparation of acid treatment Lactococcus lactis
Method：
Take a small amount of glycerine and freeze strain and rule into GM17 Solid media for plates, be placed in 30 DEG C of quiescent cultures of incubator
24h.Picking single bacterium colony is inoculated in 5mL GM17 fluid nutrient mediums in 30 DEG C of quiescent culture 12h.Take L.lactis MG1363 mistakes
Night culture, by 1:100 are inoculated into the fresh GM17 culture mediums of 500mL, 30 DEG C of quiescent culture 16h.Thalline is collected by centrifugation, uses
250mL sterilized waters be washed once, acetum (pH=1) resuspended thalline of 100mL, and 30min is boiled in boiling water.250mL
Aseptic PBS is washed 3 times, with the 50mL resuspended thalline of aseptic PBS, is placed in 4 DEG C of preservations.
2.cA-hMT albumen is backed towards and is anchored into Lactococcus lactis
Method：The recombination engineering centrifugation that 5mL is overnight induced is taken, culture supernatant is abandoned, thalline is resuspended with 1mL PBSs
After ultrasonication in ice bath (power 10%, total time 7min, 3s opens 5s passes), 12000rpm centrifugation 10min collect supernatant
Liquid, takes a small amount of supernatant plus sample-loading buffer boiling water boiling 5min.Take 5mL through acid treatment (or incubated overnight) Lactococcus lactis from
The heart, abandons supernatant, and thalline is resuspended with the supernatant after above-mentioned ultrasonication, is stored at room temperature 1h, is taken after 9000rpm centrifugations 1min a small amount of
Supernatant adds sample-loading buffer that 5min is boiled in boiling water, and other supernatants are discarded, and thalline is washed three times with PBS, 4 DEG C of placements.
As a result：As shown in figure 4, after adding lactic acid bacteria, restructuring hMT fusion protein specificity is anchored into lactic acid bacteria, in supernatant
The amount of cA-hMT is significantly reduced.
Conclusion：HMT fusion proteins can specifically be anchored into lactic acid bacteria.
3.ELISA detections are anchored on the hMT on lactic acid bacteria surface
Method：The lactic acid bacteria of grappling after coating buffer is diluted hMT is added in ELISA Plate, 100 μ L per hole, 4 DEG C of wrapper sheets
Overnight.PBST board-washings 1 time, PBST immersions 30-60s during board-washing.With the 37 DEG C of closing 2h of PBST solution containing 3%BSA.
Anti-hMT monoclonal antibodies dilute 1000 times with PBST solution, the amount by 100 μ L per hole, and diplopore control is added in ELISA Plate,
37 DEG C of incubation 1h, board-washing 3 times.Add 5000 times of goat anti-mouse secondary antibodies of the HRP marks of dilution, 37 DEG C of incubation 30min, board-washing
3 times.The μ L of tmb substrate solution 100 are added per hole, chromogenic reaction 10min.The μ L of 2M sulfuric acid 50 per hole, react, ELIASA by color development stopping
OD450 readings.
As a result：As shown in Figure 5.
Conclusion：Confirm that hMT fusion proteins can be anchored on lactic acid bacteria surface.
4. the stability after lactic acid bacteria grappling hMT is determined
Method：With step 3, determined once per 24h, METHOD FOR CONTINUOUS DETERMINATION 120h.
As a result：As shown in Figure 6.
Conclusion：HMT is anchored into lactic acid bacteria rear stability preferably, can stablize at 4 DEG C and preserve 120h.
Embodiment 4
Restructuring hMT fusion proteins are anchored into after lactic acid bacteria to Cd2+、Pb2+Adsorption capacity detection
Method：HMT is anchored into lactic acid bacteria by experimental group in the way of embodiment 3, parallel to do 3 groups, adds the Cd of 1mL2+
(100um) is resuspended, and room temperature places 2h, and period fully mixes, and makes the abundant Adsorption of Heavy Metals of albumen.8000rpm is centrifuged 1min, abandons
Clearly, thalline PBS is put into 65 DEG C of drying boxes and dries 12h after washing 3 times, after the dry mycelium for obtaining is weighed, with 60% nitric acid digestion
Overnight.Add ddH after thoroughly having digested2O is diluted to 5mL for Atomic Absorption Spectrometry.This experiment sets 6 groups altogether, and 1 group is do not have
The biodiasmin of anchorin, 2 groups is the biodiasmin of grappling GFP (the cA-GFP recombinant proteins of this seminar expression), 3
Group for grappling hMT biodiasmin, 4 groups be the acid treatment lactic acid bacteria without albumen, 5 groups of acid treatment lactic acid for grappling GFP
Bacterium, the acid treatment lactic acid bacteria that 6 groups is grappling hMT.To Cd2+、Pb2+Adsorption capacity represented with nmol/mg dry weight thalline.Absorption
Heavy metal concentration (mg/L) x5mLx10 of ability=atomic absorption detecting-6/Cd2+、Or Pb2+Molal weight (112.41 or
207.2g/mol)/dry weight thalline (mg) x10-9。
As a result：As shown in Fig. 7 (a), 7 (b).
Conclusion：The lactic acid bacteria of grappling hMT is to Cd2+、Pb2+Adsorption capacity be significantly increased.
Claims (10)
1. a kind of preparation method of the food grade lactic acid galactococcus of surface anchoring people I type metallothioneins, it is characterised in that including
Following steps：
(1) the recombination, amalgamation and expression carrier of the people's I type metallothioneins containing anchor unit is built：
CA genes are spliced with hMT genes by over-lap PCR, linker is introduced between two genes, N-terminal is introduced and promotees expression mark
Sign, obtain recombinating cA-hMT genes；The nucleotide sequence of cA genes such as SEQ ID NO:Shown in 1, the nucleotide sequence of hMT genes
Such as SEQ ID NO:Shown in 2, the nucleotide sequence such as SEQ ID NO of linker:Shown in 3；
(2) restructuring cA-hMT genes are connected to expression vector pET21a, then the connection product pET21a-cA-hMT that will be obtained turns
Change to competent escherichia coli cell, screening positive clone obtains engineering bacteria by identification；
(3) the engineering bacteria induced expression fusion protein cA-hMT for obtaining step (2), harvests ultrasonication after thalline, by supernatant
Liquid is directly incubated at room temperature with Lactococcus lactis, and fusion protein cA-hMT then anchors to Lactococcus lactis surface.
2. method according to claim 1, it is characterised in that the recombination expression of step (2) also needs to add molecular chaperones, tool
Body step is as follows：
By the connection product pET21a-cA-hMT described in step (2) and the expression vector containing molecular chaperones successively conversion to big
The expression bacterial strain of enterobacteria.
3. method according to claim 2, it is characterised in that the molecular chaperones is dnaK, dnaJ, grpE, groES and
One or more in groEL.
4. the method according to claim 1 or 2 or 3, it is characterised in that the Lactococcus lactis are also through following pretreatment：
Lactococcus lactis wet thallus are added acetic acid to process and boil 30min, anchor is carried out with recombination fusion protein cA-hMT again after PBS washings
It is fixed.
5. the method according to claim 1 or 2 or 3, it is characterised in that step (3) engineering bacteria induced expression restructuring fusion
The step of albumen cA-hMT, is as follows：
A. engineering bacteria is inoculated in the LB liquid medium containing ampicillin and chloramphenicol, is shaken overnight in 37 DEG C, 180rpm,
Then 1 is pressed：During 100 ratio is inoculated in containing ampicillin, chloramphenicol, the LB liquid medium of L-arabinose, work as OD
The isopropylthiogalactoside (IPTG) of final concentration of 0.5~1.0mM is added during up to 0.4~0.8, in 200rpm, low temperature after
Bacterium solution is collected after continuous culture 12h, centrifugation obtains thalline；
B. PBS washing step a gained thalline is used 2 times, it is resuspended with bacterial lysate, ultrasonication under ice bath is placed in, take
Homogenate centrifugation after ultrasound, collects supernatant and inclusion body respectively.
6. method according to claim 5, it is characterised in that fusion protein cA-hMT is anchored to lactic acid breast by step (3)
The step of coccus surface, is as follows：
Supernatant is directly resuspended with Lactococcus lactis, 1h is stored at room temperature, 9000g centrifugations 2min abandons supernatant, precipitation PBS solution
Washing 3 times, resuspended to 4 DEG C preservations.
7. the method according to claim 1 or 2 or 3, it is characterised in that step (3) described Lactococcus lactis be MG1363,
MG1614、LM0230。
8. method according to claim 7, it is characterised in that the incubation step of the Lactococcus lactis is as follows：
Lactococcus lactis are cultivated in GM17 fluid nutrient mediums, 30 DEG C of anaerobism quiescent cultures to exponential phase late period or platform
Phase, OD600nmUp to 3~4, room temperature 9000g centrifugation 5min, wet thallus are collected.
9. the food grade lactic acid galactococcus of surface anchoring people I type metallothioneins obtained in any one of claim 1~8 method.
10. the application of the food grade lactic acid galactococcus of surface anchoring people I type metallothioneins described in claim 9, its feature exists
In the Lactococcus lactis are used for human body heavy metal detoxification, remediating heavy metal oxidative damage, metal ion Metabolism regulation and give up
Water exhaust-gas treatment and environment remediation.
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| Cell surface display system for Lactococcus lactis: a novel development for oral vaccine;A. R. Raha et al.;《Appl Microbiol Biotechnol》;20051231;全文 * |
| Cell Wall Attachment of a Widely Distributed Peptidoglycan Binding Domain Is Hindered by Cell Wall Constituents;Anton Steen et al.;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20030613;第278卷(第26期);全文 * |
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