CN106318962B - Non-fusion expression vector capable of enhancing target gene expression and preparation method thereof - Google Patents
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Abstract
The invention discloses a non-fusion expression vector capable of enhancing target gene expression and a preparation method thereof, belonging to the technical field of biological engineering. The non-fusion expression vector capable of enhancing the expression of the target gene takes a pETduet-1 plasmid as a skeleton vector, the amino acid sequence of the cloning site 1 of the plasmid inserted into the pETduet-1 is shown as SEQ ID NO.1, and the target gene is inserted into the cloning site 2 of the plasmid inserted into the pETduet-1. The invention finds that the EGFP-CTA2-TAT expressed in a non-fusion way is 20 times higher than that expressed normally.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a non-fusion expression vector capable of enhancing target gene expression and a preparation method thereof.
Background
Glutathione S Transferase (GST) is a very well established means by which fusion to a protein of interest can increase the solubility of the protein of interest. However, in the double gene expression vector, the soluble expression of the protein of the multiple cloning site 2 in Escherichia coli is greatly improved by introducing GST into the multiple cloning site 1, and the method is not reported at present.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a non-fusion expression vector capable of enhancing the expression of a target gene. The invention provides a novel high-efficiency prokaryotic expression system for realizing protein soluble expression, which can realize the soluble expression of other intracellular foreign genes which are not fused with GST by increasing the expression of the GST.
It is still another object of the present invention to provide a method for preparing the above non-fusion expression vector capable of enhancing the expression of a target gene.
The invention aims to realize the technical scheme that a non-fusion expression vector capable of enhancing the expression of a target gene takes a pETduet-1 plasmid as a skeleton vector, the amino acid sequence of a cloning site 1 of the pETduet-1 plasmid is inserted into the plasmid is shown as SEQ ID NO.1, and the target gene is inserted into a cloning site 2 of the pETduet-1 plasmid. The target gene is a gene to be expressed. After a gene sequence corresponding to the sequence shown in SEQ ID NO.1 is inserted into the pETduet-1 plasmid cloning site 1, the gene expression level of the cloning site 2 can be obviously improved.
The preparation method of the non-fusion expression vector capable of enhancing the expression of the target gene comprises the following steps:
cloning the fragment GST-CTB into a pETduet-1 vector to construct a plasmid pETduet-GST-CTB (wherein the CTB is a cholera toxin B subunit);
secondly, cloning the segment EGFP-CTA2-TAT into a pET22b vector to construct a plasmid pET22b-EGFP-CTA2-TAT as a control plasmid;
thirdly, cloning the EGFP-CTA2-TAT fragment into a pETduet-GST-CTB vector to construct a plasmid pETDuet-GST-CTB/EGFP-CTA 2-TAT;
fourthly, transforming the two constructed plasmids into escherichia coli BL21 to perform induced expression of proteins, and analyzing and comparing expression products.
The first step specifically comprises the following steps:
(1) extracting by a plasmid extraction kit to obtain a plasmid PGEX-4T3 as a template, wherein a gene sequence of GST is on PGEX-4T 3; through designing a primer, obtaining a BamHI-CTB-XhoI fragment by PCR, carrying out BamHI/XhoI double enzyme digestion on PGEX-INSITE and BamHI-CTB-XhoI simultaneously to obtain a vector fragment and a PCR fragment, recovering enzyme digestion products by glue, connecting the enzyme digestion products by T4 ligase overnight, converting the connection products into escherichia coli BL21(DE3) competence, selecting positive clones, sending the positive clones to Shanghai bioengineering company for sequencing, wherein the sequencing result shows that the CTB is correctly connected with the vector PGEX, and newly establishing a plasmid PGEX-CTB;
(2) PGEX-CTB is taken as a template, and the primer sequence is as follows:
an upstream primer 5'-CATGCCATGGGTATGTCCCCTATACTAGGTTAT-3' and a downstream primer 5'-CCCAAGCTTTTAATTTGCCATACTAATTGCG-3', PCR are used for obtaining a target fragment GST-CTB, and a product is purified by a PCR product recovery kit;
(3) the pETduet-1 vector and the PCR product GST-CTB are subjected to double digestion by Nco I and Hind III, the digestion product is recovered, and the DNA fragment 1: 1 proportion, adding T4 ligase to react at 4 ℃ overnight, transforming the reaction product into escherichia coli BL21(DE3), carrying out double enzyme digestion identification on Nco I and Hind III, carrying out plasmid PCR, carrying out Shanghai worker sequencing, and marking the result of correct sequencing as pETduet-GST-CTB.
The second step is to construct plasmid pET22b-EGFP-CTA2-TAT, and comprises the following steps:
searching gene sequences of EGFP, CTA2 and TAT through NCBI, adding a connecting short peptide GGGGS between the EGFP and the CTA2 as a linker for modification and enhancing the expression stability in order to better fuse the EGFP with the CTA2 and prevent the termination during expression because the EGFP molecule is larger; meanwhile, enzyme cutting sites are added between the EGFP and the CTA2 so as to replace the EGFP as a target drug protein; the gene sequences are connected in series and sent to Beijing pul company for synthesis; the synthesized gene sequence is named as EGFP-CTA2-TAT and is constructed on pET22b vector, and finally the vector is marked as pET22b-EGFP-CTA 2-TAT.
The third step is specifically as follows:
(1) relying on primer design software primer 5.0, carrying out PCR amplification by using pET22b-EGFP-CTA2-TAT as a template to obtain a target fragment EGFP-CTA 2-TAT;
(2) the amplified product is subjected to enzyme digestion and enzyme connection, cloned to a pETduet-GST-CTB multiple cloning site 2 vector, transformed into competent Escherichia coli DH5 alpha, a positive transformant is screened through ampicillin resistance, digestion and sequencing are carried out to determine whether the transformant meets expectations, and the newly constructed vector is marked as pETduet-GST-CTB/EGFP-CTA 2-TAT.
The fourth step specifically includes the following steps:
a) transforming plasmid pETDuet-GST-CTB/EGF-CTA2-TAT into competence of escherichia coli BL21(DE3), screening positive transformants by an ampicillin-resistant LB plate, and preserving glycerol;
b) 1: adding 100 bacterial liquids into 400mL LB culture medium containing 100ug/mL in proportion, and culturing for 2 h;
c) adding 1mmol/L IPTG to induce expression;
d) SDS-PAGE and western blot analysis of protein expression.
Compared with the prior art, the invention has the following advantages and effects:
the GST of the invention is fused with protein CTB to form GST-CTB, and is cloned into a multiple cloning site 1 of a double-gene expression vector pETduet-1 vector to form pETduet-GST-CTB. It was found that GST introduced by the linkage of CTB to GST can induce a greatly increased soluble expression of the protein at multiple cloning site 2. Recombinant plasmids pETduet-GST-CTB/EGFP-CTA2-TAT and pET22b-EGFP-CTA2-TAT are constructed, then the recombinant plasmids are transformed into escherichia coli BL21 competent cells, positive transformants are screened, cultured, and target protein EGFP-CTA2-TAT is induced to express, and SDS PAGE and western blot analysis are compared to find that the expression of target protein EGFP-CTA2-TAT in the supernatant of the bacterium containing pETduet-GST/EGFP-CTA 2-TAT is 20 times higher than that of the expression of target protein EGFP-CTA2-TAT containing pET22b-EGFP-CTA 2-TAT.
Drawings
FIG. 1 is a diagram of pETduet-GST-CTB/EGFP-CTA2-TAT vector plasmid.
FIG. 2 is an SDS-PAGE electrophoresis of the inducible expression protein. Wherein 1 is non-induced thallus, 2 is BL21(pET22b-EGFP-CTA2-TAT) strain induced for 6 hours, and 3 is BL21(pETduet-EGFP-CTA2-TAT) strain induced for 6 hours.
FIG. 3 is a Western Blot analysis of BL21(DE3) -) pETduet-GST-CTB/EGFP-CTA2-TAT expression; wherein: a, EGFP antibody effect induces expression of protein; b, inducing expression protein by EGFP antibody; and c, inducing the expression of the protein by the CTB antibody.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 GST-containing non-fusion recombinant plasmid pETDuet-GST-CTB/EGF-CTA2-TAT was prepared as follows:
(1) plasmid PGEX-4T3 extracted by a plasmid extraction kit is used as a template, wherein the PGEX-4T3 has a GST gene sequence. A BamHI-CTB-XhoI fragment is obtained by designing a primer and carrying out PCR, a BamHI/XhoI double enzyme digestion is carried out on PGEX-INSITE and BamHI-CTB-XhoI simultaneously to obtain a vector fragment and a PCR fragment, the enzyme digestion products are recovered by glue and then are connected overnight by T4 ligase, the connection products are transformed into escherichia coli BL21(DE3) competence, positive clones are selected and sent to Shanghai bioengineering company for sequencing, the sequencing result shows that the CTB is correctly connected with the vector PGEX, and the plasmid PGEX-CTB is newly established.
(2) PGEX-CTB is taken as a template, and the primer sequence is as follows:
5'-CATGCCATGGGTATGTCCCCTATACTAGGTTAT-3' (upstream primer)
5'-CCCAAGCTTTTAATTTGCCATACTAATTGCG-3' (downstream primer) PCR to obtain the target fragment GST-CTB. The product was purified by PCR product recovery kit.
(3) The pETduet-1 vector and the PCR product GST-CTB are subjected to double digestion by Nco I and Hind III, the digestion product is recovered, and the DNA fragment 1: 1 proportion, adding T4 ligase to react at 4 ℃ overnight, transforming the reaction product into escherichia coli BL21(DE3), carrying out double enzyme digestion identification on Nco I and Hind III, carrying out plasmid PCR, carrying out Shanghai worker sequencing, and marking the result of correct sequencing as pETduet-GST-CTB.
(4) The gene sequences of EGFP, CTA2 and TAT are searched through NCBI, and because EGFP molecules are large, the EGFP molecules are better fused with CTA2 so as not to be suspended during expression, a connecting short peptide GGGGS is added between the EGFP and the CTA2 to be used as a linker for modification, and the expression stability is enhanced. Meanwhile, enzyme cutting sites are added between the EGFP and the CTA2 so as to replace the EGFP as a target drug protein. The gene sequences were concatenated and sent to Peking Poulele for synthesis. The synthesized gene sequence is named EGFP-CTA2-TAT, and is constructed on pET22b vector by Beijing pul company, and finally the vector is marked as pET22b-EGFP-CTA 2-TAT.
(5) Relying on primer design software primer 5.0, carrying out PCR amplification by using pET22b-EGFP-CTA2-TAT as a template to obtain a target fragment EGFP-CTA 2-TAT.
(6) The amplified product is subjected to enzyme digestion and enzyme ligation, cloned to a pETduet-GST-CTB polyclonal site 2 vector, transformed into competent Escherichia coli DH5 alpha, a positive transformant is screened through ampicillin resistance, digestion and sequencing are carried out to determine whether the transformant meets the expectation, and the newly constructed vector is marked as pETduet-GST-CTB/EGFP-CTA2-TAT (a plasmid construction diagram is shown in figure 1).
Wherein the results of the nucleotide sequence of the EGFP sequencing are shown below:
AACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGAATCTGGTTCCGCGTGGATCCACACCTCAAAATATTACTGATTTGTGTGCAGAATACCACAACACACAAATATATACGCTAAATGATAAGATATTTTCGTATACAGAATCTCTAGCTGGAAAAAGAGAGATGGCTATCATTACTTTTAAGAATGGTGCAATTTTTCAAGTAGAAGTACCAGGTAGTCAACATATAGATTCACAAAAAAAAGCGATTGAAAGGATGAAGGATACCCTGAGGATTGCATATCTTACTGAAGCTAAAGTCGAAAAATTATGTGTATGGAATAATAAAACGCCTCATGCGATTGCCGCAATTAGTATGGCAAATTAAAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCTAGCGAATTCGAGCTCGGAGGTGGTGGATCCATGAGCAATACGTGCGATGAGAAGACACAAAGCCTGGGCGTGAAATTCTTAGACGAATATCAAAGCAAAGTGAAACGCCAAATCTTCAGCGGATACCAAAGCGATATTGACACGCACAATCGCATCAAGGATGAGTTAGTCGACGGTCGTAAGAAACGTCGTCAGCGTCGTCGTCCACCACAGCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACTAGCGCAGCTTAATACCTAGTTGCC。
example 2 comparative analysis of EGFP-CTA2-TAT protein expression:
plasmids pETduet-GST-CTB/EGFP-CTA2-TAT and pET22b-EGFP-CTA2-TAT are respectively transformed into competent cells BL21(DE3), and positive transformants are screened by ampicillin resistant LB plates and preserved as glycerobacteria. And then, treating the bacterial liquid according to the proportion of 1: 100 portions of the protein were added into 400mL LB medium containing 100. mu.g/mL, after 2h of culture, 1mmol/L of IPTG was added to induce expression for 6h, and protein expression was detected by SDS PAGE and Western Blot (shown in FIGS. 2 and 3).
SDS PAGE and western blot analysis are compared to find that the EGFP-CTA2-TAT expressed in a non-fusion way is 20 times higher than that expressed normally.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A non-fusion expression vector for enhancing expression of a gene of interest, comprising: the pETduet-1 plasmid is taken as a skeleton vector, the nucleotide sequence of the cloning site 1 of the pETduet-1 plasmid inserted is shown as SEQ ID NO.1, and the target gene is inserted into the cloning site 2 of the pETduet-1 plasmid;
the target gene is a tandem sequence of EGFP, CTA2 and TAT genes.
2. The method for preparing a non-fusion expression vector for enhancing expression of a target gene according to claim 1, wherein: the method comprises the following steps:
cloning the fragment GST-CTB into a pETduet-1 vector to construct a plasmid pETduet-GST-CTB;
secondly, cloning the segment EGFP-CTA2-TAT into a pET22b vector to construct a plasmid pET22b-EGFP-CTA2-TAT as a control plasmid;
thirdly, cloning the EGFP-CTA2-TAT fragment into a pETduet-GST-CTB vector to construct a plasmid pETDuet-GST-CTB/EGFP-CTA 2-TAT;
fourthly, transforming the two constructed plasmids into escherichia coli BL21 to perform induced expression of proteins, and analyzing and comparing expression products.
3. The method for preparing a non-fusion expression vector for enhancing expression of a target gene according to claim 2, wherein: the first step specifically comprises the following steps:
(1) extracting by a plasmid extraction kit to obtain a plasmid PGEX-4T3 as a template, wherein a gene sequence of GST is on PGEX-4T 3; through designing a primer, obtaining a BamHI-CTB-XhoI fragment by PCR, carrying out BamHI/XhoI double enzyme digestion on PGEX-INSITE and BamHI-CTB-XhoI simultaneously to obtain a vector fragment and a PCR fragment, recovering enzyme digestion products by glue, connecting the enzyme digestion products by T4 ligase overnight, converting the connection products into escherichia coli BL21(DE3) competence, selecting positive clones, sending the positive clones to Shanghai bioengineering company for sequencing, wherein the sequencing result shows that the CTB is correctly connected with the vector PGEX, and newly establishing a plasmid PGEX-CTB;
(2) PGEX-CTB is taken as a template, and the primer sequence is as follows:
the upstream primer is shown as SEQ ID NO.2, the downstream primer is shown as SEQ ID NO.3, a target fragment GST-CTB is obtained by PCR, and a product is purified by a PCR product recovery kit;
(3) the pETduet-1 vector and the PCR product GST-CTB are subjected to double digestion by Nco I and Hind III, the digestion product is recovered, and the DNA fragment 1: 1 proportion, adding T4 ligase to react at 4 ℃ overnight, transforming the reaction product into escherichia coli BL21(DE3), carrying out double enzyme digestion identification on Nco I and Hind III, carrying out plasmid PCR, carrying out Shanghai worker sequencing, and marking the result of correct sequencing as pETduet-GST-CTB.
4. The method for preparing a non-fusion expression vector for enhancing expression of a target gene according to claim 2, wherein: the second step is to construct plasmid pET22b-EGFP-CTA2-TAT, and comprises the following steps:
searching gene sequences of EGFP, CTA2 and TAT through NCBI, adding a connecting short peptide GGGGS between the EGFP and the CTA2 as a linker for modification and enhancing the expression stability in order to better fuse the EGFP with the CTA2 and prevent the termination during expression because the EGFP molecule is larger; meanwhile, enzyme cutting sites are added between the EGFP and the CTA2 so as to replace the EGFP as a target drug protein; the gene sequences are connected in series and sent to Beijing pul company for synthesis; the synthesized gene sequence is named as EGFP-CTA2-TAT and is constructed on pET22b vector, and finally the vector is marked as pET22b-EGFP-CTA 2-TAT.
5. The method for preparing a non-fusion expression vector for enhancing expression of a target gene according to claim 2, wherein: the third step is specifically as follows:
(1) relying on primer design software primer 5.0, carrying out PCR amplification by using pET22b-EGFP-CTA2-TAT as a template to obtain a target fragment EGFP-CTA 2-TAT;
(2) the amplified product is subjected to enzyme digestion and enzyme connection, cloned to a pETduet-GST-CTB multiple cloning site 2 vector, transformed into competent Escherichia coli DH5 alpha, a positive transformant is screened through ampicillin resistance, digestion and sequencing are carried out to determine whether the transformant meets expectations, and the newly constructed vector is marked as pETduet-GST-CTB/EGFP-CTA 2-TAT.
6. The method for preparing a non-fusion expression vector for enhancing expression of a target gene according to claim 2, wherein: the fourth step specifically includes the following steps:
a) transforming plasmid pETDuet-GST-CTB/EGF-CTA2-TAT into competence of escherichia coli BL21(DE3), screening positive transformants by an ampicillin-resistant LB plate, and preserving glycerol;
b) 1: adding 100 bacterial liquids into 400mL LB culture medium containing 100ug/mL in proportion, and culturing for 2 h;
c) adding 1mmol/L IPTG to induce expression;
d) SDS-PAGE and western blot analysis of protein expression.
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