CN114672509A - Corynebacterium and escherichia coli dual-expression vector with high expression capacity and construction method thereof - Google Patents
Corynebacterium and escherichia coli dual-expression vector with high expression capacity and construction method thereof Download PDFInfo
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Abstract
The invention provides a corynebacterium and escherichia coli double expression vector with high expression capacity and a construction method thereof, aiming at improving the protein yield of foreign protein; starting from the mutation of a protein expression element, a segment of sequence in front of a target gene is mutated, and the yield of protein is improved under the conditions that a byproduct is not introduced and additional influence is not generated on a host strain; the fluorescence expression amount of the original pXMJ19-egfp is about 100000, the fluorescence expression amount of the original pXMJ19-1676-5 'UTR-egfp is about 850000, and the green fluorescence protein expression capacity of the protein expression element is improved by about 8.5 times for the addition of the 5' UTR nucleotide sequence. The invention has the effect on egfp green fluorescent protein and the effect on m-chery expression, and shows that the plasmid system can be applied to other exogenous protein expressions.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to a corynebacterium and escherichia coli double-expression vector with high expression capacity and a construction method thereof.
Background
The plasmid system is the most common system for engineering strains to express foreign proteins, and the improvement of the yield of the foreign proteins depends on the construction and optimization of the plasmid system. For example, protein expression elements such as a promoter and a terminator for expressing a protein are optimized to improve the protein yield.
In recent years, the development and application of protein expression elements have been a very hot topic. A research team performs mutation on the N-terminal sequence of the protein by using a synonymous mutation mode, and constructs a library to screen positive mutants. However, the number of mutants having the same sense of mutation is limited, and the effect is not significant because of the same amino acid composition.
Disclosure of Invention
The invention belongs to the technical field of biology, and particularly relates to a corynebacterium and escherichia coli dual-expression vector with high expression capacity and a construction method thereof, wherein a 50bp nucleotide sequence is inserted into the front end of an exogenous gene of a pXMJ19 vector, and artificial mutation is performed on the 40 bp-50 bp in the sequence, so that a corynebacterium/escherichia coli dual-purpose vector pXMJ 19-1676-5' UTR plasmid with expression capacity about 8.5 times higher than that of a pXMJ19 plasmid is successfully obtained. The fluorescence/OD value for egfp (green fluorescent protein) expressed using the pXMJ19 plasmid was around 100000, while the fluorescence/OD value for egfp expressed using the pXMJ 19-1676-5' UTR plasmid was around 850000. Meanwhile, the invention also discloses a construction method of the expression plasmid.
Both the production of pharmaceutical proteins and food-grade proteins by genetic engineering techniques are critical and safety concerns must be considered. Considering the difficulty of gene knockout and gene knock-in, the application of the plasmid system is convenient and flexible when the exogenous protein is produced in a factory. Foreign target genes are inserted into the plasmids and transformed into host bacteria, and the target proteins are produced by amplification in the host bacteria, so that the final commercial value is achieved. Currently, the most commonly used E.coli, a gram-negative bacterium, produces endotoxin while producing foreign proteins, and is not the best "factory" for producing pharmaceutical or food grade proteins. However, because of the characteristics of convenient transformation of the plasmid in the escherichia coli and short growth period of the strain, the escherichia coli host is the first choice in the plasmid construction stage. The corynebacterium glutamicum, as a gram-positive bacterium, can produce secretory proteins, and at the same time, the secretory proteins do not form inclusion bodies and are not interfered by endotoxin. Therefore, the constructed plasmid system is transformed into the corynebacterium glutamicum for protein production, and has a plurality of advantages.
The pXMJ19 plasmid is a shuttle plasmid with double expression, i.e. it can be constructed in Escherichia coli and can be expressed in Corynebacterium glutamicum.
The invention is based on pXMJ19 shuttle plasmid, through adding a section of Corynebacterium glutamicum endogenous gene 5' UTR sequence, and the sequence is artificially mutated, finally get the protein expression system that can express with high efficiency.
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above problems occurring in the prior art.
Therefore, the present invention aims at providing a dual expression vector of corynebacterium and escherichia coli with high expression capacity and a construction method thereof.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a dual-expression carrier of corynebacterium and escherichia coli with high expression ability comprises,
Taking a gene sequence of a pXMJ19 plasmid vector as a starting sequence, inserting a modified corynebacterium glutamicum endogenous gene expression promoter 5 'UTR sequence fragment at the rear end of a promoter of the plasmid for expression of exogenous genes to obtain pXMJ 19-1676-5' UTR-exogenous gene plasmid;
the pXMJ 19-1676-5' UTR-exogenous gene plasmid is transferred into BZH001 or escherichia coli competence for expression, and the expression is the corynebacterium and escherichia coli double expression vector with high expression capacity.
As a preferred embodiment of the dual expression vector of the present invention, there is provided a high expression ability coryneform bacterium and Escherichia coli dual expression vector, wherein: the nucleotide sequence of the pXMJ19 plasmid is shown in SEQ ID NO. 23.
As a preferred embodiment of the dual expression vector of Corynebacterium and Escherichia coli with high expression ability according to the present invention, wherein: also comprises the following steps of (1) preparing,
inserting a modified 5 'UTR fragment with the length of 50bp into the rear end of a promoter sequence on the basis of a pXMJ19 constitutive no-load plasmid, then inoculating a nucleotide sequence with the length of 15bp, and then inoculating an exogenous gene sequence used for expression to obtain a pXMJ 19-1676-5' UTR-exogenous gene plasmid;
wherein the exogenous gene comprises a green fluorescent protein gene segment and a red fluorescent protein gene segment.
As a preferred embodiment of the dual expression vector of the present invention, there is provided a high expression ability coryneform bacterium and Escherichia coli dual expression vector, wherein: also comprises a step of adding a new type of additive,
the modified 5' UTR fragment is 50bp in length, the SEQ ID NO.24 is used as an initial sequence, the 40 bp-50 bp are artificially mutated, and the mutated nucleotide sequence of the 40 bp-50 bp is one of the fragments 1-4:
nucleotide sequence of fragment 1: CACCTCACAC;
nucleotide sequence of fragment 2: CCGACCACCA, respectively;
nucleotide sequence of fragment 3: GTAGGTTATA, respectively;
nucleotide sequence of fragment 4: GTCCGGTATC, respectively;
the nucleotide sequence with the length of 15bp is as follows: AAAGGAGGACAACTA are provided.
As a preferred embodiment of the method for constructing the dual expression vector of corynebacterium and escherichia coli with high expression ability, the method is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
obtaining a plasmid vector: cutting the pXMJ19 plasmid vector;
obtaining a modified exogenous gene fragment: designing upstream and downstream primers, wherein the sequences of the upstream and downstream primers are respectively shown as SEQ ID NO. 2-7 and SEQ ID NO. 8-12, and carrying out amplification reaction on an exogenous gene template; obtaining an exogenous gene fragment, carrying out enzyme digestion to obtain an exogenous gene fragment product after enzyme digestion, and carrying out column recovery on the product;
Connecting: ligating the foreign gene fragment product and the cut pXMJ19 plasmid vector into a pXMJ 1919-1676-5' UTR-foreign gene plasmid;
culturing: the plasmid of exogenous gene of pXMJ 19-1676-5' UTR is transformed into escherichia coli or BZH001 competence for expression;
wherein, a 1676-5' UTR-egfp fragment 1 is obtained by an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.2, and the sequence is shown as SEQ ID NO. 19; obtaining 1676-5' UTR-egfp fragment 2 by an upstream primer SEQ ID NO.4 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 20; obtaining 1676-5' UTR-egfp fragment 3 by an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 21; obtaining 1676-5' UTR-egfp fragment 4 by an upstream primer SEQ ID NO.6 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 22;
wherein, a 1676-5' UTR-m-cherry fragment 1 is obtained by an upstream primer SEQ ID NO.8 and a downstream primer SEQ ID NO.12, and the sequence is shown as SEQ ID NO. 14; obtaining 1676-5' UTR-m-cherry fragment 2 by an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 15; obtaining 1676-5' UTR-m-cherry fragment 3 by an upstream primer SEQ ID NO.10 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 16; a1676-5' UTR-m-cherry fragment 4 is obtained by an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO.12, and the sequence is shown as SEQ ID NO. 17.
As a preferable scheme of the construction method of the high expression capacity corynebacterium and escherichia coli dual expression vector, the method comprises the following steps: also comprises a step of adding a new type of additive,
obtaining a plasmid vector: cutting the pXMJ19 plasmid vector through double enzyme digestion, wherein when the exogenous gene is a green fluorescent protein gene fragment, the enzyme digestion sites are Hind III and Ecor I; hind III and BamH I when the foreign gene is a red fluorescent protein gene fragment;
obtaining a modified exogenous gene fragment: designing upstream and downstream primers, wherein the sequences of the upstream and downstream primers are respectively shown as SEQ ID NO. 2-7 and SEQ ID NO. 8-12, and carrying out PCR amplification reaction on an exogenous gene template by using an estaq polymerase; obtaining an exogenous gene fragment, carrying out double enzyme digestion, obtaining an exogenous gene fragment product after enzyme digestion, and carrying out column recovery on the product, wherein the enzyme digestion sites are Hind III and Ecor I or Hind III and BamH I;
connecting: using T4DNA ligase to join the foreign gene fragment product and the cut Pxmj19 plasmid vector into Pxmj 19-1676-5' utr-foreign gene plasmid;
culturing: pxmj 19-1676-5' utr exogenous gene plasmid is transformed into colibacillus or BZH001 to express in competence.
As a preferred embodiment of the method for constructing the dual expression vector of corynebacterium and escherichia coli having high expression ability of the present invention, wherein: in the step of obtaining the plasmid vector, the double-enzyme cutting pXMJ19 plasmid vector system comprises 5ul of each of two fast cutting enzymes, the vector mass is 4500-5000ng, the green buffer is 10ul, and nuclease-free sterile water is added to 100 ul.
As a preferred embodiment of the method for constructing the dual expression vector of corynebacterium and escherichia coli having high expression ability of the present invention, wherein: in the step of obtaining the modified foreign gene fragment,
the amplification reaction is to use estaq polymerase to carry out PCR amplification, and the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C, performing 35 cycles of denaturation, annealing, and extension, and keeping the temperature at 72 deg.C for 2 min;
the double enzyme cutting system comprises two kinds of fast cutting enzyme each 3.5-4ul, fragment mass 3500-4000ng, green buffer 10ul, and non-nuclease sterilized water added to 100 ul.
As a preferred embodiment of the method for constructing the dual expression vector of corynebacterium and escherichia coli having high expression ability of the present invention, wherein: in the ligation step, the ligation system comprises the following fragments: the vector (0.3 pmol:0.03pmol, buffer 2ul, T4DNA ligase (1 ul) was added to 20ul in nuclease-free sterile water at a ligation temperature of 16 ℃ for 1 hour.
As a preferred embodiment of the method for constructing the dual expression vector of corynebacterium and escherichia coli having high expression ability of the present invention, wherein: in the culture step, pXMJ 19-1676-5' UTR-exogenous gene plasmid is transformed into BZH001 competence, ice bath is carried out for 10-15mim, 1800v electric shock is carried out twice, the mixed solution of the plasmid and the competence is injected into LBHIS culture medium, water bath at 46 ℃ is carried out for 6min, shaking table at 30 ℃ is carried out for recovery culture for 1.5-2.5h, a LBHIS + chloramphenicol resistant solid culture medium plate is coated, and culture is carried out in an incubator at 30 ℃ for 24 h.
The invention has the beneficial effects that:
the invention provides a corynebacterium and escherichia coli double expression vector with high expression capacity and a construction method thereof, aiming at improving the protein yield of foreign protein; starting from the mutation of a protein expression element, a segment of sequence in front of a target gene is mutated, and the yield of protein is improved under the conditions that no by-product is introduced and no additional influence is generated on a host strain; the fluorescence expression quantity of the original pXMJ19-egfp is about 100000, the fluorescence expression quantity of the pXMJ19-1676-5 'UTR-egfp is about 850000, and the green fluorescence protein expression capacity of the protein expression element is improved by about 8.5 times for the addition of 5' UTR nucleotide sequence;
the invention has effects on egfp green fluorescent protein and m-chery expression, and the plasmid system can be applied to other exogenous protein expression.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a constitutive plasmid map of pXMJ 19-egfp;
FIG. 2 is a pXMJ 19-1676-5' UTR-egfp constitutive plasmid map;
FIG. 3 shows the verification of egfp (green fluorescent protein) expression in pXMJ 19-1676-5' UTR-egfp constitutive plasmid system; wherein, the pXMJ19-egfp plasmid system, the pXMJ 19-1676-5' UTR-egfp1, the 2, 3 and 4 plasmid systems are arranged from left to right in sequence;
FIG. 4 shows the validation of expression of m-cherry (red fluorescent protein) in pXMJ 19-1676-5' UTR-m-cherry constitutive plasmid system; wherein, the plasmid systems of pXMJ19-m-cherry, pXMJ 19-1676-5' UTR-m-cherry1, 2, 3 and 4 are arranged from left to right.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, the references herein to "one embodiment" or "an embodiment" refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The chemical reagents used in the examples of the present invention are generally commercially available unless otherwise specified.
The plasmid sequence of the pXMJ19 vector used in the embodiment of the invention is shown in SEQ ID NO. 22; the fast-cutting enzyme is purchased from Takara Bio Tad doctor, Inc., and the enzyme cutting sites are Hind III and Ecor I or Hind III and BamH I; the product column was purchased from Jiangsukang, a century Biotechnology GmbH, model DNAClean-up Kit.
The BZH001 competence preparation used in the examples was as follows:
culturing BZH001 single colony in LBB liquid culture medium at 30 deg.C and 220r/min for 12 h; transferring the bacterial liquid into an EPO culture medium after 12h, enabling the initial OD of transfer to reach 0.3, and continuing to culture the transferred bacterial liquid in a shaking table at the temperature of 30 ℃ and the speed of 220r/min for 3-5h, so that the final OD reaches 0.6-0.9; carrying out ice-bath on the bacterial liquid for 25-30min, subpackaging into 50 ml/tube, and centrifuging at 4500rpm at 4 ℃ for 10 min; discarding the supernatant, adding 30ml of precooled 10% glycerol, resuspending the precipitated thallus, centrifuging at 4500rpm at 4 ℃ for 10min, and repeating for three times; after the last centrifugation, 400ul of 10% precooled glycerol is added, and after the heavy suspension, the mixture is subpackaged according to the amount of 80-100 ul/tube; storing in a refrigerator at-80 deg.C for use.
Example 1:
construction of Pxmj19-egfp plasmid:
1. obtaining a plasmid vector: the vector was cut by double digestion with fast cutter enzymes Hind III and Ecor I using pXMJ19 constitutive no-load plasmid. 5ul of each of the two fast cutting enzymes, the quality of the carrier controlled between 4500-.
2. Obtaining egfp fragments: designing an upstream primer and a downstream primer, wherein the sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and inserting the upstream primer and the downstream primer into a restriction enzyme site through the primers; reacting egfp template by PCR using estaq polymerase under the following conditions: pre-denaturation at 94 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C, and performing 35 cycles of denaturation, annealing, and extension, and keeping the temperature at 72 deg.C for 2 min.
The egfp fragment is obtained, and the sequence is shown as SEQ ID NO. 18. Then, fast cutting enzymes of Takara Bio Nippon physician company are applied, enzyme cutting sites are Hind III and Ecor I, the two fast cutting enzymes are respectively 3.5-4ul, the fragment mass is 3500 and 4000ng, and the green buffer is 10ul, finally, nuclease-free sterilized water is added to 100ul, and the egfp fragment product after enzyme cutting is obtained, and the product is used for column recovery.
3. Connecting: the egfp fragment product and the plasmid vector were ligated into a Pxmj19-egfp plasmid using T4DNA ligase from Takara Bio Tahiti physician's products, wherein the egfp fragment product: carrier
0.3pmol:0.03pmol, buffer 2. mu.l, T4DNA ligase was added to 1. mu.l, and finally, nuclease-free sterile water was added to 20. mu.l, followed by ligation at 16 ℃ for one hour to obtain a plasmid map Pxmj19-egfp as shown in FIG. 1.
4. Culturing: the Pxmj19-egfp plasmid was transformed into DH 5. alpha. E.coli competence supplied by assist in san Diego, ice-washed for 25min, heat-shocked in a water bath at 42 ℃ for 45s-1min, ice-washed again for 2-3min, and transferred to a shaker at 37 ℃ for recovery culture for 45-60 min. After the recovery culture, plates of LBB medium (LBB medium: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl, 10g/L brain heart extract, 20g/L agar powder) resistant to chloramphenicol were applied. And (4) performing plate culture for 12-16h, performing primary screening on the single colony under a blue light instrument, and performing liquid culture medium culture on the fluorescent single colony. (liquid medium LBB: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl, 10g/L brain-heart extract) for 12-16h, extracting 2ml bacterial liquid for plasmid extraction, and performing plasmid sequence sequencing on the extracted and purified plasmids by Jinzhi corporation.
Example 2:
construction of pXMJ 19-1676-5' UTR-egfp plasmid:
the principle and method for constructing pXMJ 19-1676-5' UTR-egfp plasmid are consistent with the construction method of pXMJ19-egfp plasmid, only the difference between the upstream primer and the downstream primer exists in step 2, and the mutation1676 sequence fragment is added to the upstream primer, which is specifically as follows.
Designing an upstream primer and a downstream primer, wherein the sequences of the upstream primer are respectively shown as SEQ ID NO. 3-6, the sequence of the downstream primer is shown as SEQ ID NO.2, and the upstream primer and the downstream primer are inserted into a restriction enzyme site through the primers; reacting egfp template by PCR using estaq polymerase under the following conditions: pre-denaturation at 94 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃, and carrying out 35 cycles of denaturation, annealing and extension, and keeping the temperature at 72 ℃ for 2 min.
Wherein, a 1676-5' UTR-egfp fragment 1 is obtained by an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.2, and the sequence is shown as SEQ ID NO. 19; obtaining 1676-5' UTR-egfp fragment 2 by an upstream primer SEQ ID NO.4 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 20; obtaining 1676-5' UTR-egfp fragment 3 by an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 21; obtaining 1676-5' UTR-egfp fragment 4 by an upstream primer SEQ ID NO.6 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 22;
after product recovery and ligation, pXMJ 19-1676-5' UTR-egfp plasmids 1, 2, 3 and 4 were obtained, respectively. The plasmid map of pXMJ 19-1676-5' UTR-egfp is shown in FIG. 2.
Example 3:
the plasmids prepared in examples 1 and 2 were transformed into competent cultures of C.glutamicum BZH 001:
Respectively taking pXMJ19-egfp plasmids, pXMJ 19-1676-5' UTR-egfp plasmids 1, 2, 3 and 4 each 5ul, and BZH001 competence 80-100ul, respectively injecting the plasmids into the BZH001 competence, performing ice bath for 10-15mim and 1800v electric shock twice, injecting the mixture of the plasmids and the BZH001 competence into an LBHIS culture medium, performing water bath at 46 ℃ for 6min, recovering and culturing at 30 ℃ for 1.5-2.5h by a shaking table, coating a LBHIS + chloramphenicol resistant solid culture medium plate, and culturing at 30 ℃ in an incubator for 24 h.
LBHIS medium: 2.5g/L of yeast extract, 5g/L of peptone, 5g/L of NaCl, 18.5g/L of brain-heart leaching solution and 91g/L of sorbitol.
Example 4:
fermentation-fluorescent quantitative analysis:
after the plate bacteria are cultured for 24 hours, single bacterial colonies expressing green fluorescence are preliminarily screened under a blue light instrument, the single bacterial colonies are selected to be cultured in a liquid culture medium with LBB + chloramphenicol resistance for 12 hours and used as an activation stage of the bacterial colonies, after 12 hours, the activated bacterial liquid is transferred to a new liquid culture medium with LBB + chloramphenicol resistance for fermentation for 24 hours, and the fluorescence after fermentation is tested through a high-throughput fluorescence detector. The experimental values are shown in fig. 3.
Compared with the pXMJ19-egfp plasmid without the addition of the mutation1676 sequence, the fluorescence bacteria of the four pXMJ 19-1676-5' UTR-egfp plasmids are improved to different degrees, the ratio of fluorescence/OD after fermentation of the plasmid without the addition of the mutation1676 sequence is about 1000000, the ratio of fluorescence/OD after fermentation of the plasmid with the addition of the mutation1676 sequence is about 850000, and the highest fluorescence is enhanced by 8.5 times compared with that before mutation.
Example 5:
pXMJ 19-m-chery plasmid construction:
the construction principle and method of pXMJ 19-m-chery plasmid are the same as those of the construction method of pXMJ19-egfp plasmid, and only the difference of upstream and downstream primers and enzyme cutting sites exists in step 2, which is specifically as follows.
Obtaining m-cherry fragments: designing an upstream primer and a downstream primer, wherein the sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.7 and SEQ ID NO.12, and inserting enzyme cutting sites Hind III and BamH I through the primers; reacting the m-chery template by using an estaq polymerase through PCR under the following reaction conditions: pre-denaturation at 94 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃, and carrying out 35 cycles of denaturation, annealing and extension, and keeping the temperature at 72 ℃ for 2 min.
Obtaining an m-cherry fragment, and the sequence is shown as SEQ ID NO. 13. Then, Takara Bio Tanshiji physician's company is used to cut the enzyme, the enzyme cutting sites are Hind III and BamH I, the two kinds of cut-fast enzymes are 3.5-4ul respectively, the fragment mass is 3500 and 4000ng, the green buffer is 10ul, finally, the nuclease-free sterilized water is added to 100ul, the m-cherry fragment product after enzyme cutting is obtained, and the product is used for column recovery.
Then obtaining pXMJ19-m-cherry plasmid through connection culture.
Example 6:
pXMJ 19-1676-5' UTR-m-cherry plasmid construction:
The principle and method for constructing pXMJ 19-1676-5' UTR-m-cherry plasmid are the same as those of the construction method of pXMJ19-m-cherry plasmid described above, and only the difference between the upstream primer and the downstream primer exists in step 2, and a mutation1676 sequence fragment is added to the upstream primer, as described in detail below.
Designing an upstream primer and a downstream primer, wherein the upstream primer sequences are respectively shown as SEQ ID NO. 8-11, the downstream primer sequences are shown as SEQ ID NO.12, and the upstream primer and the downstream primer are inserted into enzyme cutting sites Hind III and BamH I through the primers; reacting egfp template by PCR using estaq polymerase under the following conditions: pre-denaturation at 94 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C, and performing 35 cycles of denaturation, annealing, and extension, and keeping the temperature at 72 deg.C for 2 min.
Wherein, a 1676-5' UTR-m-cherry fragment 1 is obtained by an upstream primer SEQ ID NO.8 and a downstream primer SEQ ID NO.12, and the sequence is shown as SEQ ID NO. 14; obtaining 1676-5' UTR-m-cherry fragment 2 by an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 15; obtaining 1676-5' UTR-m-cherry fragment 3 by an upstream primer SEQ ID NO.10 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 16; obtaining 1676-5' UTR-m-chery segment 4 by an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 17;
After product recovery and ligation, pXMJ 19-1676-5' UTR-m-chery plasmids 1, 2, 3 and 4 are obtained respectively.
Example 7:
the plasmids prepared in examples 5 and 6 were transformed into competent cultures of C.glutamicum BZH 001:
respectively taking 5ul of pXMJ19-m-cherry plasmids, 5ul of pXMJ 19-1676-5' UTR-m-cherry plasmids 1, 2, 3 and 4 and 80-100ul of BZH001 competence, respectively pumping the plasmids into the BZH001 competence, shocking twice by ice bath 10-15mim and 1800v, pumping the mixed solution of the plasmids and the BZH001 competence into an LBHIS culture medium, carrying out water bath at 46 ℃ for 6min, carrying out shake cultivation at 30 ℃ for 1.5-2.5h, coating a LBHIS + chloramphenicol resistant solid culture medium plate, and carrying out cultivation in an incubator at 30 ℃ for 24 h.
LBHIS medium: 2.5g/L of yeast extract, 5g/L of peptone, 5g/L of NaCl, 18.5g/L of brain-heart leaching solution and 91g/L of sorbitol.
Example 8:
fermentation-fluorescence quantitative analysis:
and (3) recovering the plate bacteria after 24h of culture, primarily screening single colonies expressing green fluorescence under a blue light instrument, selecting the single colonies, culturing the single colonies in an LBB + chloramphenicol resistant liquid culture medium for 12h as an activation stage of the colonies, transferring the activated bacteria liquid into a new LBB + chloramphenicol resistant liquid culture medium for fermentation for 24h after 12h, and testing the fluorescence after fermentation through a high-throughput fluorescence detector. The experimental values are shown in fig. 4.
Compared with the pXMJ 19-m-chery plasmid without the mutation1676 sequence, the fluorescence amount of the four pXMJ 19-1676-5' UTR-m-chery plasmids is improved to different degrees, and compared with the examples 1-4, the fluorescence amount of egfp (green fluorescent protein) is not improved by 8.5 times, but is improved by 4.5 times. The verification experiment shows that the protein expression element of the plasmid system is favorable for improving the protein expression yield to a certain extent, and has certain robustness, and the plasmid system can be applied to other exogenous protein expressions in an attempt.
The invention provides a corynebacterium and escherichia coli double expression vector with high expression capacity and a construction method thereof, aiming at improving the protein yield of foreign protein; starting from the mutation of a protein expression element, a segment of sequence in front of a target gene is mutated, and the yield of protein is improved under the conditions that no by-product is introduced and no additional influence is generated on a host strain; the fluorescence expression quantity of the original pXMJ19-egfp is about 100000, the fluorescence expression quantity of the pXMJ19-1676-5 'UTR-egfp is about 850000, and the green fluorescence protein expression capacity of the protein expression element is improved by about 8.5 times for the addition of 5' UTR nucleotide sequence.
The invention has effects on egfp green fluorescent protein and m-chery expression, and the plasmid system can be applied to other exogenous protein expression.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Sequence listing
<110> university of south of the Yangtze river
<120> corynebacterium and escherichia coli dual expression vector with high expression ability and construction method thereof
<141> 2022-02-22
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 1
aaaggaggac aactaatggt gagca 25
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence
<400> 2
ccggaattct tacttgtaca gctcgtccat gcc 33
<210> 4
<211> 92
<212> DNA
<213> Artificial Sequence
<400> 4
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc acctcacaca 60
aaggaggaca actaatggtg agcaagggcg ag 92
<210> 4
<211> 92
<212> DNA
<213> Artificial Sequence
<400> 4
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc cgaccaccaa 60
aaggaggaca actaatggtg agcaagggcg ag 92
<210> 5
<211> 92
<212> DNA
<213> Artificial Sequence
<400> 5
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg taggttataa 60
aaggaggaca actaatggtg agcaagggcg ag 92
<210> 6
<211> 92
<212> DNA
<213> Artificial Sequence
<400> 6
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg tccggtatca 60
aaggaggaca actaatggtg agcaagggcg ag 92
<210> 7
<211> 47
<212> DNA
<213> Artificial Sequence
<400> 7
cccaagctta aaggaggaca actaatggtg agcaagggcg aggagga 47
<210> 8
<211> 85
<212> DNA
<213> Artificial Sequence
<400> 8
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc acctcacaca 60
aaggaggaca actaatggtg agcaa 85
<210> 9
<211> 85
<212> DNA
<213> Artificial Sequence
<400> 9
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc cgaccaccaa 60
aaggaggaca actaatggtg agcaa 85
<210> 10
<211> 85
<212> DNA
<213> Artificial Sequence
<400> 10
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg taggttataa 60
aaggaggaca actaatggtg agcaa 85
<210> 11
<211> 85
<212> DNA
<213> Artificial Sequence
<400> 11
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg tccggtatca 60
aaggaggaca actaatggtg agcaa 85
<210> 12
<211> 35
<212> DNA
<213> Artificial Sequence
<400> 12
cgcggatcct tacttgtaca gctcgtccat gccgc 35
<210> 13
<211> 744
<212> DNA
<213> Artificial Sequence
<400> 13
cccaagctta aaggaggaca actaatggtg agcaagggcg aggaggataa catggccatc 60
atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc 120
gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag 180
gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac 240
ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc 300
cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg 360
acccaggact cctccctgca ggacggcgaa ttcatctaca aggtgaagct gcgcggcacc 420
aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc 480
gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg 540
aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg 600
cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac 660
tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac 720
gagctgtaca agtaaggatc cgcg 744
<210> 14
<211> 794
<212> DNA
<213> Artificial Sequence
<400> 14
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc acctcacaca 60
aaggaggaca actaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 120
tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 180
gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 240
gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 300
cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 360
tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 420
cctccctgca ggacggcgaa ttcatctaca aggtgaagct gcgcggcacc aacttcccct 480
ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 540
accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 600
gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 660
gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 720
tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 780
agtaaggatc cgcg 794
<210> 15
<211> 794
<212> DNA
<213> Artificial Sequence
<400> 15
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc cgaccaccaa 60
aaggaggaca actaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 120
tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 180
gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 240
gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 300
cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 360
tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 420
cctccctgca ggacggcgaa ttcatctaca aggtgaagct gcgcggcacc aacttcccct 480
ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 540
accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 600
gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 660
gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 720
tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 780
agtaaggatc cgcg 794
<210> 16
<211> 794
<212> DNA
<213> Artificial Sequence
<400> 16
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg taggttataa 60
aaggaggaca actaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 120
tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 180
gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 240
gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 300
cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 360
tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 420
cctccctgca ggacggcgaa ttcatctaca aggtgaagct gcgcggcacc aacttcccct 480
ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 540
accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 600
gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 660
gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 720
tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 780
agtaaggatc cgcg 794
<210> 17
<211> 794
<212> DNA
<213> Artificial Sequence
<400> 17
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg tccggtatca 60
aaggaggaca actaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt 120
tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg 180
gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg 240
gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg 300
cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct 360
tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact 420
cctccctgca ggacggcgaa ttcatctaca aggtgaagct gcgcggcacc aacttcccct 480
ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt 540
accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg 600
gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg 660
gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg 720
tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca 780
agtaaggatc cgcg 794
<210> 18
<211> 753
<212> DNA
<213> Artificial Sequence
<400> 18
cccaagctta aaggaggaca actaatggtg agcaagggcg aggagctgtt caccggggtg 60
gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 120
gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 180
aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 240
agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 300
tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 360
gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 420
gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 480
atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc 540
gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 600
cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 660
aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc 720
ggcatggacg agctgtacaa gtaagaattc cgg 753
<210> 19
<211> 803
<212> DNA
<213> Artificial Sequence
<400> 19
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc acctcacaca 60
aaggaggaca actaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 120
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 180
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 240
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 300
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 360
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 420
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 480
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 540
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 600
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 660
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 720
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 780
agctgtacaa gtaagaattc cgg 803
<210> 20
<211> 803
<212> DNA
<213> Artificial Sequence
<400> 20
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacc cgaccaccaa 60
aaggaggaca actaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 120
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 180
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 240
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 300
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 360
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 420
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 480
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 540
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 600
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 660
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 720
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 780
agctgtacaa gtaagaattc cgg 803
<210> 21
<211> 803
<212> DNA
<213> Artificial Sequence
<400> 21
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg taggttataa 60
aaggaggaca actaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 120
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 180
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 240
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 300
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 360
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 420
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 480
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 540
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 600
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 660
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 720
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 780
agctgtacaa gtaagaattc cgg 803
<210> 22
<211> 803
<212> DNA
<213> Artificial Sequence
<400> 22
cccaagcttg acactaagtt attacattta ttatatgatt ggttaggacg tccggtatca 60
aaggaggaca actaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 120
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 180
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 240
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 300
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 360
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 420
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 480
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 540
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 600
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 660
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 720
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 780
agctgtacaa gtaagaattc cgg 803
<210> 23
<211> 2048
<212> DNA
<213> Artificial Sequence
<400> 23
aattaagctt ggatccccgg gtaccgagct cgaattcagc ttggctgttt tggcggatga 60
gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag 120
aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg 180
aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag 240
gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 300
gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa 360
gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa 420
gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt 480
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 540
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 600
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 660
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 720
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt gcttcctcgc 780
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 840
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 900
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 960
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 1020
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 1080
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 1140
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 1200
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 1260
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 1320
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 1380
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 1440
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 1500
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 1560
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 1620
aaaggatctt cacctagatc cttttggggt gggcgaagaa ctccagcatg agatccccgc 1680
gctggaggat catccagcca ttcggggtcg ttcactggtt cccctttctg atttctggca 1740
tagaagaacc cccgtgaact gtgtggttcc gggggttgct gatttttgcg agacttctcg 1800
cgcaattccc tagcttaggt gaaaacacca tgaaacacta gggaaacacc catgaaacac 1860
ccattagggc agtagggcgg cttcttcgtc tagggcttgc atttgggcgg tgatctggtc 1920
tttagcgtgt gaaagtgtgt cgtaggtggc gtgctcaatg cactcgaacg tcacgtcatt 1980
taccgggtca cggtgggcaa agagaactag tgggttagac attgttttcc tcgttgtcgg 2040
tggtggtg 2048
<210> 24
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 24
gacactaagt tattacattt attatatgat tggttaggac tatggacatg 50
Claims (10)
1. A dual expression vector of corynebacterium and escherichia coli with high expression capacity is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
taking a gene sequence of a pXMJ19 plasmid vector as a starting sequence, inserting a modified 5 'UTR sequence fragment of a corynebacterium glutamicum endogenous gene expression promoter at the rear end of the promoter of the plasmid for expression of a foreign gene to obtain a pXMJ 19-1676-5' UTR-foreign gene plasmid;
The pXMJ 19-1676-5' UTR-exogenous gene plasmid is transferred into BZH001 or escherichia coli competence for expression, and the expression is the corynebacterium and escherichia coli double expression vector with high expression capacity.
2. The high expression ability dual expression vector for coryneform bacteria and Escherichia coli according to claim 1, wherein: the pXMJ19 plasmid has a nucleotide sequence shown in SEQ ID NO. 23.
3. The high expression ability dual expression vector for coryneform bacteria and Escherichia coli according to claim 1, wherein: also comprises the following steps of (1) preparing,
inserting a modified 5 'UTR fragment with the length of 50bp into the rear end of a promoter sequence on the basis of a pXMJ19 constitutive no-load plasmid, then inoculating a nucleotide sequence with the length of 15bp, and then inoculating an exogenous gene sequence used for expression to obtain a pXMJ 19-1676-5' UTR-exogenous gene plasmid;
wherein the exogenous gene comprises a green fluorescent protein gene segment and a red fluorescent protein gene segment.
4. The high expression ability coryneform bacterium and Escherichia coli double expression vector according to claim 1 or 3, wherein: also comprises the following steps of (1) preparing,
the modified 5' UTR fragment is 50bp in length, the SEQ ID NO.24 is used as an initial sequence, the 40 bp-50 bp are artificially mutated, and the mutated nucleotide sequence of the 40 bp-50 bp is one of the fragments 1-4:
Nucleotide sequence of fragment 1: CACCTCACAC, respectively;
nucleotide sequence of fragment 2: CCGACCACCA, respectively;
nucleotide sequence of fragment 3: GTAGGTTATA, respectively;
nucleotide sequence of fragment 4: GTCCGGTATC, respectively;
the nucleotide sequence with the length of 15bp is as follows: AAAGGAGGACAACTA are provided.
5. The method for constructing a dual expression vector of coryneform bacteria and Escherichia coli according to claim 1 to 4, wherein: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
obtaining a plasmid vector: cutting the pXMJ19 plasmid vector;
obtaining a modified exogenous gene fragment: designing upstream and downstream primers, wherein the sequences of the upstream and downstream primers are respectively shown as SEQ ID NO. 2-7 and SEQ ID NO. 8-12, and carrying out amplification reaction on an exogenous gene template; obtaining an exogenous gene fragment, carrying out enzyme digestion to obtain an exogenous gene fragment product after enzyme digestion, and carrying out column recovery on the product;
connecting: ligating the foreign gene fragment product and the cut pXMJ19 plasmid vector into a pXMJ 19-1676-5' UTR-foreign gene plasmid;
culturing: transforming pXMJ 19-1676-5' UTR-exogenous gene plasmid into escherichia coli or BZH001 competence for expression;
wherein, a 1676-5' UTR-egfp fragment 1 is obtained by an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.2, and the sequence is shown as SEQ ID NO. 19; obtaining 1676-5' UTR-egfp fragment 2 by an upstream primer SEQ ID NO.4 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 20; obtaining 1676-5' UTR-egfp fragment 3 by an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 21; obtaining 1676-5' UTR-egfp fragment 4 by an upstream primer SEQ ID NO.6 and a downstream primer SEQ ID NO.2, wherein the sequence is shown as SEQ ID NO. 22;
Wherein, a 1676-5' UTR-m-chery segment 1 is obtained by an upstream primer SEQ ID NO.8 and a downstream primer SEQ ID NO.12, and the sequence is shown in SEQ ID NO. 14; obtaining 1676-5' UTR-m-chery fragment 2 by an upstream primer SEQ ID NO.9 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 15; obtaining 1676-5' UTR-m-chery fragment 3 by an upstream primer SEQ ID NO.10 and a downstream primer SEQ ID NO.12, wherein the sequence is shown as SEQ ID NO. 16; a1676-5' UTR-m-chery segment 4 is obtained by an upstream primer SEQ ID NO.11 and a downstream primer SEQ ID NO.12, and the sequence is shown as SEQ ID NO. 17.
6. The method for constructing a high expression ability dual expression vector of Corynebacterium and Escherichia coli according to claim 5, wherein: also comprises a step of adding a new type of additive,
obtaining a plasmid vector: cutting the pXMJ19 plasmid vector by double enzyme digestion, wherein when the exogenous gene is a green fluorescent protein gene fragment, the enzyme digestion sites are Hind III and Ecor I; when the exogenous gene is a red fluorescent protein gene fragment, enzyme cutting sites are Hind III and BamH I;
obtaining a modified exogenous gene fragment: designing upstream and downstream primers, wherein the sequences of the upstream and downstream primers are respectively shown as SEQ ID NO. 2-7 and SEQ ID NO. 8-12, and carrying out PCR amplification reaction on an exogenous gene template by using an estaq polymerase; obtaining an exogenous gene fragment, carrying out double enzyme digestion, obtaining an exogenous gene fragment product after enzyme digestion, and carrying out column recovery on the product, wherein the enzyme digestion sites are Hind III and Ecor I or Hind III and BamH I;
Connecting: ligating the foreign gene fragment product and the cut pXMJ19 plasmid vector into a pXMJ 19-1676-5' UTR-foreign gene plasmid using T4DNA ligase;
culturing: pXMJ 19-1676-5' UTR-foreign gene plasmid is transformed into Escherichia coli or BZH001 to be expressed in competence.
7. The method for constructing a coryneform bacterium and Escherichia coli double expression vector according to claim 5 or 6, wherein: in the step of obtaining the plasmid vector, the double-enzyme cutting pXMJ19 plasmid vector system comprises 5ul of each of two fast cutting enzymes, the vector mass is 4500-5000ng, the green buffer is 10ul, and nuclease-free sterile water is added to 100 ul.
8. The method for constructing a coryneform bacterium and Escherichia coli double expression vector according to claim 5 or 6, wherein: in the step of obtaining the modified foreign gene fragment,
the amplification reaction is to use estaq polymerase to carry out PCR amplification, and the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C, performing 35 cycles of denaturation, annealing, and extension, and keeping the temperature at 72 deg.C for 2 min;
the double enzyme cutting system comprises two kinds of fast cutting enzyme each 3.5-4ul, fragment mass 3500-4000ng, green buffer 10ul, and non-nuclease sterilized water added to 100 ul.
9. The method for constructing a high expression ability dual expression vector of Corynebacterium and Escherichia coli according to claim 5 or 6, wherein: in the ligation step, the ligation system comprises, fragments: the vector (0.3 pmol:0.03pmol, buffer 2ul, T4DNA ligase (1 ul) was added to 20ul in nuclease-free sterile water at a ligation temperature of 16 ℃ for 1 hour.
10. The method for constructing a coryneform bacterium and Escherichia coli double expression vector according to claim 5 or 6, wherein: in the culture step, pXMJ 19-1676-5' UTR-exogenous gene plasmid is transformed into BZH001 competence, ice bath is carried out for 10-15mim, 1800v electric shock is carried out twice, the mixed solution of the plasmid and the competence is injected into LBHIS culture medium, water bath at 46 ℃ is carried out for 6min, shaking table at 30 ℃ is carried out for recovery culture for 1.5-2.5h, a LBHIS + chloramphenicol resistant solid culture medium plate is coated, and culture is carried out in an incubator at 30 ℃ for 24 h.
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| CN116574749A (en) * | 2022-10-20 | 2023-08-11 | 江南大学 | Method for improving expression level of exogenous protein based on protein N-terminal modification |
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| CN109913490A (en) * | 2019-03-13 | 2019-06-21 | 江南大学 | A kind of enhanced expression vector suitable for Corynebacterium glutamicum |
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