CN103409360A - Recombinant large yellow croaker cysteine proteinase inhibitor, and preparation method and application thereof - Google Patents
Recombinant large yellow croaker cysteine proteinase inhibitor, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant large yellow croaker cysteine proteinase inhibitor, and a preparation method and application thereof, and relates to a cysteine proteinase inhibitor. The collection number of Escherichia coli BL21/pET-his-Lyccystatin B is CCTCC NO:M2013225. The recombinant large yellow croaker cysteine proteinase inhibitor cystatin B is prepared from a recombinant expression product of the Escherichia coli BL21/pET-his-Lyccystatin B expressing a large yellow croaker cysteine proteinase inhibitor cystatin B gene. After imidazole removal through dislysis, the prepared recombinant large yellow croaker cysteine proteinase inhibitor cystatin B can be used in preparation of aquatic product processing additives.
Description
Technical field
The present invention relates to a kind of cystatin, especially relate to a kind of restructuring large yellow croaker cystatin and preparation method thereof and application.
Background technology
Cystatin (cystatins) is that a class suppresses the supressor that the protease hydrolysis effect of cysteine residues is contained in active centre specially, to halfcystine class kethepsin inhibited (1. Zheng often the literary composition, Dong Changjiang, Tang Hongli etc. the separation and purification of porcine blood plasma thiol protease inhibitor and property research. Sichuan University's journal (natural science edition), 1993,3:015).
Processing of aquatic products; especially in the rotten course of processing of fish, residual halfcystine class kethepsin is understood the myosin in the gruel of degraded fish in the heating preparation process of minced fish gel; cause deteriorated (the 2.Olafsdottir G of gel; Martinsd ó ttir E, et al.Methods to evaluate fish freshness in research and industry.Trends in Food Science& Technology, 1997,8 (8): 258-265), reduce the quality of gefillte fish.Cause the serine protease that the deteriorated halfcystine class kethepsin of minced fish gel mainly contains cathepsin B, L, L-like and be combined with sarcostyle (3. Hou Luna, Chen Xueyun, Nie Xiaohua; Liu's Lin. the progress of the deteriorated related tissue of minced fish gel proteolytic enzyme. food science and technology, 2010,35:164-167).At present, often deteriorated by adding some chemical reagent or suppressing gel from the cystatin made pig, Ox blood plasma and Ovum Gallus domesticus album, potato in the processing of aquatic products industry, strengthen the product gel-strength.Although the use of the chemical reagent such as antifreezing agent, reductive agent, oxygenant, gel toughener can help to a certain extent the formation of gel and strengthen gel-strength, can not fundamentally solve the deteriorated problem of the gel caused by endogenous protease.Expensive from the cystatin made pig, Ox blood plasma and Ovum Gallus domesticus album, potato, in the course of processing, can make product produce bad local flavor and color and luster, and popular due to bird flu, mad cow disease, make people feel misgivings to the food safety that contains these sources, therefore in the urgent need to finding food grade cystatin novel, safe, efficient, with low cost.The cystatin that derives from fish self can efficiently solve the deteriorated problem of gel in the gefillte fish course of processing targetedly by the activity that obligate suppresses fish halfcystine proteinoid enzyme, utilize the restructuring fish cystatin of gene engineering expression can greatly reduce the product preparation cost, have broad application prospects in the rotten processing industry of fish.
Summary of the invention
The first purpose of the present invention is to provide a kind of intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B.
The second purpose of the present invention is to provide restructuring large yellow croaker cystatin cystatin B and preparation method thereof.
The 3rd purpose of the present invention is to provide the application of restructuring large yellow croaker cystatin cystatin B in preparation processing of aquatic products additive.
Described intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B has been preserved in Chinese Typical Representative culture collection center on May 20th, 2013, deposit number is CCTCC NO:M2013225, the address at Chinese Typical Representative culture collection center is China, Wuhan, Wuhan University.
Described restructuring large yellow croaker cystatin cystatin B(rLyccystatin) recombination expression product by intestinal bacteria (Escherichia coli) the BL21/pET-his-Lyccystatin B that expresses large yellow croaker cystatin cystatin B gene makes, the about 11kDa of its theoretical molecular.
The preparation method of described restructuring large yellow croaker cystatin cystatin B, comprise the steps:
1) the picking intestinal bacteria mono-bacterium colony of (Escherichia coli) BL21/pET-his-Lyccystatin B shaking culture in substratum is spent the night, second day is inoculated in this overnight culture in the 100mL substratum according to 1% ratio, shaking culture reaches 0.4～0.6 to OD600, add inductor IPTG(Isopropyl β-D-1-Thiogalctopyranoside, sec.-propyl-β-D-sulfo-galactopyranoside) to final concentration be 0.1～1mM, continue after shaking culture the centrifugal collection thalline of culture, with after binding buffer liquid washing thalline 3 times, use again the resuspended thalline of binding buffer liquid, obtain the resuspended thalline solution of binding buffer liquid, the gained sample carries out the expression that polyacrylamide gel electrophoresis SDS-PAGE detects large yellow croaker Cystatin B gene,
2) the resuspended thalline solution of binding buffer liquid step 1) obtained carries out ultrasonication in ice bath, ultrasonic after product centrifugation supernatant and precipitation prepare respectively sample through SDS-PAGE analyze to confirm the expression product of large yellow croaker cystatin cystatin B gene be present in a large number ultrasonic after in supernatant, illustrate that it obtains solubility expression in intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B;
3) utilize the recombination expression product of large yellow croaker cystatin cystatin B gene in immobilized metal ion affinity chromatography medium purification appeal abduction delivering culture: by step 2) in preparation ultrasonic rear supernatant liquor by every 10mL add 1mL immobilized metal ion affinity chromatography medium in 4 ℃ in conjunction with crossing post, coutroi velocity is at 2～5mL/min, with 5～10 times of column volumes, contains after the binding buffer liquid washing of 10～20mM imidazoles with the binding buffer liquid washing foreign protein that contains 20～40mM imidazoles to OD again
280Be less than 0.01, add 1mL to contain the elution buffer wash-out target protein of 500～1000mM imidazoles, repeat 1 time, the purified product obtained is large yellow croaker Cystatin B gene e. coli expression product after SDS-PAGE detects, large yellow croaker cystatin cystatin B namely recombinates.
In step 1), the composition of described substratum is calculated as 1% NaCl, 1% peptone, 0.5% yeast extract, the penbritin of 100 μ g/mL, pH7.0 by mass volume ratio;
The described condition that culture is centrifugal can be in 6000r/min, 4 ℃ of centrifugal 10min;
The condition of described shaking culture can be in 37 ℃ with 180～250r/min shaking culture;
The condition of described continuation shaking culture can be spent the night with 180～250r/min shaking culture in 16～25 ℃;
Described binding buffer liquid consist of 20mM NaH
2PO
4, 0.5M NaCl, pH7.4;
In step 2) in, the condition of described ultrasonication can be in output rating 50～70W, and 5S is ultrasonic/the ultrasonic 10min of cycling condition of 5S tranquillization; The centrifugal condition of described ultrasonic after product can be in 12000r/min, 4 ℃ of centrifugal 20min;
In step 3), described elution buffer consist of 20mM NaH
2PO
4, 0.5M NaCl, pH7.4; Described dialysis process can be: the every 10mL of protein purification product is placed in dialysis tubing, being placed in successively the beaker that 2L dialyzate I, dialyzate II and dialyzate III are housed dialyses and progressively removes imidazoles, in dialyzate I and dialyzate II in 4 ℃ the dialysis 6h, finally in dialyzate III in 4 ℃ of dialysed overnight; Described dialyzate I consists of 20mM NaH
2PO
4, 0.5M NaCl, the 500mM imidazoles, described dialyzate II consists of 20mM NaH
2PO
4, 0.5M NaCl, the 200mM imidazoles, described dialyzate III consists of 20mM NaH
2PO
4, 0.5M NaCl.
Prepared restructuring large yellow croaker cystatin cystatin B can apply in preparation processing of aquatic products additive after imidazoles is removed in dialysis, described fishery products comprise fish gruel, fish slurry, shrimp slurry, shrimp gruel, crab slurry, crab gruel etc.
The present invention relates generally to the recombination expression product of a kind of large yellow croaker cystatin cystatin B in intestinal bacteria, and the application in the rotten processing of processing of aquatic products especially fish.The expression product (rLyccystatin B) of described large yellow croaker Cystatin B gene in intestinal bacteria is the protein band of a big or small about 17KD, when protein concentration is 1 μ g/g, 5 μ g/g, suppress the deteriorated effect of the rotten isogel of fish the strongest, demonstrate its applications well prospect in the processing of aquatic products processes such as fish gruel.
The accompanying drawing explanation
To be intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B analyze in the spend the night SDS-PAGE of rear bacterial protein of 25 ℃ of shaking culture Fig. 1.In Fig. 1, the M swimming lane is the molecular weight of albumen standard; 1,2 swimming lanes are the blank group; 3,4 swimming lanes are large yellow croaker Cystatin B gene e. coli expression product.
Fig. 2 is the soluble analysis of intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B expression product.In Fig. 2, the M swimming lane is the molecular weight of albumen standard; 1 swimming lane is large yellow croaker Cystatin B gene e. coli expression product; 2 swimming lanes are the ultrasonic rear supernatant of expression product; 3 swimming lanes are the ultrasonic postprecipitation of expression product.
Fig. 3 is the restructuring large yellow croaker Cystatin B of purifying.In Fig. 3, the M swimming lane is the molecular weight of albumen standard; 1 swimming lane is the large yellow croaker Cystatin B of purifying.
Fig. 4 is the gel-strength that adds seawater trash fish gruel after the rLyccystatin B of different concns.In Fig. 4, mark a is K, and b is EW, and c is rLyccystatin B; K is blank, i.e. negative control; EW is for adding the egg-white powder of 6000 μ g/g, i.e. positive control; 1,10 be respectively interpolation 1 μ g/g, the Lyccystatin B of 10 μ g/g; Error line means the standard deviation of different treatment group; Mark
*Mean to exist significant difference (P<0.05).
Fig. 5 adds the gel-strength that after the rLyccystatin B of different concns, the silver carp gruel/hairtail gruel mixes the fish gruel.In Fig. 5, mark a is K, and b is EW, and c is the rLyccystatin B of different concns; K is blank, i.e. negative control; EW is for adding the egg-white powder of 6000 μ g/g, i.e. positive control; 1,5,10,30 be respectively 1 μ g/g, 5 μ g/g, 10 μ g/g, the rLyccystatin B of 30 μ g/g; Error line means the standard deviation of different treatment group.
Fig. 6 adds the rotten total protein that mixes minced fish gel of silver carp gruel/hairtail after the rLyccystatin B of different concns, mainly in order to the content difference of myosin Myosin wherein.In Fig. 6, the M swimming lane is the molecular weight of albumen standard; 1 swimming lane is the blank group; 2 swimming lanes are for adding the egg-white powder of 6000 μ g/g, i.e. positive control; 3～6 swimming lanes mean respectively to have added 1 μ g/g, 5 μ g/g, 10 μ g/g, the rLyccystatin B of 30 μ g/g.
Embodiment
The present invention is further illustrated in connection with accompanying drawing for following examples.
Restructuring large yellow croaker cystatin cystatin B(rLyccystatin B) preparation method comprises following concrete steps:
1. the abduction delivering of recombinant protein
By the mono-colony inoculation of intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B in the 5mL substratum, in 37 ℃ of 200r/min shaking culture, spend the night, second day is inoculated in this overnight culture in the 100mL substratum in 1: 100 ratio, and shaking culture is to culture OD
600Reach at 0.4～0.6 o'clock, add IPTG to final concentration be 1mM, in 25 ℃ of 200r/min shaking culture, spend the night, culture is proceeded in centrifuge tube, with 6000r/min, in 4 ℃ of centrifugal 10min, abandon supernatant; Bacterial sediment is used the resuspended thalline of about 10mL binding buffer liquid after with binding buffer liquid, washing 3 times, the expression of preparation sample detection large yellow croaker cystatin cystatin B gene.
Culture medium prescription: 1% NaCl, 1% peptone, 0.5% yeast extract, the penbritin of 100 μ g/mL, pH7.0.
2.SDS-PAGE analyze the expression of recombinant protein
In thalline solution after resuspended, add equal-volume 2 * protein electrophoresis sample-loading buffer (20mM Tris.HCl, 2mM EDTA, 2%SDS, 10% beta-mercaptoethanol, 16% glycerine, 0.05% tetrabromophenol sulfonphthalein, pH8.2) boil 5min and prepare protein sample, carry out the SDS-PAGE electrophoresis.Result shows, the protein band that has occurred an about 17kDa of size in the bacterial protein of e. coli bl21/after pET-his-Lyccystatin B induces, and do not have corresponding band to occur in the bacterial protein of control strain BL21/pET-his after inducing, illustrate that large yellow croaker cystatin cystatin B gene has obtained expression (Fig. 1).
For analyzing further whether large yellow croaker cystatin cystatin B gene is solubility expression in BL21/pET-his-Lyccystatin B, the thalline solution that binding buffer liquid in step 1 is resuspended carries out ultrasonication in ice bath, ultrasonic after product is in 12000r/min, 4 ℃ of centrifugal 20min, the expression that centrifugal rear supernatant and precipitation prepare respectively sample detection large yellow croaker cystatin cystatin B gene, result shows, in supernatant sample after ultrasonic, contain a large amount of purpose bands, illustrate that cystatin B gene is solubility expression in BL21/pET-his-Lyccystatin B, available ultrasonic supernatant of inducing rear thalline carries out the purifying (Fig. 2) of its expression product.
3. affinity chromatography purifying large yellow croaker cystatin cystatin B recombinant protein
1) prepare the immobilized metal ion affinity chromatography post
(GE company product, commodity are called Ni Sepharrose by the ultrasonic rear supernatant liquor of every 10mL/1mL metal ion affinity chromatography medium
TM6Fast Flow) ratio dress post, first wash post with 5 column volume distilled waters, then use 5 column volume binding buffer liquid (20mM NaH
2PO
4, 0.5M NaCl, pH7.4) and the balance chromatography column, wait for loading.
2) upper column purification
By the ultrasonic supernatant of BL21/pET-his-Lyccystatin B expression product of preparation in step 2 in 4 ℃ of whole upper prop combinations, coutroi velocity is at 3～4mL/mim, then with 5 times of column volumes, contains after the binding buffer liquid washing of 20mM imidazoles with the binding buffer liquid washing foreign protein that contains the 40mM imidazoles to OD again
280Be less than 0.01, add 1mL to contain elution buffer (the 20mM NaH of 1M imidazoles
2PO
4, 0.5M NaCl, pH7.4) and the wash-out target protein, repeat 1 time.Take a morsel and prepare sample and carry out SDS-PAGE electrophoresis evaluation (Fig. 3).
3) imidazoles is removed in dialysis
The every 10mL of protein purification product is placed in dialysis tubing, is placed in successively 2L dialyzate I(20mM NaH is housed
2PO
4, 0.5M NaCl, 500mM imidazoles), dialyzate II(20mM NaH
2PO
4, 0.5M NaCl, 200mM imidazoles), dialyzate III(20mM NaH
2PO
4, 0.5M NaCl) beaker in dialyse and progressively remove imidazoles.In dialyzate I, II in 4 ℃ the dialysis 6h, finally in dialyzate III in 4 ℃ of dialysed overnight.Protein solution after dialysis is measured concentration through the BradFord method, namely can be used for follow-up processing of aquatic products.
Below provide the application of large yellow croaker cystatin cystatin B DNA recombinant expression product (rLyccystatin B) in the rotten course of processing of fish, its step is as follows:
1. the preparation of gefillte fish
Get the 500g frozen minced fillets, 4 ℃ of placements are spent the night, and thaw to chopping after approximately-2.5 ℃ of rotten integrated temperatures of fish, to put choppingmachine and cut and mix 1～2min (all cut the processes of mixing by peripheral frozen water control temperature below 10 ℃); Add 3% salt to cut and mix 4～6min, add the rLyccystatin B of different final concentrations, cut and mix about 1min and mix, discharging; Bowel lavage, insert said mixture in the Polyvinylidene bag that diameter is 30mm, and the bag two ends are tamping with aluminium wire; Gefillte fish adds the deteriorated heating mode of thermal recovery gel: after 55 ℃ of heating in water bath 30min, 90 ℃ are continued heating in water bath 30min.Obtained minced fish gel is deteriorated gel (modori gel).
Separately get the rotten 500g of fish, operate the samely, rLyccystatin B is replaced with to the egg-white powder (egg white, EW) of 6000 μ g/g, working method and deteriorated gel phase together, are used as the positive control gel.
Separately get the rotten 500g of fish, operate the samely, additionally do not add any material, working method and deteriorated gel phase together, are used as negative control gel (K).
After 90 ℃ of heating of all gels, cooling 30min in ice-water bath at once, 4 ℃ of placements are in order to the use of analysis.
2. the mensuration of gel-strength
Adopt Britain SMS matter structure instrument to analyze the intensity of gel.Before test, take out gel from 4 ℃ of refrigerators, be cut into the long gel column of 30mm, each experimental group prepares five gel columns.Use the P/5s probe, test pattern is compression, and before surveying, speed is 2mm/s, and test speed is 1mm/s, and after surveying, speed is 10mm/s; Target pattern is displacement, and size is 15mm; Triggering power is automatic mode, and size is 5g.Detect breaking force (Breaking force, g) and the deflection (deformation, cm) of gel.
Breaking force * the deflection of gel-strength=gel
After adding different rLyccystatin B to process, the minced fish gel intensity results of seawater trash fish gruel is shown in Fig. 4.Under the gel deterioration mode, rLyccystatin B treatment group minced fish gel strength increase is to 1.4 times of blank group (K, i.e. negative control), and has significant difference in P<0.05 o'clock.
After adding different rLyccystatin B to process, the rotten mixing of silver carp gruel/hairtail minced fish gel intensity the results are shown in Figure 5.Under the gel deterioration mode, rLyccystatin B suppresses to mix the deteriorated successful of minced fish gel, when add-on is 1.5 times that the rotten gel-strength of 1 μ g/g, 5 μ g/g macrura reevesiis all can reach the blank group, and the rotten gel-strength of the positive control egg-white powder macrura reevesii that adds 6000 μ g/g can only be brought up to 1.3 times of the blank group, successful is weaker than rLyccystatin B.
3. the test of gel TPA (hardness, elasticity, chewiness)
Adopt Britain SMS matter structure instrument to analyze the TPA of gel column.Use the P/20 probe; Before test, speed is 2mm/s; Test speed is 1mm/s; After test, speed is 5mm/s; Target pattern is displacement, and the displacement size is 8mm.With reference to the people such as Zhou Jianyong (8. Zhou Jianyong, Wang Yufen, Luo Fei etc. the impact of compression ratio on ham sausage TPA characteristic value. food science and technology, 2003 (z1)) the compression ratio (be displacement of targets than gel length) of document selected 27%; Triggering power is automatic mode, and size is 5g, carries out 3 repeated tests.
With blank, compare, while adding the rLyccystatin B of different concns, can significantly improve the TPA of minced fish gel, comprise gel hardness, elasticity, chewiness (P<0.05).While adding the rLyccystatin B of 10 μ g/g, the TPA value of minced fish gel reaches maximum, wherein the hardness of minced fish gel, elasticity and chewiness all reach 1.2 times (P<0.05) of blank group, the positive controls (table 1) that is better than adding egg-white powder EW.
4. the detection of gel whiteness
According to International Commission on Illumination (Commission International d ' Eclairage, CIE) definition, use the WSC-S colour examining colour-difference-metre to detect the Blue Whiteness of gel sample, operate as follows:
The first preheating 30min of instrument before using, then instrument reaches and manually carries out zero point and blank correction through self check.During test, gel is paved with sample pool bottom and is placed on the load sample platform and measures, and space can not be arranged at gel and sample pool bottom, presses white II key reading, i.e. Blue Whiteness (the results are shown in Table 2).
With blank, compare, the rLyccystatin B that adds different concns all can significantly improve the whiteness (P<0.05) of minced fish gel, and when to add concentration be the rLyccystatin B of 5 μ g/g, the whiteness of minced fish gel reached maximum value.
Table 1
Annotate:
*Mean to exist significant difference.
Table 2
Annotate:
*Mean to exist significant difference.
5. the detection of gel water retention property
In reference literature (9.Ng C.Measurement of free and expressible drips.Laboratory manual on analytical methods and procedure for fish and fish products1987:1-2), detect in gel the method for moisture (expressible drip) content of can squeezing out and do suitable improvement.After being cut into to the thin slice that 5mm is thick, gel weighs (X).Then gel slice is picked up with double-deck filter paper, the flat counterweight that 5kg is heavy is placed in top, after static squeezing 2min, and the weight of weighing gel slice (Y) again.Calculate according to the following equation moisture content (table 3): water retention property (%)=1 – 100 * [(X – the Y)/X] that can squeeze out
While adding the rLyccystatin B of different concns, the water retention property of minced fish gel all has significance to improve (P<0.05), and when to add concentration be the rLyccystatin B of 5 μ g/g, the water retention property of minced fish gel was best.
Table 3
Annotate:
*Mean to exist significant difference.
6. the detection of minced fish gel myosin content
Get the SDS solution of 18mL5%, add the 2g minced fish gel.This mixture, with electric homogenate machine homogeneous 1min under the speed of 11000r/min, is made to homogenizing fluid.Homogenizing fluid is heated to 1h in the water-bath of 85 ℃ with solubilising protein, then under room temperature the centrifugal 5min of 10000r/min to remove indissolvable component.Supernatant liquor is mixed with the ratio of electrophoresis sample-loading buffer by 1: 1, after boiling 5min, get 5 μ L loadings and carry out electrophoresis.Result shows to be compared with the blank group, when the amount that adds rLyccystatin B is 5 μ g/g, 10 μ g/g, can obviously suppress the degraded (Fig. 6) of myosin.
7. data analysis
All experiments all repeat 3 times, and result is expressed as mean+SD.The rendering error analysis chart is used GraphPad Prism5 software, and the SPSS18.0 software package is used in the significance of difference analysis of data, and analytical procedure is one-way analysis of variance (ONE-WAY ANOVA), and program thereby is Duncan.
Above method can be applicable to the processing of fish slurry, shrimp slurry, shrimp gruel, crab slurry, crab gruel etc. equally.
Claims (10)
1. intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B, be preserved in Chinese Typical Representative culture collection center on May 20th, 2013, and deposit number is CCTCC NO:M2013225.
2. large yellow croaker cystatin cystatin B recombinates, it is characterized in that being made by the recombination expression product of intestinal bacteria (Escherichia coli) the BL21/pET-his-Lyccystatin B that expresses large yellow croaker cystatin cystatin B gene the about 11kDa of its theoretical molecular.
3. recombinate the as claimed in claim 2 preparation method of large yellow croaker cystatin cystatin B, is characterized in that comprising the steps:
1) the picking intestinal bacteria mono-bacterium colony of (Escherichia coli) BL21/pET-his-Lyccystatin B shaking culture in substratum is spent the night, and second day is inoculated in this overnight culture in the 100mL substratum according to 1% ratio, and shaking culture is to OD
600Reach 0.4～0.6, add inductor IPTG to final concentration be 0.1～1mM, continue after shaking culture the centrifugal collection thalline of culture, with after binding buffer liquid washing thalline 3 times, use again the resuspended thalline of binding buffer liquid, obtain the resuspended thalline solution of binding buffer liquid, the gained sample carries out the expression that polyacrylamide gel electrophoresis SDS-PAGE detects large yellow croaker Cystatin B gene;
2) the resuspended thalline solution of binding buffer liquid step 1) obtained carries out ultrasonication in ice bath, ultrasonic after product centrifugation supernatant and precipitation prepare respectively sample through SDS-PAGE analyze to confirm the expression product of large yellow croaker cystatin cystatin B gene be present in a large number ultrasonic after in supernatant, illustrate that it obtains solubility expression in intestinal bacteria (Escherichia coli) BL21/pET-his-Lyccystatin B;
3) utilize the recombination expression product of large yellow croaker cystatin cystatin B gene in immobilized metal ion affinity chromatography medium purification appeal abduction delivering culture: by step 2) in preparation ultrasonic rear supernatant liquor by every 10mL add 1mL immobilized metal ion affinity chromatography medium in 4 ℃ in conjunction with crossing post, coutroi velocity is at 2～5mL/min, with 5～10 times of column volumes, contains after the binding buffer liquid washing of 10～20mM imidazoles with the binding buffer liquid washing foreign protein that contains 20～40mM imidazoles to OD again
280Be less than 0.01, add 1mL to contain the elution buffer wash-out target protein of 500～1000mM imidazoles, repeat 1 time, the purified product obtained is large yellow croaker Cystatin B gene e. coli expression product after SDS-PAGE detects, large yellow croaker cystatin cystatin B namely recombinates.
4. the preparation method of large yellow croaker cystatin cystatin B as claimed in claim 3 recombinates, it is characterized in that in step 1), the composition of described substratum is calculated as 1% NaCl by mass volume ratio, 1% peptone, 0.5% yeast extract, the penbritin of 100 μ g/mL, pH7.0.
5. recombinate the as claimed in claim 3 preparation method of large yellow croaker cystatin cystatin B is characterized in that in step 1) the described condition that culture is centrifugal is in 6000r/min, 4 ℃ of centrifugal 10min; The condition of described shaking culture is with 180～250r/min shaking culture in 37 ℃; The condition of described continuation shaking culture is to spend the night with 180～250r/min shaking culture in 16～25 ℃.
6. recombinate the as claimed in claim 3 preparation method of large yellow croaker cystatin cystatin B, is characterized in that in step 1), described binding buffer liquid consist of 20mM NaH
2PO
4, 0.5M NaCl, pH7.4.
7. the preparation method of large yellow croaker cystatin cystatin B as claimed in claim 3 recombinates, it is characterized in that in step 2) in, the condition of described ultrasonication is in output rating 50～70W, 5S is ultrasonic/and the ultrasonic 10min of cycling condition of 5S tranquillization; The centrifugal condition of described ultrasonic after product can be in 12000r/min, 4 ℃ of centrifugal 20min.
8. recombinate the as claimed in claim 3 preparation method of large yellow croaker cystatin cystatin B, is characterized in that in step 3), described elution buffer consist of 20mM NaH
2PO
4, 0.5M NaCl, pH7.4.
9. the preparation method of large yellow croaker cystatin cystatin B as claimed in claim 3 recombinates, it is characterized in that in step 3), described dialysis process is: the every 10mL of protein purification product is placed in dialysis tubing, being placed in successively the beaker that 2L dialyzate I, dialyzate II and dialyzate III are housed dialyses and progressively removes imidazoles, in dialyzate I and dialyzate II in 4 ℃ the dialysis 6h, finally in dialyzate III in 4 ℃ of dialysed overnight; Described dialyzate I consists of 20mM NaH
2PO
4, 0.5M NaCl, the 500mM imidazoles, described dialyzate II consists of 20mM NaH
2PO
4, 0.5M NaCl, the 200mM imidazoles, described dialyzate III consists of 20mM NaH
2PO
4, 0.5M NaCl.
10. the application of large yellow croaker cystatin cystatin B in preparation processing of aquatic products additive of recombinating as claimed in claim 2, described fishery products can comprise that fish is rotten, fish slurry, shrimp slurry, shrimp are rotten, crab slurry, crab gruel; Described restructuring large yellow croaker cystatin cystatin B removes imidazoles through dialysis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773794A (en) * | 2014-01-15 | 2014-05-07 | 辽宁大学 | Recombinant wild type chicken cystatin and application thereof |
| CN112226378A (en) * | 2020-10-31 | 2021-01-15 | 福建农林大学 | Large yellow croaker cysteine protease inhibitor Cystatin recombinant protein and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851627A (en) * | 2010-03-10 | 2010-10-06 | 中国科学院海洋研究所 | Gene encoding protein of eriocheir sinensis cysteine protease inhibitor Escystatin and application |
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2013
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| CN101851627A (en) * | 2010-03-10 | 2010-10-06 | 中国科学院海洋研究所 | Gene encoding protein of eriocheir sinensis cysteine protease inhibitor Escystatin and application |
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| 李淑英: "大黄鱼两种半胱氨酸蛋白酶抑制剂的分子特征与功能研究", 《中国优秀硕士学位论文全文数据库 基础科技辑》 * |
| 杨志军: "大黄鱼组织蛋白酶B、L、S的分子克隆和半胱氨酸蛋白酶抑制剂cystatins的初步功能研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773794A (en) * | 2014-01-15 | 2014-05-07 | 辽宁大学 | Recombinant wild type chicken cystatin and application thereof |
| CN112226378A (en) * | 2020-10-31 | 2021-01-15 | 福建农林大学 | Large yellow croaker cysteine protease inhibitor Cystatin recombinant protein and application thereof |
| CN112226378B (en) * | 2020-10-31 | 2022-07-12 | 福建农林大学 | Large yellow croaker cysteine protease inhibitor Cystatin recombinant protein and application thereof |
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