CN105647959B - A method of building yeast multi-copy expression vector - Google Patents
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Abstract
The invention discloses a kind of methods for constructing yeast multi-copy expression vector, the first vector and Second support with target gene are chosen first, then digestion is carried out to first vector and Second support using a pair of of isocaudarner and a reference enzyme, obtain the subsequence with target gene and auxiliary sequence, obtain the destination carrier having with reference to segment, it is finally quickly screened using referring to segment, obtains the multicopy expression vector.Through the invention, the multicopy number carrier containing specific copy number and specific gene can fast and accurately be constructed, reduce the probability that expression cassette in conventional building multicopy expression vector method connects the generation false positive factors such as direction is random, carrier is direct-connected or digestion process is insufficient, it is easy to operate, it is high-efficient.
Description
Technical field
The invention belongs to genetic engineering fields, more particularly, to a kind of method for constructing yeast multi-copy expression vector.
Background technique
In recent years, yeast expression system is widely used in the expression of various exogenous proteins, and obtains higher egg
White expression.There are many factor for influencing Yeast expression level, selection, foreign gene such as the characteristic, promoter of foreign gene
Copy number, glycosylation, condition of culture and the fermentation processes of foreign gene etc..
The copy number for increasing target gene tends to the expression for effectively improving destination protein, however target gene
Copy number is excessively high, can cause endoplasmic reticulum pressure to engineering bacteria instead and lead to the reduction of expression.Therefore, gene appropriate is copied
Shellfish number is conducive to the expression of foreign protein.Artificial constructed external multicopy expression vector is currently utilized, transformed yeast obtains copy more
Shellfish bacterial strain is one of common technology.Common, the method for the outer multicopy expression vector of construct can be public using Invitrogen
The pAO815 serial carrier of exploitation is taken charge of, utilizes expression cassette both ends isocaudarner restriction enzyme site Bgl II and BamH I in this method, together
Tail enzyme digestion is inserted into BamH I site after separating expression cassette to generate tandem sequence repeats expression cassette.This method is able to achieve the structure of multicopy
It builds, but the reasons such as the direction of expression cassette insertion is random, carrier connects certainly cause false positive rate in building process very high, and are difficult to
Screen specific copy number expression vector.
In recent years, the application of " biological brick " method can quickly and effectively construct multicopy expression vector, and this method can be effective
The direction of expression cassette insertion is controlled, while being avoided that carrier from connecting, can greatly improve building efficiency.However, conventional " biology
In brick " method such as patent document CN200410011044, when restriction enzyme site is close, due to after digestion segment and inabundant enzyme
The carrier lengths cut are close, it is difficult to be screened, can not quickly and effectively judge and screening positive clone is sub, can only pass through the later period
Plasmid extraction, restriction analysis carry out the verifying of multicopy expression vector, process very complicated.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of expression of building yeast multi-copy to carry
The method of body, its object is to using with reference to segment carry out destination carrier screening, to improve the structure of multicopy expression vector
Build efficiency.
To achieve the above object, according to one aspect of the present invention, a kind of building yeast multi-copy expression vector is provided
Method, comprising the following steps:
(1) first vector and Second support are chosen, wherein first vector includes the first expression cassette and refers to segment, the
Two carriers are including the second expression cassette and refer to segment；
First expression cassette has m first object gene, and second expression cassette has n the second target genes, m
With the integer that n is more than or equal to 1；It is described that there is the reference restriction enzyme site that can be referenced enzyme digestion, and the reference with reference to segment
Restriction enzyme site is all unique sequence code in the first vector and the gene order of Second support respectively；
In the first vector, the 3 ' ends with reference to segment to the gene sequence between 5 ' ends of first expression cassette
Column have can be by the first restriction enzyme site of the first enzyme digestion, and first restriction enzyme site is in the downstream of first restriction enzyme site
It is unique in gene order between end and the upstream end with reference to restriction enzyme site；
On the Second support, the 3 ' of second expression cassette are held to the gene sequence between the 5 ' ends with reference to segment
Column have can be by the second restriction enzyme site of second enzyme digestion, and second restriction enzyme site is in the downstream with reference to restriction enzyme site
It is unique in gene order between end and the upstream end of second restriction enzyme site；
First enzyme and second enzyme are isocaudarner；
(2) the first enzyme and reference enzyme digestion first vector are utilized, downstream and the institute of first restriction enzyme site are recycled
Stating the gene order between the upstream end with reference to restriction enzyme site is subsequence；
Meanwhile using second enzyme and reference enzyme digestion Second support, recycle the downstream with reference to restriction enzyme site and
Gene order between the upstream end of second restriction enzyme site is auxiliary sequence；
(3) subsequence and auxiliary sequence are connected, so that the downstream of the first restriction enzyme site of the subsequence and female sequence
The upstream end connection of second restriction enzyme site of column, the upstream end of the reference restriction enzyme site of the subsequence and the ginseng of the auxiliary sequence
The upstream end connection for examining restriction enzyme site, obtains destination carrier；
(4) it filters out with the destination carrier with reference to segment, obtaining has m first object gene and n the second mesh
Mark the multicopy expression vector of gene.
Preferably, 3 ' ends of first expression cassette are to the gene order between 5 ' ends of first expression cassette, with institute
3 ' the ends for stating the second expression cassette are identical to the gene order between 5 ' ends of second expression cassette.
Preferably, the first object gene is identical as the second target gene, and the multicopy expression vector has m+n
First object gene.
Preferably, the yeast is Pichia pastoris.
Preferably, the length of the expression cassette is less than or equal to 100bp.
Preferably, the method screened in the step (4) is bacterium colony PCR identification.
Preferably, the reference segment is antibiotic resistance gene, and the screening technique in the step (4) is antibiotic plate
Method.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, due in multicopy table
Up to introducing in the building of carrier with reference to segment, can achieve the following beneficial effects:
1, the present invention introduces one and is used as label with reference to segment in building process, can be better in screening process
Judge whether that digestion is abundant, reduce false positive rate, reduces the workload of screening positive clone, pass through letter on its basis
Single digestion verification is that can verify that the accuracy of constructed multicopy expression vector；
2, in the range of plasmid can accommodate, method of the invention can be not only used for the table of any number of multicopy
Up to the rapid build of carrier, it can be used for the building of the coexpression vector of several genes multicopy, have a wide range of application；
3, it is preferably used as by the segment with functional gene and refers to segment, so that screening process is easier quickly, into
One step reduces false positive rate；
4, the present invention is based on biological brick methods, can fast and accurately construct the expression vector containing specific copy number, reduce
The probability for the generation false positive factors such as expression cassette connection direction is random in conventional method or carrier is direct-connected, does not need using dephosphorization
Good effect can be obtained in the operations such as acidification, and experimental implementation is simple.
Detailed description of the invention
Fig. 1 is the method flow schematic diagram of present invention building yeast multi-copy expression vector, wherein the first digestion of A- position
Point, the second restriction enzyme site of B-, C- refer to restriction enzyme site；
Fig. 2 is that single copy expression vector pAO α-rol constructs schematic diagram；
Fig. 3 is that double copy expression vector pAO α -2rol schematic diagrames are constructed using the method for the present invention；
Fig. 4 is expression vector pAO α-rol, pAO α -2rol, pAO α -3rol and pAO α -4rol pass through Bgl II and BamH
I double digestion rear electrophoresis detection figure.Wherein swimming lane 1: sample is pAO α-rol, and swimming lane 2: sample is pAO α -2rol, swimming lane 3: sample
For pAO α -3rol, swimming lane 4: sample is pAO α -4rol；
Fig. 5 is expression vector pAO α-rol, pAO α -2rol, pAO α -3rol and pAO α -4rol after BamH I digestion
Electrophoresis detection figure.Wherein swimming lane 1: sample is pAO α-rol, and swimming lane 2: sample is pAO α -2rol, and swimming lane 3: sample is pAO α -
3rol, swimming lane 4: sample is pAO α -4rol；
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
One aspect of the present invention provides a kind of method for constructing yeast multi-copy expression vector, includes the following steps,
Its flow diagram is as shown in Figure 1, wherein the first restriction enzyme site of A-, the second restriction enzyme site of B-, C- refer to restriction enzyme site；
(1) first vector and Second support are chosen, wherein first vector includes the first expression cassette and refers to segment, the
Two carriers are including the second expression cassette and refer to segment；
First expression cassette has m first object gene, and second expression cassette has n the second target genes, m
With the integer that n is more than or equal to 1；When first object gene and identical the second target gene, it can be used for constructing multicopy expression
Carrier can be used for constructing first object gene and the second target gene when first object gene and the second target gene difference
Coexpression vector；
It is described that there is the reference restriction enzyme site that can be referenced enzyme digestion with reference to segment, and the restriction enzyme site that refers to exists respectively
It is all unique sequence code in the first vector and the gene order of Second support；
In the first vector, the 3 ' ends with reference to segment to the gene sequence between 5 ' ends of first expression cassette
Column have can be by the first restriction enzyme site of the first enzyme digestion, and first restriction enzyme site is in the downstream of first restriction enzyme site
It is unique in gene order between end and the upstream end with reference to restriction enzyme site；
On the Second support, the 3 ' of second expression cassette are held to the gene sequence between the 5 ' ends with reference to segment
Column have can be by the second restriction enzyme site of second enzyme digestion, and second restriction enzyme site is in the downstream with reference to restriction enzyme site
It is unique in gene order between end and the upstream end of second restriction enzyme site；Usually, in building multicopy expression
During carrier, usually using identical carrier, i.e., the 3 ' of described first expression cassette, which are held to the 5 ' of first expression cassette, holds it
Between gene order, the gene order between the 5 ' ends at the 3 ' of second expression cassette ends to second expression cassette is identical；
It is more convenient in this way in subsequent needs further building；
First enzyme and second enzyme are isocaudarner；
(2) the first enzyme and reference enzyme digestion first vector are utilized, downstream and the institute of first restriction enzyme site are recycled
Stating the gene order between the upstream end with reference to restriction enzyme site is subsequence；The subsequence includes the first expression cassette and reference plate
The upstream portion of section；
Meanwhile using second enzyme and reference enzyme digestion Second support, recycle the downstream with reference to restriction enzyme site and
Gene order between the upstream end of second restriction enzyme site is auxiliary sequence；The auxiliary sequence includes the second expression cassette and reference
The downstream part of segment；
(3) subsequence and auxiliary sequence are connected, since the first enzyme, second enzyme and reference enzyme are different, and the first enzyme and second
Enzyme is isocaudarner；At this point, the second restriction enzyme site of the downstream of the first restriction enzyme site of the subsequence and the auxiliary sequence
Upstream end connection, the upstream end of the reference restriction enzyme site of the upstream end and auxiliary sequence of the reference restriction enzyme site of the subsequence
Connection obtains destination carrier；
(4) destination carrier necessarily has with reference to segment, and false positive clones does not have then with reference to segment, at this point, utilizing
Bacterium colony PCR identification can be filtered out with the destination carrier with reference to segment, can be obtained a with m first object gene and n
The multicopy expression vector of second target gene.
If using antibiotic resistance gene as refer to segment, at this point, in this step if can also be directly with antibiotic flat band method
It is screened, to simplify screening step.
In step (2), digestion is avoided to have insufficient part, needs to isolate the subsequence in first vector, non-enzyme
Complete first vector is cut, and other sequences that digestion obtains in addition to subsequence, needs the genetic fragment between three long
Degree has obvious gap；It can be before step (1), using to the first restriction enzyme site, the second restriction enzyme site on carrier and referring to digestion
The selection in site is adjusted；For example, the distance between the first restriction enzyme site and reference enzyme restriction enzyme site and the second digestion position
Maximum distance between point and reference enzyme restriction enzyme site is preferably 100bp or more, convenient for effectively judging whether digestion is abundant.Together
Sample, the separation for auxiliary sequence, is applied equally to the above method.
In addition, the first restriction enzyme site is usual with a distance from nearest first object gene when choosing the first restriction enzyme site
For it is more closer better, to avoid have when constructing multicopy expression vector excessive amorph to influence expression effect
Rate；When choosing the second restriction enzyme site, the second restriction enzyme site is typically also to get over a distance from the second nearest target gene
Close better, reason is same as above.When actually choosing restriction enzyme site, the distance of restriction enzyme site can be integrated, it is unique in gene order
The composite factors such as property are considered, to obtain optimal scheme.
In actual multi-copy vector building process, first it can go out double copy expression loads by singly copying expression vector establishment
Body recycles the above method to construct repeatedly, until obtaining the expression vector of required gene and copy number.
The following contents is embodiment:
Embodiment 1 constructs Rhizopus oryzae lipase multicopy expression vector pAO α -2rol
Bacterial strain and plasmid: Escherichia coli Top10 competent cell and carrier T-rol containing Rhizopus oryzae lipase gene from
It makes and saves；PPICZ α A and pAO815 expression vector is purchased from Invitrogen company.
Enzyme and kit etc.: the purchase such as enzyme preparation used in restriction enzyme, PCR, connection enzyme reagent kit Solution I
In Takara company；Plasmid extraction kit and DNA QIAquick Gel Extraction Kit are purchased from Omega company.
Experimental material preparation:
Building Rhizopus oryzae lipase list copies expression vector pAO α-rol first, as shown in Figure 2:
A. design synthesis clone Rhizopus oryzae lipase gene rol primer ROL-F and ROL-R, sequence information are as follows:
Rol-F:5 '-CTGAATTCGTTCCTGTTTCTGGTAAATC-3 '
Rol-R:5 '-CCGCTCGAGTTACAAACAGCTTCCTTCGT-3 '
B. using the carrier T-rol containing Rhizopus oryzae lipase gene as template, using primer Rol-F and Rol-R, PCR expands
Increasing obtains target fragment, recovery purifying after agarose gel electrophoresis；
C. to recycle after EcoR I and Xho I double digestion, then pass through EcoR I and Sal with commercial carrier pPICZ α A
The carrier that glue recycles after I digestion (using the feature of Xho I and Sal I isocaudarner, is eliminated in pPICZ α A with this and is deposited with 3:1 mixing
The site SalI), convert Escherichia coli after connecting 1h at 16 DEG C with the connection kit Solution I of Takara company
Top10 competent cell；
D. the LB plate screening clone containing ammonia benzyl mycin is passed through, is carried out with primer Rol-F and Rol-R to competent cell
Bacterium colony PCR is carried out, pPICZ α A-rol is named as after sequence verification is correct.
E. carrier pPICZ α A-rol and pAO815 using Sac I and BamH I double digestion and are recycled into target product, connected
Target product and to convert Escherichia coli Top10 competence thin, carries out positive gram of bacterium colony PCR verifying with primer Rol-F and Rol-R
It is grand, pAO α-rol is named as after sequence verification is correct.
PCR response procedures in step d and e are as follows: 95 DEG C of 4min；94 DEG C of 1min, 56 DEG C of 30s, 72 DEG C of 1min, circulation 30
It is secondary；72℃7min.
Rhizopus oryzae lipase multicopy expression vector pAO α -2rol is constructed, as shown in Figure 3:
S1. since pAO α-rol carrier is in the Sal I site of His4 Gene Partial existence anduniquess, and rol expression cassette both ends
Containing Bgl II and BamH I isocaudarner restriction enzyme site, genetic fragment length between the downstream of BamH I and the upstream end of SalI
About 1600bp；
S2. plasmid pAO α-rol is divided for two parts, a part glue recycling carrier portion after BamH I and Sal I double digestion
Point, a part glue after Bgl II and Sal I double digestion recycles expression cassette part, and two parts product separately includes His4 gene
Upstream and downstream part；
S3. two recycling segments are connected at 16 DEG C after being mixed with 1:1 with the connection kit Solution I of Takara company
Escherichia coli Top10 competent cell is converted after meeting 1h；
S4. PCR primer (His-F:5 '-should be designed comprising complete His4 gene by constructing correct pAO α -2rol
ATGACATTTCCCTTGCTACCTG-3 ', His-R:5 '-TTAAATAAGTCCCAGTTTCTCC-3 ') 10 clones are selected at random
Son carries out bacterium colony PCR identification, PCR response procedures are as follows: 95 DEG C of 4min；94 DEG C of 1min, 56 DEG C of 30s, 72 DEG C of 75s are recycled 30 times；72
℃7min。
Continue to use Bgl II and BamH I after PCR totally 6 clones for obtaining purpose band (about 2500bp) are taken out plasmid
Digestion verification is carried out, digestion size meets theory expectation, it is known that the success of pAO α -2rol vector construction.
Embodiment 2 constructs Rhizopus oryzae lipase multicopy expression vector pAO α -3rol
Embodiment 1 is repeated with the same steps, difference is, in the step 2, plasmid pAO α-rol is passed through
After BamH I and Sal I double digestion glue recycle carrier part, by plasmid pAO α -2rol after Bgl II and Sal I double digestion glue
Recycle expression cassette part.
Embodiment 3 constructs Rhizopus oryzae lipase multicopy expression vector pAO α -4rol
Embodiment 1 is repeated with the same steps, difference is, replaces pAO α-rol with pAO α -2rol.
With pAO α-rol, pAO α -2rol after BamH I and Sal I double digestion glue recycle carrier part, respectively with pAO
By Bgl II, the glue recycling expression cassette part after Sal I double digestion connects conversion to α -2rol, selects each 10 clones and passes through
Bacterium colony PCR identification, there is 4 respectively and 6 clones can obtain purpose band, takes wherein each 2 clones progress digestion verification,
Digestion result meets theory expectation, that is, constructs successfully pAO α -3rol and pAO α -4rol.
Fig. 4 is expression vector pAO α-rol, pAO α -2rol, pAO α -3rol and pAO α -4rol pass through Bgl II and BamH
I double digestion rear electrophoresis detection figure.Wherein swimming lane 1: sample is pAO α-rol, and clip size respectively may be about 2400bp, 4030bp and
2650bp, swimming lane 2: sample is pAO α -2rol, and clip size respectively may be about 2400bp, 4030bp and 5300bp, swimming lane 3: sample
For pAO α -3rol, clip size respectively may be about 2400bp, 4030bp and 7950bp, swimming lane 4: sample is pAO α -4rol, segment
Size respectively may be about 2400bp, 4030bp and 10600bp；
Fig. 5 is expression vector pAO α-rol, pAO α -2rol, pAO α -3rol and pAO α -4rol after BamH I digestion
Electrophoresis detection figure.Wherein swimming lane 1: sample is pAO α-rol, about 9090bp, swimming lane 2: sample is pAO α -2rol, about 11750bp,
Swimming lane 3: sample is pAO α -3rol, and about 14400bp, swimming lane 4: sample is pAO α -4rol, about 17050bp.
It can be seen that from the result of the above electrophoresis photographs, pAO α -2rol, pAO α -3rol and pAO α -4rol hold shellfish expression more
Vector construction success.
Embodiment 4 constructs multicopy expression vector pPICZ α A-2rol
S1. analysis carrier sequence knows the existence anduniquess digestion at antibiotic marker genes zeocin of pPICZ α A-rol carrier
Site Aat II intends choosing reference segment of the zeocin gene as building multi-copy vector, the downstream of BamH I and Aat
The length of genetic fragment is about 530bp between the upstream end of II.
S2. 2 parts of pPICZ α A-rol carriers are taken, portion recycles expression cassette part after Bgl II and Aat II digestion, another
Carrier part is recycled in part after BamH I and Aat II digestion, and two parts product separately includes the upstream of zeocin gene under
Swim part；
S3. after two recovery product 1:1 mixing after 16 DEG C of connection 1h of Solution I ligase, conversion to Escherichia coli
Top10 competent cell；
S4. it is screened with the LLB resistant panel containing bleomycin.It can be broken in the clone explanation that resistant panel is grown
Bad zeocin gene restores complete after connection, select at random 3 clones take out it is bis- with BamH I and Bgl II after plasmids
Digestion verification, verification result show that 3 clones are all correct.Successfully two copies expression vector is constructed, pPICZ is named as
αA-2rol。
Embodiment 5 constructs Rhizopus oryzae lipase multicopy expression vector pPICZ α A-3rol
Embodiment 4 is repeated with the same steps, difference is, in the step 2, by plasmid pPICZ α A-rol
Carrier recycles expression cassette part after Bgl II and Aat II digestion, and plasmid pPICZ α A-2rol carrier is through BamH I and Aat II
Carrier part is recycled after digestion.
Embodiment 6 constructs Rhizopus oryzae lipase multicopy expression vector pPICZ α A-4rol
Embodiment 4 is repeated with the same steps, difference is, replaces pPICZ α A-rol with pPICZ α A-2rol.
With pPICZ α A-rol, pPICZ α A-2rol, glue recycles expression cassette part after Bgl II and Aat II double digestion,
Respectively with pAO α -2rol after BamH I and Aat II double digestion glue recycling carrier part mixed by 1:1 after, process
Escherichia coli Top10 competent cell is converted after 16 DEG C of connection 1h of Solution I ligase, by the LLB containing bleomycin
Resistant panel is incubated overnight screening at 37 DEG C, from being selected at random respectively after plasmid is taken out in 3 cultures in the clone grown with BamH
The verifying of I and Bgl II double digestion, wherein 3 clone digestion verifications are correct, as pPICZ α A-3rol and pPICZ α A-4rol.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (7)
1. a kind of method for constructing yeast multi-copy expression vector, which comprises the following steps:
(1) choose first vector and Second support: the first vector is including the first expression cassette and refers to segment, and described second
Carrier is including the second expression cassette and refers to segment；
First expression cassette has m first object gene, and second expression cassette has n the second target genes, m and n
For the integer more than or equal to 1；It is described that there is the reference restriction enzyme site that can be referenced enzyme digestion, the reference digestion position with reference to segment
Point is all unique sequence code in the first vector and the gene order of Second support；
In the first vector, it is described with reference to segment 3 ' end to first expression cassette 5 ' end between have can be by first
First restriction enzyme site of enzyme digestion, and downstream and the reference of first restriction enzyme site in first restriction enzyme site
It is unique between the upstream end of restriction enzyme site；
On the Second support, second expression cassette 3 ' end to it is described with reference to segment 5 ' end between have can be by second
Second restriction enzyme site of enzyme digestion, and second restriction enzyme site is in the downstream with reference to restriction enzyme site and described second
It is unique between the upstream end of restriction enzyme site；
First enzyme and second enzyme are isocaudarner；
The distance between first restriction enzyme site and reference enzyme restriction enzyme site and the second restriction enzyme site and reference enzyme digestion position
The distance between point is greater than or equal to 100bp；
(2) the first enzyme and reference enzyme digestion first vector, the downstream of recycling first restriction enzyme site and the ginseng are utilized
Examining the gene order between the upstream end of restriction enzyme site is subsequence；
Meanwhile using second enzyme and reference enzyme digestion Second support, the downstream with reference to restriction enzyme site and described is recycled
Gene order between the upstream end of second restriction enzyme site is auxiliary sequence；
(3) subsequence and auxiliary sequence are connected, so that the downstream of the first restriction enzyme site of the subsequence and the auxiliary sequence
The upstream end of second restriction enzyme site connects, the upstream end of the reference restriction enzyme site of the subsequence and the reference enzyme of the auxiliary sequence
The upstream end of enzyme site connects, and obtains destination carrier；
(4) it filters out with the destination carrier with reference to segment, obtaining has m first object gene and n the second target bases
The multicopy expression vector of cause.
2. the method as described in claim 1, which is characterized in that the yeast is Pichia pastoris.
3. the method as described in claim 1, which is characterized in that 3 ' ends of first expression cassette to first expression cassette
Gene order between 5 ' ends is held with the 3 ' of second expression cassette to the gene order between 5 ' ends of second expression cassette
It is identical.
4. the method as described in claim 1, which is characterized in that the first object gene is identical as the second target gene, institute
Multicopy expression vector is stated with m+n first object gene.
5. the method as described in claim 1, which is characterized in that the method screened in the step (4) is bacterium colony PCR identification.
6. the method as described in claim 1, which is characterized in that the reference segment is antibiotic resistance gene.
7. method as claimed in claim 6, which is characterized in that the screening technique in the step (4) is antibiotic flat band method.
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