CN115786340A - Method for improving activity of leader less promoter and application thereof - Google Patents
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Abstract
The invention discloses a method for improving the activity of a leader less promoter and application thereof, which comprises the step of inserting a 62bp nucleic acid sequence shown in SEQ ID NO.2, a 13bp SD sequence shown in SEQ ID NO.3 and a 5bp translation coupling structure shown in SEQ ID NO.4 between the leader less promoter and a target gene to prepare the high-activity promoter. The method provided by the invention has good universality for improving the activity of the leader less promoter; in the other two leaderless promoters tested, H36 and Cg0124, a significant promoter activity enhancing effect was also observed.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and biology, and particularly relates to a method for improving the activity of a leader less promoter and application thereof.
Background
In bacteria, archaea and extreme microorganisms, the mRNA of some genes lacks the 5' UTR sequence, translation begins at the transcription start site (TSP). In recent years, many synthetic promoters lacking 5'UTR sequence have been developed by researchers, and these promoters lacking 5' UTR sequence are collectively called leader loss promoter. Compared with the traditional promoter containing 5' UTR, the leader mill less promoter avoids the influence of 5' UTR sequence on gene expression due to the lack of a 5' UTR structure, and reduces the variability of gene expression.
Therefore, a gene expression cassette constructed based on the leader less promoter is theoretically more favorable for reliable control of gene expression.
Although the leader less promoter lacking the 5'UTR circumvents the effect of the 5' UTR on gene expression, it is more suitable for reliable control of gene expression. However, the leader less promoter generally has a lower expression strength relative to the conventional promoter (containing 5' UTR).
Disclosure of Invention
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 title of the 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 and/or other problems occurring in the prior art.
Therefore, the present invention aims to overcome the defects in the prior art and provide a method for improving the activity of a leader less promoter.
In order to solve the technical problems, the invention provides the following technical scheme: a method of increasing the activity of a leader less promoter comprising,
a section of 62bp nucleic acid sequence shown in SEQ ID NO.2, a 13bp SD sequence shown in SEQ ID NO.3 and a 5bp translation coupling structure shown in SEQ ID NO.4 are inserted between the leader loss promoter and the target gene to prepare the high-activity promoter.
As a preferable aspect of the method of the present invention, wherein: the leader less promoter comprises promoters with nucleic acid sequences shown as SEQ ID NO.1, SEQ ID NO.9 and SEQ ID NO. 10.
It is still another object of the present invention to overcome the disadvantages of the prior art and to provide an expression vector of a high activity promoter, which includes a corynebacterium glutamicum recombinant vector.
The invention further aims to overcome the defects in the prior art and provide a recombinant bacterium, which comprises a corynebacterium glutamicum recombinant bacterium.
It is another object of the present invention to overcome the disadvantages of the prior art and provide the use of the method of claim 1 for increasing gene expression, the use of the expression vector of any one of claims 3 or 4 for increasing gene expression, and the use of the recombinant strain of any one of claims 5 or 6 for increasing gene expression.
As a preferable aspect of the application of the present invention, wherein: the gene is a green fluorescent protein EGFP coding gene shown in SEQ ID NO.5, a VHH coding gene shown in SEQ ID NO.7 or a LacZ coding gene shown in SEQ ID NO. 8.
The invention has the beneficial effects that:
the leader Beless promoter without 5' UTR is constructed in a Bicistronic (BCD) form, and a coding sequence (a pre-cistron sequence) of a short peptide, a conserved SD sequence and a translation coupling structure are inserted between the promoter and a target gene to obtain a leader Beless BCD expression cassette; the construction mode can obviously improve the activity of a leader loss promoter in corynebacterium glutamicum, and when the strategy is applied to the leader loss promoter H36, the expression levels of proteins EGFP, VHH and LacZ are respectively improved by 15.45 times, 7.19 times and 8.80 times;
the method provided by the invention has good universality for improving the activity of the leader less promoter; in the other two leaderless promoters tested, H36 and Cg0124, a significant promoter activity enhancing effect was also observed.
Drawings
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 schematic diagram of a construction method for improving the activity of a leader less promoter in the embodiment of the present invention.
FIG. 2 is a schematic diagram of an EGFP expression cassette based on a leader class H36 promoter and a graph analyzing the expression level of EGFP in the example of the present invention.
FIG. 3 is a schematic diagram of a VHH expression cassette based on the leader less H36 promoter and an SDS-PAGE analysis of the expression level of VHH in the example of the present invention.
FIG. 4 is a schematic diagram of a LacZ expression cassette based on the leader less H36 promoter and a diagram of the LacZ (. Beta. -galactosidase) activity assay in the examples of the present invention.
FIG. 5 is a schematic diagram of an EGFP expression cassette based on a leader less H30 promoter and an analysis diagram of EGFP expression level in the present example.
FIG. 6 is a schematic diagram of an EGFP expression cassette and an EGFP expression level analysis based on an endogenous leader erless promoter Cg0142 in the examples of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
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.
Example 1
Construction of leader class BCD based on H36 promoter:
the invention carries out molecular construction on the basis of a shuttle plasmid pXMJ19 vector skeleton (GenBank: AJ 133195.1) of corynebacterium glutamicum/escherichia coli.
A95 bp leader loss H36 promoter (the nucleic acid sequence is referred to as SEQ ID NO. 1), a 62bp short peptide coding sequence (the nucleic acid sequence is referred to as SEQ ID NO. 2), a 13bp conserved SD sequence (the nucleic acid sequence is referred to as SEQ ID NO. 3) and a 5bp translation coupling structure (the nucleic acid sequence is referred to as SEQ ID NO. 4) are constructed into a skeleton vector pXMJ19 through EcoRV and Hind III to obtain a leader loss BCD expression vector H36-BCD based on the H36 promoter.
Meanwhile, the control vector was constructed in a conventional construction manner.
The construction process is as follows: directly constructing the leader-less H36 promoter (the nucleic acid sequence is referred to as SEQ ID NO. 1) into an EcoR V and HindIII digested linearized vector pXMJ19 by means of homologous recombination to obtain a H36 promoter-based single cistron expression vector H36-MCD of the leader-less.
Then, a reporter gene EGFP (nucleic acid sequence is referred to as SEQ ID NO. 5) is constructed into the above leader bicistronic and monocistronic expression vector after HindIII and EcoRI digestion by means of homologous recombination.
A leaderless monocistronic design (MCD) H36-MCD-EGFP and a bicistronic expression vector H36-BCD-EGFP based on EGFP expression of an H36 promoter are obtained by removing an upstream Hind III enzyme cutting site by using amplification primers EGFP-BCD-F/EGFP-R or EGFP-MCD-F/EGFP-R (Table 1), and the schematic diagram of an expression cassette is shown in figure 2.
TABLE 1 primers used in this example
Example 2
H36 leader desk cell DNA (BCD) expression intensity analysis based on EGFP fluorescence intensity:
in order to test the expression strength of leader circle H36 constructed by the bicistronic strategy, 500ng of two successfully constructed expression plasmids H36-BCD-EGFP and H36-MCD-EGFP are respectively transformed into Corynebacterium glutamicum CGMCC 1.15647.
After culturing in LBB solid medium supplemented with 10mg/L chloramphenicol at 30 ℃ for 48 hours, the correct single clones were picked up and inoculated into 10mL LBB medium, respectively, and incubated at 220rpm at 30 ℃ for 24 hours.
Then, the two strains were inoculated at 2% inoculum size into 2 bottles of LBB liquid medium containing 10mL, and the culture was continued at 220rpm at 30 ℃ for 24 hours.
Centrifuging 1mL of the above culture solution at low temperature, discarding the supernatant, and treating OD with PBS 600 Diluting to about 0.5, and detecting OD with fluorescence spectrophotometer 600 And fluorescence intensity. The EGFP fluorescence detection condition is set to be 488nm of excitation wavelength and 507nm of emission wavelength.
OD 600 Indicating amount of bacteria, fluorescence measurement value/OD 600 Used to represent the unit fluorescence level of EGFP.
The results show that: the EGFP unit OD fluorescence intensity of the expression vector (H36-MCD-EGFP) of the leader erless H36 constructed in the traditional manner is 247, the OD fluorescence intensity of the EGFP unit of the expression vector (H36-BCD-EGFP) constructed in the leader erless BCD manner is 3813 (figure 2), and the activity of the leader erless promoter H36 for expressing the EGFP is enhanced by 15.45 times.
Example 3
Expression of VHH and LacZ:
the CspB signal peptide (nucleic acid sequence referred to SEQ ID NO. 6) and the VHH gene (GenBank: JQ 068760.1) (nucleic acid sequence referred to SEQ ID NO. 7) were constructed by homologous recombination into the vectors H36-MCD and H36-BCD after digestion with endonuclease Hind III and EcoRI.
The corresponding expression vectors H36-MCD-VHH and H36-BCD-VHH based on the leadersss H36 promoter were obtained by removing the upstream HindIII cleavage site using the amplification primers VHH-BCD-F/VHH-R or VHH-MCD-F/VHH-R (Table 2).
The two groups of expression vectors are transferred into a corynebacterium glutamicum expression strain CGMCC 1.15647 by an electrotransformation method, correct monoclonals are respectively picked and inoculated into 10mL of LBB, and then the two groups of expression vectors are inoculated into a fresh 10mL of LBB culture medium by 2 percent of inoculation amount. The supernatant of the fermentation broth was collected after culturing at 220rpm at 30 ℃ for 24 hours. 12% SDS-PAGE to detect VHH expression and grayscale analysis of the SDS-PAGE result using Image J software.
The results show that: compared with a VHH expression vector obtained by a traditional construction mode, the construction method provided by the invention obviously improves the activity of the leader loss H36 promoter. The expression level of VHH was enhanced 7.19 fold (fig. 3).
The gene LacZ (the nucleic acid sequence is referred to as SEQ ID NO. 8) for coding beta-galactosidase is constructed into vectors H36-MCD and H36-BCD after digestion by endonuclease Hind III and EcoRI through homologous recombination. LacZ expression vectors H36-MCD-LacZ and H36-BCD-LacZ based on the leaderless H36 promoter were obtained by removing the upstream hindIII cleavage site using amplification primers LacZ-BCD-F/LacZ-R or LacZ-MCD-F/LacZ-R (Table 2).
The two groups of expression vectors are transferred into a corynebacterium glutamicum expression strain CGMCC 1.15647 by an electrotransformation method, correct monoclonals are respectively picked and inoculated into 10mL of LBB, and then the two groups of expression vectors are inoculated into a fresh 10mL of LBB culture medium by 2 percent of inoculation amount.
After culturing at 220rpm at 30 ℃ for 24 hours, 500. Mu.L of the culture broth was collected by centrifugation. Centrifuging and discarding cultureNutrient medium, and 500. Mu.L of precooled Z buffer (60 mM Na) 2 HPO 4 、40mM NaH 2 PO 4 ·H2O、10mM KCl、1mM MgSO 4 50 mM. Beta. -mercaptoethanol, pH 7.0) was added to the suspension.
By measuring OD 600 To assess cell density. Then, 100. Mu.L of the cell suspension was taken out, 100. Mu.L of chloroform and 50. Mu.L of 0.1% SDS (sodium dodecyl sulfate) were added thereto, and mixed in a vortex shaker for 10 seconds. 200. Mu.L of ONPG (4 mg/mL) was added and reacted at 30 ℃ for 1 hour, and then 500. Mu.L of 1M sodium carbonate was added to terminate the reaction.
Measurement A 420 And A 550 The value was used to calculate the activity of beta-galactosidase.
The results show that: the construction method provided by the invention obviously improves the activity of the leader less H36 promoter, so that the expression level of beta-galactosidase is improved by 8.80 times (figure 4).
TABLE 2 primers used in this example
Example 4
Construction and expression intensity analysis of leader BCD based on H30 and Cg0124 promoters:
in order to verify the universality of the method for enhancing the activity of the leader less promoter, the improvement effect of the construction method on the activities of the other two leader less promoters H30 (the nucleic acid sequence is referred to as SEQ ID NO. 9) and Cg0124 (the nucleic acid sequence is referred to as SEQ ID NO. 10) is continuously evaluated. In reference to the construction manner in the example, a monocistronic expression vector H30-MCD-EGFP and a bicistronic expression vector H30-BCD-EGFP based on the leader erless H30 promoter were constructed.
The construction method comprises the following steps: a leader less H30 promoter (the nucleic acid sequence is referred to as SEQ ID NO. 9) of 95bp, a short peptide coding sequence (the nucleic acid sequence is referred to as SEQ ID NO. 2) of 62bp, a conserved SD sequence (the nucleic acid sequence is referred to as SEQ ID NO. 3) of 13bp and a translation coupling structure (the nucleic acid sequence is referred to as SEQ ID NO. 4) of 5bp are constructed into a skeleton vector pXMJ19 digested by EcoRV and Hind III in a homologous recombination mode, and a leader less BCD expression vector H30-BCD based on the H30 promoter is obtained. The monocistronic control vector H30-MCD is a linearized vector pXMJ19 which is prepared by directly constructing the leader less H30 promoter into EcoRV and HindIII digested vectors through homologous recombination. After obtaining the two expression vectors, the reporter gene EGFP (the nucleic acid sequence is referred to as SEQ ID NO. 5) is respectively constructed into the leader bicistronic and monocistronic expression vectors digested by HindIII and EcoRI in a homologous recombination mode. The expression cassette shown in FIG. 5 was finally obtained. The result shows that the construction method provided by the invention obviously improves the activity of the leader loss H30 promoter, so that the expression level of EGFP is improved by 16.25 times compared with the traditional construction mode (figure 5).
Then, based on endogenous LEADERLESS Cg0124 promoters in Corynebacterium glutamicum, monocistronic expression vectors Cg0124-MCD-EGFP and bicistronic expression vectors Cg0124-BCD-EGFP are constructed. The construction of the monocistronic vector Cg0124-MCD-EGFP directly recombines a Cg0124 promoter of 100bp to pXMJ19 digested by EcoR V and HindIII, and then constructs the EGFP to the monocistronic vector digested by HindIII and EcoRI in a homologous recombination mode. The bicistronic mRNA expression vector Cg0124-BCD-EGFP is prepared by firstly recombining a Cg0124 promoter, a 62bp short peptide coding sequence, a conserved SD sequence and a TAATG translation coupling structure to EcoRV and HindIII digested pXMJ19, and then constructing EGFP to HindIII and EcoRI digested bicistronic mRNA skeleton Cg0124-BCD.
The schematic diagram of the finally obtained expression cassette is shown in fig. 6, and the result shows that the construction method provided by the invention is also suitable for the endogenous LEADERLESS promoter Cg0124 of Corynebacterium glutamicum, and the expression level of EGFP in the Cg0124 expression cassette constructed by the method of the invention is improved by 2.70 times compared with the traditional construction mode (fig. 6).
In conclusion, the method for improving the activity of the leader less promoter provided by the invention has good universality and can obviously improve the activity of the leader less promoter.
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.
Claims (10)
1. A method for increasing the activity of a leader less promoter, which is characterized by comprising the following steps: comprises the steps of (a) preparing a substrate,
a62 bp nucleic acid sequence shown in SEQ ID NO.2, a 13bp SD sequence shown in SEQ ID NO.3 and a 5bp translation coupling structure shown in SEQ ID NO.4 are inserted between the leader less promoter and the target gene to prepare the high-activity promoter.
2. The method of claim 1, wherein: the leader less promoter comprises promoters with nucleic acid sequences shown as SEQ ID NO.1, SEQ ID NO.9 and SEQ ID NO. 10.
3. An expression vector comprising the highly active promoter of claim 1.
4. The expression vector of claim 3, wherein: the expression vector comprises a corynebacterium glutamicum recombinant vector.
5. A recombinant bacterium comprising the promoter according to claim 1 or the expression vector according to claim 4.
6. The recombinant bacterium of claim 5, wherein: the recombinant bacteria comprise corynebacterium glutamicum recombinant bacteria.
7. Use of the method of claim 1 for increasing gene expression.
8. Use of the expression vector of any one of claims 3 or 4 to increase gene expression.
9. Use of the recombinant bacterium of any one of claims 5 or 6 for increasing gene expression.
10. Use according to any one of claims 7 to 9, characterized in that: the gene is a green fluorescent protein EGFP coding gene shown in SEQ ID NO.5, a VHH coding gene shown in SEQ ID NO.7 or a LacZ coding gene shown in SEQ ID NO. 8.
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| CN107338267A (en) * | 2016-05-03 | 2017-11-10 | 中国科学院深圳先进技术研究院 | Two-cistron expression vector and its construction method and application |
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