CN104031853A - Construction method used for large scale production of MSTN saccharomycopsis - Google Patents
Construction method used for large scale production of MSTN saccharomycopsis Download PDFInfo
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- CN104031853A CN104031853A CN201310429660.7A CN201310429660A CN104031853A CN 104031853 A CN104031853 A CN 104031853A CN 201310429660 A CN201310429660 A CN 201310429660A CN 104031853 A CN104031853 A CN 104031853A
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Abstract
The invention discloses a construction method used for large scale production of MSTN saccharomycopsis, which comprises the following steps: amplification of a KanMX4 gene fragment and construction of a pBlue-KanMX4 carrier; insertion of yeast gene homologous arm; construction of pBlue-Kan-NTS2-5'3'-MSTN integration vector; integration of the MSTN carrier in yeast genome; and removal of resistant gene KanMX4. According to the invention, Myostatin integrated yeast strain without containing of screening gene is constructed, The bacterial classification can be produced in an enrichment-type medium, and has the advantages of safety, reliability and low cost, target gene loss is not generated, and the bacterial classification is suitable for large scale production of recombinant yeast, The yeast strain through large scale production can be directly used for feeding animals, and can generate high-level MSTN specific antibody in animal body, can release the inhibition effect and promote muscle growth, which has important meaning for animal production.
Description
Technical field
The invention belongs to gene engineering technology field, relate in particular to a kind of structure of the MSTN barms that can be used for scale operation.
Background technology
It is a kind of negative regulatory factor of muscle growth that flesh generates statin (Myostatin, MSTN), is under the jurisdiction of TGF-beta superfamily, has all features of TGF-beta superfamily.
Two kuhne's phenomenons that MSTN sudden change produces bring tremendous influence to herding production and pharmaceutical sector.Mcpherron research finds that this genetically deficient mouse muscle increases, and skeletal muscle muscle group distributes more extensive, and body weight is approximately the more than 2 times of wild mouse.Kambadur finds that sudden change has occurred this gene of two flesh oxen, and under same feeding condition, two flesh oxen can provide 30% meat product more than common ox.
2011, zhang etc. found the yeast of the oral expression of mouse MSTN, can produce high-caliber MSTN specific antibody, and this antibody can be removed the inhibition of MSTN gene pairs muscle growth, and then the carcass weight and the muscle growth that improve.
But adopt the numerous meeting of long-term expansion of nonconformity plasmid expression vector to cause the loss of carrying goal gene plasmid, and need screening culture medium to select the sustained resistance screening of pressing, cost is high, and complicated operation is not suitable for large-scale industrial production.
Summary of the invention
The object of the embodiment of the present invention is to provide a kind of construction process of the MSTN barms that can be used for scale operation, the numerous meeting of long-term expansion that is intended to solve current employing nonconformity plasmid expression vector causes the loss of carrying goal gene plasmid, and need screening culture medium to select the sustained resistance screening of pressing, cost is high, complicated operation, is not suitable for the problem of large-scale industrial production.
The present invention is achieved in that a kind of construction process of the MSTN barms that can be used for scale operation, and this construction process that can be used for the MSTN barms of scale operation comprises the following steps:
The structure of the amplification of step 1, KanMX4 gene fragment and pBlue-KanMX4 carrier;
The insertion of step 2, yeast genes homology arm;
The structure of step 3, pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector;
Step 4, MSTN carrier is integrated into Yeast genome;
Step 5, resistant gene KanMX4 knock out.
Further, the structure concrete steps of the amplification of step 1 KanMX4 gene fragment and pBlue-KanMX4 carrier are:
Design primer LoxP-Kan F, the LoxP-Kan R KanMX4 gene fragment that increases in the Yeast genome extracting;
Add LoxP sequence and EcoRI two sections of primers, two restriction enzyme sites of BamHI increase KanMX4 gene fragment out in Yeast genome, PCR program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After PCR, carry out solution recovery, carry out double digestion with EcoRI and BamHI, on 1% agarose gel electrophoresis, run glue, glue reclaims enzyme and cuts product and obtain purer fragment, with same enzyme double digestion pBluescript II SK+, then by two fragments 16 DEG C of connections of spending the night;
The connection product of acquisition is transformed in intestinal bacteria, the mono-clonal of selecting after transforming successfully wherein extracts bacterial plasmid, first use BamHI and EcoRI double digestion, the agargel electrophoresis of race 1% is determined the length of endonuclease bamhi, tentatively determine whether positive plasmid, and whether order-checking qualification plasmid successfully transforms.
Further, step 2 by the insertion concrete steps of Yeast genome homology arm is:
Be four primers that design NST2-5 ' end and two homology arms of NST2-3 ' end in NST2 fragment at the non-transcriptional of Yeast genome space2;
In NTS2-5 ' primer, insert the restriction enzyme site of XhoI and two restriction enzymes of KpnI;
In NTS2-3 ' primer, insert NotI, and the restriction enzyme site of two restriction enzymes of SacII;
NTS2-5 ' R and two primers of NTS2-5 ' F are added in PCR damping fluid, cooling 5min under 94 DEG C of room temperatures, directly obtain homology arm NTS2-5 ', with XhoI and KpnI double digestion NTS2-5 ' homology arm and pBlue-kan respectively, glue recovery two portions connect respectively again, connecting product is transformed in intestinal bacteria, after transforming successfully, the mono-clonal of selecting wherein extracts bacterial plasmid PCR qualification, order-checking is further determined, by identical method, NTS2-3 ' homology arm is inserted into, finally obtain pBlue-kan-NTS5 ' 3 ' carrier.
Further, the concrete steps of the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector are:
From JMB667, amplify ADH1 promotor and Cyc3 terminator, from pET32a-MSTN, amplify MSTN gene;
By three parts respectively glue reclaim purifying, design primer connects ADH1 promotor by the method for overlap PCR, Cyc3 terminator and MSTN gene are assembled into the MSTN gene fragment of 2890bp;
After PCR, carry out glue recovery, carry out double digestion with Xho I and EcoR I again, with same enzyme double digestion pBlue-kan-NTS5 ' 3 ' carrier, then carry out two-part connection, connection product is transformed in intestinal bacteria, obtains MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN;
The mono-clonal that picking transforms extracts plasmid, uses enzyme cutting method preliminary evaluation, and checks order.
Further, in the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector, in the time being Overlap PCR, MSTN and Cyc-3 two portions first increase;
Connect ADH1, MSTN, Cyc-3 tri-parts, first will be a Pool;
PCR program: 1. denaturation: 94 DEG C of 3m; 2. 94 DEG C of 1m of 8Cycle sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After completing, PCR program adds again primer ADH1 and the each 1ul of Cyc-3, continuation program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 3m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
Further, the concrete steps that MSTN carrier is integrated into Yeast genome by step 4 are:
By pBlue-kan-NTS2-5 ' 3'-MSTN carrier KpnI linearization for enzyme restriction, be transformed into yeast JMY1, due to the existence of the homology arm of NTS2-5 ' in yeast 3 ', make carrier and yeast strain generation high-level efficiency homologous recombination, after conversion, on the solid culture plate that contains G418, grown mono-clonal, picking wherein several mono-clonals extracts genome, by the sequence in MSTN primer PCR amplification integrative vector, whether qualification is integrated successful, thereby obtains positive colony, called after JMY11;
Further, in step 4, MSTN carrier is integrated into the PCR that checks MSTN to integrate in Yeast genome: 1. denaturation: 94 DEG C of 3m; 2. 25Cycle, 94 DEG C of 30s of sex change; 55 DEG C of 30s anneal; Extend 72 DEG C of 1m30s; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
Further, the concrete steps that knock out of step 5 resistant gene KanMX4 are:
Yeast strain JMB943 containing Cre enzyme carrier is proceeded in JMY11, due to the effect of Cre-LoxP system, KanMX4 resistant gene is knocked out, bacterium liquid after transforming is coated on the culture plate that lacks Ure, JMB943, containing Ure, can grow on the culture plate that lacks Ure, chooses mono-clonal to containing in the substratum of G418 resistance, on scarce Ure culture plate, grow, and the proof KanMX4 carrier of not growing in G418 resistance culture base knocks out;
Verify by the method for PCR, 4 mono-clonals of picking are carried genome, and pcr amplification KanMX4 expression casette fragment is 1826bp if do not knock out PCR product, be 346bp if knocked out PCR product, result demonstration obtains the MSTN integration bacterial strain that reporter gene KanMX4 knocks out.Called after JMB12;
Extract JMY12 yeast total protein, the expression with Western Blot detection MSTN in yeast JMY12.
The construction process of the MSTN barms that can be used for scale operation provided by the invention has built the integrated yeast strain of Myostatin (MSTN) that does not contain screening-gene, this bacterial classification can be produced in concentration type substratum, safe and reliable cost is low, can not cause goal gene to lose, be suitable for scale operation recombination yeast.First the present invention has built pBlue-kan-NTS2-5 ' the 3 '-MSTN carrier that contains KanMX4 resistance screening, MSTN is integrated directly in Yeast genome, adopt again Cre/LoxP system-kill resistance reporter gene, thereby obtain the integrated yeast expression bacterial strain of safe MSTN.
The yeast strain Direct-fed animal that adopts scale operation of the present invention, can produce high-level MSTN specific antibody in animal body, removes its restraining effect, promotes muscle growth, and this produces significant to animal.
Brief description of the drawings
Fig. 1 is the construction process schema of the MSTN barms that can be used for scale operation provided by the invention.
Fig. 2 is the amplification schematic diagram of the KanMX4 expression casette that provides of the embodiment of the present invention, and in figure, KanMX4 length is 1438bp.
Fig. 3 is that the enzyme of the plasmid pBlue-KanMX4 that provides of the embodiment of the present invention is cut qualification result schematic diagram.
Fig. 4 is that the 3NST2-5 ' that provides of the embodiment of the present invention and the enzyme of NST2-3 ' are cut qualification result schematic diagram; In figure, 1 is 2000Plus Maker, and 2,3,4 cut qualification result for the enzyme of pBlue-Kan-NTS2-5 ' 3 ' d; In figure, 2 is the result that KpnI and XhoI enzyme are cut NTS2-5 ' end homology arm and pBlue-Kan-NTS2-3 ' carrier framework; In figure, 3 is NotI, after SacII double digestion pBlue-Kan-NTS2-5 ' 3 ', and NTS2-3, the qualification result of end homology arm and pBlue-Kan-NTS2-5 '; In figure, 4 is KpnI and SacII double digestion carrier, and the enzyme that obtains pBlue skeleton and Kan4-NTS2-5 ' 3 ' is cut qualification result.
Fig. 5 is the amplification schematic diagram of the embodiment of the present invention MSTN gene, Cyc3 terminator and the ADH1 promotor that provide; 1:MSTN gene in figure; 2:Cyc3 terminator; ADH1 promotor.
Fig. 6 is the overlap pcr amplification result schematic diagram of the MSTN expression casette that provides of the embodiment of the present invention.
Fig. 7 is pBlue-Kan-NTS2-5 ' the 3'-MSTN vector construction collection of illustrative plates that the embodiment of the present invention provides.
Fig. 8 is that the enzyme of MSTN integrative vector pBlue-kan-NTS2-5 ' the 3 '-MSTN that provides of the embodiment of the present invention is cut qualification collection of illustrative plates.
Fig. 9 is that pBlue-kan-NTS2-5 ' 3'-MSTN that the embodiment of the present invention provides proceeds to yeast and obtains MSTN positive strain JMY11, and in figure 1,7,8PCR detects MSTN fragment and confirms to transform successfully.
Figure 10 is the deletion schematic diagram of the PCR qualification KanMX4 expression casette that provides of the embodiment of the present invention, and in figure, #1, #2, #3, #4 are the yeast strain that qualification KanMX4 knocks out, and clip size is 346bp.The positive control group of Control, is the bacterial strain that KanMX4 does not knock out, and clip size is 1826bp.
Figure 11 is the expression of MSTN in the Westren Blot qualification JMY12 yeast strain that provides of the embodiment of the present invention and protokaryon bacterial strain, in figure: (A) before 2 years, Western Blot detects, (B) be that after 2 years, Wetem Blot detects, confirmed that JMY12 yeast strain can preserve stably express for a long time.Ctl (-) is yeast negative control, and Ctl (+) is MSTN prokaryotic expression carrier, and JMY12 is the safely expressed carrier of MSTM that knocks out resistant gene.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
As shown in Figure 1, the construction process of the MSTN barms that can be used for scale operation provided by the invention comprises the following steps:
The structure of the amplification of S101:KanMX4 gene fragment and pBlue-KanMX4 carrier;
S102: the insertion of yeast genes homology arm;
The structure of S103:pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector;
S104: MSTN carrier is integrated into Yeast genome;
S105: resistant gene KanMX4 knocks out.
As a prioritization scheme of the embodiment of the present invention, the structure concrete steps of the amplification of step 1 KanMX4 gene fragment and pBlue-KanMX4 carrier are:
Design primer LoxP-Kan F, the LoxP-Kan R KanMX4 gene fragment that increases in the Yeast genome extracting;
Add LoxP sequence and EcoRI two sections of primers, two restriction enzyme sites of BamHI increase KanMX4 gene fragment out in Yeast genome, PCR program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After PCR, carry out solution recovery, carry out double digestion with EcoRI and BamHI, on 1% agarose gel electrophoresis, run glue, glue reclaims enzyme and cuts product and obtain purer fragment, with same enzyme double digestion pBluescript II SK+, then by two fragments 16 DEG C of connections of spending the night;
The connection product of acquisition is transformed in intestinal bacteria, the mono-clonal of selecting after transforming successfully wherein extracts bacterial plasmid, first use BamHI and EcoRI double digestion, the agargel electrophoresis of race 1% is determined the length of endonuclease bamhi, tentatively determine whether positive plasmid, and whether order-checking qualification plasmid successfully transforms.
As a prioritization scheme of the embodiment of the present invention, step 2 by the insertion concrete steps of Yeast genome homology arm is:
Be four primers that design NST2-5 ' end and two homology arms of NST2-3 ' end in NST2 fragment at the non-transcriptional of Yeast genome space2;
In NTS2-5 ' primer, insert the restriction enzyme site of XhoI and two restriction enzymes of KpnI;
In NTS2-3 ' primer, insert NotI, and the restriction enzyme site of two restriction enzymes of SacII;
NTS2-5 ' R and two primers of NTS2-5 ' F are added in PCR damping fluid, cooling 5min under 94 DEG C of room temperatures, directly obtain homology arm NTS2-5 ', with XhoI and KpnI double digestion NTS2-5 ' homology arm and pBlue-kan respectively, glue recovery two portions connect respectively again, connecting product is transformed in intestinal bacteria, after transforming successfully, the mono-clonal of selecting wherein extracts bacterial plasmid PCR qualification, order-checking is further determined, by identical method, NTS2-3 ' homology arm is inserted into, finally obtain pBlue-kan-NTS5 ' 3 ' carrier.
As a prioritization scheme of the embodiment of the present invention, the concrete steps of the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector are:
From JMB667, amplify ADH1 promotor and Cyc3 terminator, from pET32a-MSTN, amplify MSTN gene;
By three parts respectively glue reclaim purifying, design primer connects ADH1 promotor by the method for overlap PCR, Cyc3 terminator and MSTN gene are assembled into the MSTN gene fragment of 2890bp;
After PCR, carry out glue recovery, carry out double digestion with Xho I and EcoR I again, with same enzyme double digestion pBlue-kan-NTS5 ' 3 ' carrier, then carry out two-part connection, connection product is transformed in intestinal bacteria, obtains MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN;
The mono-clonal that picking transforms extracts plasmid, uses enzyme cutting method preliminary evaluation, and checks order.
As a prioritization scheme of the embodiment of the present invention, in the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector, in the time being Overlap PCR, MSTN and Cyc-3 two portions first increase;
Connect ADH1, MSTN, Cyc-3 tri-parts, first will be a Pool;
PCR program: 1. denaturation: 94 DEG C of 3m; 2. 94 DEG C of 1m of 8Cycle sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After completing, PCR program adds again primer ADH1 and the each 1ul of Cyc-3, continuation program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 3m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
As a prioritization scheme of the embodiment of the present invention, the concrete steps that MSTN carrier is integrated into Yeast genome by step 4 are:
By pBlue-kan-NTS2-5 ' 3'-MSTN carrier KpnI linearization for enzyme restriction, be transformed into yeast JMY1, due to the existence of the homology arm of NTS2-5 ' in yeast 3 ', make carrier and yeast strain generation high-level efficiency homologous recombination, after conversion, on the solid culture plate that contains G418, grown mono-clonal, picking wherein several mono-clonals extracts genome, by the sequence in MSTN primer PCR amplification integrative vector, whether qualification is integrated successful, thereby obtains positive colony, called after JMY11;
As a prioritization scheme of the embodiment of the present invention, in step 4, MSTN carrier is integrated into the PCR that checks MSTN to integrate in Yeast genome: 1. denaturation: 94 DEG C of 3m; 2. 25Cycle, 94 DEG C of 30s of sex change; 55 DEG C of 30s anneal; Extend 72 DEG C of 1m30s; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
As a prioritization scheme of the embodiment of the present invention, the concrete steps that knock out of step 5 resistant gene KanMX4 are:
Yeast strain JMB943 containing Cre enzyme carrier is proceeded in JMY11, due to the effect of Cre-LoxP system, KanMX4 resistant gene is knocked out, bacterium liquid after transforming is coated on the culture plate that lacks Ure, JMB943, containing Ure, can grow on the culture plate that lacks Ure, chooses mono-clonal to containing in the substratum of G418 resistance, on scarce Ure culture plate, grow, and the proof KanMX4 carrier of not growing in G418 resistance culture base knocks out;
Further verify by the method for PCR, 4 mono-clonals of picking are carried genome, and pcr amplification KanMX4 expression casette fragment is 1826bp if do not knock out PCR product, be 346bp if knocked out PCR product, result demonstration obtains the MSTN integration bacterial strain that reporter gene KanMX4 knocks out.Called after JMB12;
Extract JMY12 yeast total protein, the expression with Western Blot detection MSTN in yeast JMY12.
For making technical scheme of the present invention be convenient to understand, below in conjunction with embodiment, the present invention is further illustrated.
The present invention is intended to MSTN to be incorporated in Yeast genome, builds the yeast strain of the MSTN with certain security can be mass-produced.First carried out the structure of MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN, LoxP sequence is inserted to the reporter gene KanMX4 of MSTN integrative vector, the homologous recombination effect of recycling NTS2-5 ' 3 ' homology arm is incorporated into integrative vector in yeast strain, finally applied Cre/LoxP system-kill resistant gene KanMX4, acquisition can be applied to the integrated yeast strain of MSTN of scale operation.
The structure of the amplification of 1.KanMX4 gene fragment and pBlue-KanMX4 carrier
First, design primer LoxP-Kan F, the LoxP-Kan R KanMX4 gene fragment that increases in the Yeast genome extracting.LoxP-Kan F, LoxP-Kan R design of primers is as table 1.1:
Table 1.1LoxP-KanMX4 primer sequence
Add LoxP sequence and EcoRI two sections of primers, two restriction enzyme sites of BamHI increase KanMX4 gene fragment out in Yeast genome.PCR system is as shown in table 1.2.
Table 1.2KanMX4 amplification PCR system
PCR program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle (94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m); 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever
After PCR, carry out solution recovery, carry out double digestion with EcoRI and BamHI, on 1% agarose gel electrophoresis, run glue, glue reclaims enzyme and cuts product and obtain purer fragment, with same enzyme double digestion pBluescript IISK+, then by two fragments 16 DEG C of connections of spending the night.
The connection product of acquisition is transformed in intestinal bacteria, and the mono-clonal of selecting after transforming successfully wherein extracts bacterial plasmid.First use BamHI and EcoRI double digestion, run 1% agargel electrophoresis and determine the length of endonuclease bamhi, tentatively determine whether positive plasmid, then send order-checking qualification plasmid whether successfully to transform.
Table 1.3KanMX4 enzyme is cut system
2. by the insertion of Yeast genome homology arm
Four primers that design NST2-5 ' end and two homology arms of NST2-3 ' end in the non-transcriptional of Yeast genome space2 (NST2) fragment, homology arm sequences Design is as follows:
Table 2.1NTS2-5 ' and NTS2-3 ' homology arm sequence
In NTS2-5 ' primer, insert the restriction enzyme site of XhoI and two restriction enzymes of KpnI, the design of NTS2-5 ' primer sequence is as follows:
Table 2.2NTS2-5 ' primer sequence
In NTS2-3 ' primer, insert NotI, and the restriction enzyme site of two restriction enzymes of SacII, two primer sequence designs of NTS2-3 ' are as follows:
Table 2.3NTS2-3 ' primer sequence
NTS2-5 ' R and two primers of NTS2-5 ' F are added in PCR damping fluid, cooling 5min under 94 DEG C of room temperatures, directly obtain homology arm NTS2-5 ', with XhoI and KpnI double digestion NTS2-5 ' homology arm and pBlue-kan respectively, glue recovery two portions connect respectively again, connect product and are transformed in intestinal bacteria, after transforming successfully, the mono-clonal of selecting wherein extracts bacterial plasmid PCR qualification, then order-checking is further determined.By identical method, NTS2-3 ' homology arm is inserted into, finally obtain pBlue-kan-NTS5 ' 3 ' carrier.
The structure of 3.pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector
The JMB667 preserving from the present invention, amplify ADH1 promotor and Cyc3 terminator, from pET32a-MSTN, amplify MSTN gene.By three parts respectively glue reclaim purifying, design primer connects ADH1 promotor by the method for overlap PCR, Cyc3 terminator and MSTN gene are assembled into the MSTN gene fragment of 2890bp.After PCR, carry out glue recovery, carry out double digestion with Xho I and EcoR I again, with same enzyme double digestion pBlue-kan-NTS5 ' 3 ' carrier, then carry out two-part connection, connection product is transformed in intestinal bacteria, obtains MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN.The mono-clonal that picking transforms extracts plasmid, uses enzyme cutting method preliminary evaluation, then checks order.
In the time being Overlap PCR, first increase MSTN and Cyc-3 two portions, PCR system is as follows:
The PCR reaction system of table 3.1MSTN+Cyc-3
Connect ADH1, MSTN, Cyc-3 tri-parts.First will be a Pool, system is as follows:
The PCR reaction system of table 3.2 ADH1+MSTN+Cyc-3
PCR program: 1. denaturation: 94 DEG C of 3m; 2. 8 cycle (94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m); 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever
After completing, this program adds again primer ADH1 and the each 1ul of Cyc-3, continuation program:
1. denaturation: 94 DEG C of 3m; 2. 20 Cycle (94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 3m); 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever
4. MSTN carrier is integrated into Yeast genome
By pBlue-kan-NTS2-5'3'-MSTN carrier KpnI linearization for enzyme restriction, be transformed into yeast JMY1, due to the existence of the homology arm of NTS2-5 ' in yeast 3 ', make carrier and yeast strain generation high-level efficiency homologous recombination, after conversion, on the solid culture plate that contains G418, grown mono-clonal, picking wherein several mono-clonals extracts genome, by the sequence in MSTN primer PCR amplification integrative vector, whether qualification is integrated successful, thereby obtains positive colony, called after JMY11.
The PCR system that table 4.1 checks MSTN to integrate
PCR: 1. denaturation: 94 DEG C of 3m; 2. 25Cycle (94 DEG C of 30s of sex change; 55 DEG C of 30s anneal; Extend 72 DEG C of 1m30s); 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever
5. knocking out of resistant gene KanMX4
Yeast strain JMB943 containing Cre enzyme carrier is turned in JMY11, due to the effect of Cre-LoxP system, KanMX4 resistant gene is knocked out.Bacterium liquid after transforming is coated in and lacks that on the culture plate of Ure, (JMB943 is containing Ure, can on the culture plate that lacks Ure, grow), choose mono-clonal to containing in the substratum of G418 resistance, on scarce Ure culture plate, grow, and the proof KanMX4 carrier of not growing in G418 resistance culture base knocks out.
Further verify by the method for PCR, 4 mono-clonals of picking are carried genome, and pcr amplification KanMX4 expression casette fragment is 1826bp if do not knock out PCR product, be 346bp if knocked out PCR product, result demonstration obtains the MSTN integration bacterial strain that reporter gene KanMX4 knocks out.Called after JMB12.
Table 5.1 is checked the PCR reaction system of KanMX4
PCR: 1. denaturation: 94 DEG C of 3m; 2. 35Cycle (94 DEG C of 30s of sex change; 47 DEG C of 30s anneal; Extend 72 DEG C of 2m);
3. extend 72 DEG C of 5m; 4. 4 DEG C of forever
Extract JMY12 yeast total protein, the expression with Western Blot detection MSTN in yeast JMY12.Result demonstration yeast strain has been expressed the MSTN of about 54KD.
The thinking of entirety of the present invention is: build the basic integrative vector pBlue-Kan-NTS2-5 ' 3 ' that contains reporter gene KanMX4, LoxP sequence, NTS2-5 ' end and two homology arms of NTS2-3 ' end.
The amplification of KanMX4 expression casette as shown in Figure 2, KanMX4 length is 1438bp.
The enzyme of plasmid pBlue-KanMX4 is as shown in Figure 3 cut qualification result.
NST2-5 ' as shown in Figure 4 and the enzyme of NST2-3 ' are cut qualification result, and in figure, 1 is 2000Plus Maker, and 2,3,4 cut qualification result for the enzyme of pBlue-Kan-NTS2-5 ' 3 ' d; 2 is the result that KpnI and XhoI enzyme are cut NTS2-5 ' end homology arm and pBlue-Kan-NTS2-3 ' carrier framework; 3 is NotI, after SacII double digestion pBlue-Kan-NTS2-5 ' 3 ', the qualification result of NTS2-3 ' end homology arm and pBlue-Kan-NTS2-5 ', 4 is KpnI and SacII double digestion carrier, the enzyme that obtains pBlue skeleton and Kan4-NTS2-5 ' 3 ' is cut qualification result.
ADH1 promotor, Cyc3 terminator and MSTN gene are assembled into the MSTN expression casette of 2890bp with overlap PCR.Be inserted into again in pBlue-Kan-NTS2-5 ' 3 ' carrier, obtain pBlue-Kan-NTS2-5 ' 3'-MSTN integrative vector.
The amplification of MSTN gene, Cyc3 terminator and ADH1 promotor as shown in Figure 5,1:MSTN gene in figure; 2:Cyc3 terminator; ADH1 promotor.
The overlap pcr amplification result of MSTN expression casette as shown in Figure 6.
The final carrier building as shown in Figure 7, i.e. pBlue-Kan-NTS2-5 ' 3'-MSTN vector construction collection of illustrative plates.
The enzyme of MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN is as shown in Figure 8 cut qualification collection of illustrative plates.
MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN is proceeded to Yeast genome JMY1, obtain positive yeast expression bacterial strain JMY11.Cre enzyme carrier JMB943 is proceeded in JMY11, knock out KanMX4 resistance, obtain safe yeast strain JMY12.Identify by the method for resistance culture base and PCR, determine that finally obtaining positive yeast expression bacterial strain has pounded out KanMX4 resistant gene.Expression with Western Blot detection MSTN in eukaryotic cell.Finally obtain the security integration carrier of MSTN.
Fig. 9 is that pBlue-kan-NTS2-5 ' 3'-MSTN proceeds to yeast and obtains MSTN positive strain JMY11, and in figure 1,7,8PCR detects MSTN fragment and confirms to transform successfully.
Figure 10 is the deletion of PCR qualification KanMX4 expression casette, and in figure, #1, #2, #3, #4 are the yeast strain that qualification KanMX4 knocks out, and clip size is 346bp.The positive control group of Control, is the bacterial strain that KanMX4 does not knock out, and clip size is 1826bp.
Figure 11 is the expression of MSTN in Westren Blot qualification JMY12 yeast strain and protokaryon bacterial strain, (A) before 2 years, Western Blot detects, (B) be that after 2 years, Wetem Blot detects, confirmed that JMY12 yeast strain can preserve stably express for a long time.Ctl (-) is yeast negative control, and Ctl (+) is MSTN prokaryotic expression carrier, and JMY12 is the safely expressed carrier of MSTM that knocks out resistant gene.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. a construction process that can be used for the MSTN barms of scale operation, is characterized in that, this construction process that can be used for the MSTN barms of scale operation comprises the following steps:
The structure of the amplification of step 1, KanMX4 gene fragment and pBlue-KanMX4 carrier;
The insertion of step 2, yeast genes homology arm;
The structure of step 3, pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector;
Step 4, MSTN carrier is integrated into Yeast genome;
Step 5, resistant gene KanMX4 knock out.
2. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 1, is characterized in that, the structure concrete steps of the amplification of step 1 KanMX4 gene fragment and pBlue-KanMX4 carrier are:
Design primer LoxP-Kan F, the LoxP-Kan R KanMX4 gene fragment that increases in the Yeast genome extracting;
Add LoxP sequence and EcoRI two sections of primers, two restriction enzyme sites of BamHI increase KanMX4 gene fragment out in Yeast genome, PCR program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After PCR, carry out solution recovery, carry out double digestion with EcoRI and BamHI, on 1% agarose gel electrophoresis, run glue, glue reclaims enzyme and cuts product and obtain purer fragment, with same enzyme double digestion pBluescript II SK+, then by two fragments 16 DEG C of connections of spending the night;
The connection product of acquisition is transformed in intestinal bacteria, the mono-clonal of selecting after transforming successfully wherein extracts bacterial plasmid, first use BamHI and EcoRI double digestion, the agargel electrophoresis of race 1% is determined the length of endonuclease bamhi, tentatively determine whether positive plasmid, and whether order-checking qualification plasmid successfully transforms.
3. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 1, is characterized in that, step 2 by the insertion concrete steps of Yeast genome homology arm is:
Be four primers that design NST2-5 ' end and two homology arms of NST2-3 ' end in NST2 fragment at the non-transcriptional of Yeast genome space2;
In NTS2-5 ' primer, insert the restriction enzyme site of XhoI and two restriction enzymes of KpnI;
In NTS2-3 ' primer, insert NotI, and the restriction enzyme site of two restriction enzymes of SacII;
NTS2-5 ' R and two primers of NTS2-5 ' F are added in PCR damping fluid, cooling 5min under 94 DEG C of room temperatures, directly obtain homology arm NTS2-5 ', with XhoI and KpnI double digestion NTS2-5 ' homology arm and pBlue-kan respectively, glue recovery two portions connect respectively again, connecting product is transformed in intestinal bacteria, after transforming successfully, the mono-clonal of selecting wherein extracts bacterial plasmid PCR qualification, order-checking is further determined, by identical method, NTS2-3 ' homology arm is inserted into, finally obtain pBlue-kan-NTS5 ' 3 ' carrier.
4. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 1, is characterized in that, the concrete steps of the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector are:
From JMB667, amplify ADH1 promotor and Cyc3 terminator, from pET32a-MSTN, amplify MSTN gene;
By three parts respectively glue reclaim purifying, design primer connects ADH1 promotor by the method for overlap PCR, Cyc3 terminator and MSTN gene are assembled into the MSTN gene fragment of 2890bp;
After PCR, carry out glue recovery, carry out double digestion with Xho I and EcoR I again, with same enzyme double digestion pBlue-kan-NTS5 ' 3 ' carrier, then carry out two-part connection, connection product is transformed in intestinal bacteria, obtains MSTN integrative vector pBlue-kan-NTS2-5 ' 3'-MSTN;
The mono-clonal that picking transforms extracts plasmid, uses enzyme cutting method preliminary evaluation, and checks order.
5. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 4, it is characterized in that, in the structure of step 3 pBlue-kan-NTS2-5 ' 3'-MSTN integrative vector, in the time being Overlap PCR, MSTN and Cyc-3 two portions first increase;
Connect ADH1, MSTN, Cyc-3 tri-parts, first will be a Pool;
PCR program: 1. denaturation: 94 DEG C of 3m; 2. 94 DEG C of 1m of 8Cycle sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 2m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever;
After completing, PCR program adds again primer ADH1 and the each 1ul of Cyc-3, continuation program: 1. denaturation: 94 DEG C of 3m; 2. 20Cycle, 94 DEG C of 1m of sex change; 45 DEG C of 30s anneal; Extend 72 DEG C of 3m; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
6. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 1, is characterized in that, the concrete steps that MSTN carrier is integrated into Yeast genome by step 4 are:
By pBlue-kan-NTS2-5 ' 3'-MSTN carrier KpnI linearization for enzyme restriction, be transformed into yeast JMY1, due to the existence of the homology arm of NTS2-5 ' in yeast 3 ', make carrier and yeast strain generation high-level efficiency homologous recombination, after conversion, on the solid culture plate that contains G418, grown mono-clonal, picking wherein several mono-clonals extracts genome, by the sequence in MSTN primer PCR amplification integrative vector, whether qualification is integrated successful, thereby obtains positive colony, called after JMY11.
7. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 6, is characterized in that, in step 4, MSTN carrier is integrated into the PCR that checks MSTN to integrate in Yeast genome: 1. denaturation: 94 DEG C of 3m; 2. 25Cycle, 94 DEG C of 30s of sex change; 55 DEG C of 30s anneal; Extend 72 DEG C of 1m30s; 3. extend 72 DEG C of 5m; 4. 4 DEG C of forever.
8. the construction process of the MSTN barms that can be used for scale operation as claimed in claim 1, is characterized in that, the concrete steps that knock out of step 5 resistant gene KanMX4 are:
Yeast strain JMB943 containing Cre enzyme carrier is proceeded in JMY11, due to the effect of Cre-LoxP system, KanMX4 resistant gene is knocked out, bacterium liquid after transforming is coated on the culture plate that lacks Ure, JMB943, containing Ure, can grow on the culture plate that lacks Ure, chooses mono-clonal to containing in the substratum of G418 resistance, on scarce Ure culture plate, grow, and the proof KanMX4 carrier of not growing in G418 resistance culture base knocks out:
Further verify by the method for PCR, 4 mono-clonals of picking are carried genome, and pcr amplification KanMX4 expression casette fragment is 1826bp if do not knock out PCR product, be 346bp if knocked out PCR product, result demonstration obtains the MSTN integration bacterial strain that reporter gene KanMX4 knocks out.Called after JMB12;
Extract JMY12 yeast total protein, the expression with Western Blot detection MSTN in yeast JMY12.
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| CN1569230A (en) * | 2003-11-05 | 2005-01-26 | 华南农业大学 | Preparation and application of a gene engineering vaccine of muscle growth retarder |
| US20060216279A1 (en) * | 2005-03-22 | 2006-09-28 | Glass David J | Myostatin inhibiting fusion polypeptides and therapeutic methods thereof |
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| CN1569230A (en) * | 2003-11-05 | 2005-01-26 | 华南农业大学 | Preparation and application of a gene engineering vaccine of muscle growth retarder |
| US20060216279A1 (en) * | 2005-03-22 | 2006-09-28 | Glass David J | Myostatin inhibiting fusion polypeptides and therapeutic methods thereof |
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