CN115418370B - Helicobacter pylori-escherichia coli shuttle expression vector and construction method and application thereof - Google Patents
Helicobacter pylori-escherichia coli shuttle expression vector and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a helicobacter pylori-escherichia coli shuttle expression vector and a construction method and application thereof, belonging to the technical field of biology. The invention uses the high-brightness fluorescent protein as a transcription reporter gene, and successfully constructs and obtains the helicobacter pylori-escherichia coli shuttle expression vector by adding the replication origin of escherichia coli, the helicobacter pylori, the penicillin and streptomycin double-resistance box, the transcription initiation region and the promoter, and the expression vector has good passage stability, and can ensure that an exogenous gene is expressed in the escherichia coli and the helicobacter pylori under the control of related elements of the vector, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a helicobacter pylori-escherichia coli shuttle expression vector, and a construction method and application thereof.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Helicobacter pylori (Helicobacter pylori) is a gram-negative bacterium that colonizes gastric epithelial cells, and can cause diseases such as gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Although the infection is chronic and often asymptomatic, this bacterial infection accounts for over 50% of the world's population and has been recognized by the world health organization as a class I carcinogen.
At present, the green fluorescent protein is utilized to mark helicobacter pylori, and the carrier for researching genetic regulation is very limited. The first generation of vectors, designed for example in pHel2, heuermann & Haas (1998), is an expression vector which can be shuttled in E.coli, H.pylori. The vector element comprises an Escherichia coli replication initiation sequence, a helicobacter pylori specific replication initiation sequence, oriT and a multiple cloning site. The vector takes green fluorescent protein as a reporter gene and simultaneously carries chloramphenicol resistance genes and kanamycin resistance genes. However, both chloramphenicol and kanamycin have strong cytotoxicity, and meanwhile, the pHel vector and the bacterial chromosome have more homologous sections, are easy to integrate into the bacterial chromosome, and have poor passage stability.
The second generation vector pTM117 was designed by Beth M.C. et al (2007), and consists of the H.pylori endogenous plasmid (pHP 666 (45)) with the addition of an E.coli replication origin, a kanamycin resistance cassette, a multiple cloning site, and a promoterless green fluorescent protein mutant gene (gfp mut 3). However, the vector has a simple structure, the fluorescence of the green fluorescent protein is weak, and helicobacter pylori is not easy to detect after entering a host cell. The study of H.pylori has been somewhat limited due to the relative lack of genetic manipulation tools for studying this bacterium.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a helicobacter pylori-escherichia coli shuttle expression vector and a construction method and application thereof. The invention uses the gene expressing high brightness fluorescent protein (AausFP 1) as transcription report gene, synthesizes optimized designed plasmid element through whole gene, and integrates the plasmid element on pCDFDuet-1 carrier by using double enzyme digestion, thereby successfully constructing and obtaining the helicobacter pylori-escherichia coli shuttle expression carrier, the expression carrier has good passage stability, and can lead the exogenous gene to be expressed in escherichia coli and helicobacter pylori under the control of related elements of the carrier, thereby having good value of practical application.
Specifically, the technical scheme of the invention is as follows:
the first aspect of the invention provides a helicobacter pylori-escherichia coli shuttle expression vector, and the nucleotide sequence of the shuttle expression vector is shown in SEQ ID NO. 1.
Specifically, the shuttle expression vector comprises the following elements: the coding sequence of the replication origin of Escherichia coli and helicobacter pylori, the gene of the penicillin and streptomycin double resistance cassette, the transcription initiation region and promoter of a molecular chaperone gene (co-chaperonin groES), and the reporter gene of high light green fluorescent protein (AausFP 1).
In still another embodiment of the present invention, the shuttle expression vector is modified by using pCDFDuet-1 vector as the backbone vector.
In a second aspect of the present invention, there is provided a method for constructing the above helicobacter pylori-escherichia coli shuttle expression vector, wherein the method comprises:
the recombinant vector is obtained by synthesizing a green fluorescent protein (AausFP 1) optimized based on codon usage preference of helicobacter pylori, a penicillin and streptomycin dual-resistance cassette, a replication origin, a promoter, a transcription initiation site and a ribosome binding site into a whole gene, integrating the gene into a fragment, connecting the fragment to a pCDFDuet-1 vector by using a dual-enzyme digestion method, and connecting the green fluorescent protein (AausFP 1) optimized based on the codon usage preference of escherichia coli to a plasmid pCDFDuet-1 by using the dual-enzyme digestion method.
In a third aspect of the present invention, there is provided the use of the aforementioned helicobacter pylori-escherichia coli shuttle expression vector in any one or more of the following:
1) Expression of exogenous genes in H.pylori;
2) Expressing the exogenous gene in escherichia coli;
3) Genetic regulation and control research of helicobacter pylori and/or escherichia coli;
4) Research and development of helicobacter pylori and/or escherichia coli related medicaments.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme provides a helicobacter pylori-escherichia coli shuttle expression vector and a construction method and application thereof. The invention uses the high-brightness fluorescent protein as a transcription reporter gene, and successfully constructs and obtains the helicobacter pylori-escherichia coli shuttle expression vector by adding the escherichia coli, the replication origin of the helicobacter pylori, the penicillin and streptomycin double-resistance box, the transcription initiation region and the promoter, wherein the expression vector has good passage stability, and can ensure that an exogenous gene is expressed in the escherichia coli and the helicobacter pylori under the control of related elements of the vector, thereby having good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a diagram of the skeleton of a helicobacter pylori-Escherichia coli shuttle expression vector of the present invention;
FIG. 2 is a photograph of DH 5. Alpha. Escherichia coli observed by fluorescence microscope in the examples of the present invention;
FIG. 3 is a diagram showing the observation of helicobacter pylori by a fluorescence microscope in an example of the present invention;
FIG. 4 is a 4 th passage of E.coli streaked in the present example;
FIG. 5 is a flat drawing of the H.pylori passage 4 coating in the example of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention will now be further described with reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
In a typical embodiment of the invention, the invention provides a helicobacter pylori-escherichia coli shuttle expression vector, and the nucleotide sequence of the shuttle expression vector is shown in SEQ ID NO. 1.
Specifically, the shuttle expression vector comprises the following elements: the coding sequence of the replication origin of Escherichia coli and helicobacter pylori, the penicillin and streptomycin double-resistance box gene, the transcription initiation region and promoter of a molecular chaperone gene (co-chaperonin groES), and a high-brightness green fluorescent protein (AausFP 1) reporter gene.
In still another embodiment of the present invention, the shuttle expression vector is modified by using pCDFDuet-1 vector as the backbone vector.
Wherein, the replication origin coding sequence of the Escherichia coli adopts a CloDF13 ori replicon gene, which can realize self-replication in the Escherichia coli.
The said helicobacter pylori replication origin coding sequence adopts the sequence of helicobacter pylori natural plasmid, so that the self-replication in helicobacter pylori can be realized. In particular, in one embodiment of the present invention, a native plasmid (ACCESSION DQ198799; helicobacter pylori strain CCUG 17874plasmid pHP666) is used, which enables the copy number of the plasmid to be controlled, and also increases the stability of the vector. The origin of replication in H.pylori has an AT-rich region and multiple repeats (repeats), to which the Rep, the replication initiation protein encoded by the native plasmid pHP666, can bind and control the copy number of the plasmid.
The traditional fluorescent protein has low brightness, and after the helicobacter pylori is transferred into the helicobacter pylori, although the thallus can emit green fluorescence under the irradiation of ultraviolet light, when the helicobacter pylori enters host cells, the common fluorescent protein has low fluorescence brightness, and the host cells carrying the helicobacter pylori are difficult to be separated by a flow cytometer. Therefore, in the invention, green fluorescent protein (AausFP 1) newly found in Aequorea australis in 2020 is selected as a reporter gene, and the brightness of the protein is more than 5 times of that of the traditional GFP. The invention also designs the gene of AausFP1 based on the codon usage preference of helicobacter pylori, so that the gene is easier to express in helicobacter pylori with high efficiency. This design facilitates flow cytometry for positive seed detection.
The present invention uses a promoter of a molecular chaperone gene (co-chaperonin groES) to effect gene expression in helicobacter pylori. It was found by annotating the data of the H.pylori proteome that the protein translated from the molecular chaperone gene (co-chaperonin groES) had the highest protein abundance in H.pylori, and it was presumed that the gene promoter was a strong promoter. The transcription initiation region of the gene is predicted to be a region downstream of the promoter by bioinformatic means. The Ribosome Binding Site (RBS) is AGGAGA, 6 bases apart from the initiation codon (ATG) (ACTAAT).
The vector uses a penicillin and streptomycin dual-resistance box as a screening marker, and compared with chloramphenicol and kanamycin, penicillin and streptomycin are low in cytotoxicity, and the cell survival rate can be improved.
In another embodiment of the present invention, there is provided a method for constructing the aforementioned helicobacter pylori-escherichia coli shuttle expression vector, the method comprising:
the recombinant vector is obtained by synthesizing a green fluorescent protein (AausFP 1) optimized based on codon usage preference of helicobacter pylori, a penicillin and streptomycin dual-resistance cassette, a replication origin, a promoter, a transcription initiation site and a ribosome binding site into a whole gene, integrating the gene into a fragment, connecting the fragment to a pCDFDuet-1 vector by using a dual-enzyme digestion method, and connecting the green fluorescent protein (AausFP 1) optimized based on the codon usage preference of escherichia coli to a plasmid pCDFDuet-1 by using the dual-enzyme digestion method.
In yet another embodiment of the present invention, the method comprises:
s1, green fluorescent protein (AausFP 1) optimized based on Escherichia coli codon preference and used for expressing in Escherichia coli; after the whole gene is synthesized, the DNA fragment is connected to a pCDFDuet-1 vector by utilizing two enzyme cutting sites of Bgl II (5 ') and Kpn I (3');
s2, constructing an ampicillin resistance expression cassette; the enzyme cutting sites are Xba I (5 ') and Sal I (3'); the sequence is shown in SEQ ID NO. 3;
s3, a replication origin in helicobacter pylori, wherein a double enzyme cutting site is Smal; the sequence is shown in SEQ ID NO. 4;
s4, a promoter, a transcription initiation site and a ribosome binding site in helicobacter pylori, wherein the double enzyme cutting site is EcoR V; the sequence is shown as SEQ ID NO. 5;
s5, helicobacter pylori codon preference-optimized green fluorescent protein (AausFP 1) for expression in helicobacter pylori; the restriction enzyme sites are EcoR V (5 ') and Not I (3'); the sequence is shown in SEQ ID NO. 6;
the fragments obtained in steps S2 to S5 were integrated into one fragment, and then ligated to the pCDFDuet-1 vector containing the green fluorescent protein gene obtained in step S1 using Xba I, not I.
It should be noted that, the invention adds blunt-end enzyme cutting sites at the two ends of the elements such as replication origin, promoter, reporter gene, screening marker gene, etc., to realize modular design, and provide convenience for researching the expression regulation mechanism of helicobacter pylori. Wherein the replication origin in the helicobacter pylori, and the enzyme cutting sites at two ends are Smal; a promoter, a transcription initiation site and a ribosome binding site in helicobacter pylori, wherein enzyme cutting sites at two ends of the promoter, the transcription initiation site and the ribosome binding site are EcoR V; the optimized green fluorescent protein (AausFP 1) enzyme cutting sites based on the helicobacter pylori codon preference are EcoR V and Not I; the restriction sites of the green fluorescent protein (AausFP 1) optimized based on the codon preference of Escherichia coli are Bgl II and Kpn I.
Adding blunt-end enzyme cutting sites at two ends of the element provides a convenient way for element replacement, and the modular design is helpful for researching the strength of the helicobacter pylori gene promoter, the verification of a constitutive/inducible promoter and other expression regulation mechanisms.
In another embodiment of the present invention, there is provided a use of the aforementioned helicobacter pylori/escherichia coli shuttle expression vector in any one or more of the following:
1) Expression of exogenous genes in H.pylori;
2) Expressing the exogenous gene in escherichia coli;
3) Genetic regulation and control research of helicobacter pylori and/or escherichia coli;
4) Research and development of related medicaments of helicobacter pylori and/or escherichia coli.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.
Examples
1. Materials and methods of research
Reagent required by the experiment
The main reagents involved in this experiment are shown in table 1 below.
TABLE 1 information of the main reagents required for this experiment
(II) instruments required by the experiment
The main instruments involved in this experiment are shown in table 2 below.
Table 2 main instrument information required for this experiment
(III) sequence screening design
The vector takes plasmid pCDFDuet-1 as a framework, synthesizes an optimized element sequence by a whole gene, and connects the synthesized sequence to the plasmid pCDFDuet-1 by using a double enzyme digestion method. The original sequences of the gene segments involved in the experiment are all obtained from GeneBank. The sequences, plasmids and strains used in this experiment are as follows in table 3.
TABLE 3 sequence, plasmid, strain information used in this experiment
1. Design of expressed Gene sequences in helicobacter pylori
The origin of replication (ori) used in H.pylori, the promoter for gene expression, and the transcription initiation region are the gene sequences of H.pylori itself. The origin of replication (ori) in H.pylori is the sequence of the H.pylori native plasmid (plasmid pHP 666) which contains a replicon construct that can regulate the copy number of the plasmid by binding to Rep proteins produced in the bacterium. Promoters for effecting gene expression in helicobacter pylori employ the promoter of a chaperone gene (co-chaperonin groES), the transcription initiation region of which is predicted by bioinformatic means to be in the downstream region of the promoter. The Ribosome Binding Site (RBS) is AGGAGA, 6 bases apart from the initiation codon (ATG) (ACTAAT).
2. Implementation of modular design
After obtaining the gene sequence of the desired element, the gene sequence was optimized according to the codon usage preference of E.coli and H.pylori, and blunt-ended enzyme cutting sites were added to both ends of each element sequence, as shown in Table 4 below.
TABLE 4 enzyme cleavage sites at both ends of each element
(IV) Total Gene Synthesis
Integrating II, III, IV and V segments into a sequence VI, and carrying out whole gene synthesis on the sequence and the sequence I.
(V) ligation of vector and element sequence
1. Vector double digestion and fragment ligation
(1) Connection of green fluorescent protein (AausFP 1) optimized based on Escherichia coli codon preference and carrier
The pCDFDuet-1 empty vector was provided by the laboratory. The reaction was carried out using bgl II at 37 ℃ for 1h, as shown in Table 5 below. Reaction product a is obtained.
TABLE 5 Bgl II reaction System
The reaction product a was digested with Kpn I at 37 ℃ for 1h, and the reaction system is shown in Table 6 below. Reaction product b is obtained.
TABLE 6 Kpn I reaction System
Agarose gel electrophoresis, cutting gel and recovering, preparing a reaction system (10 mu L in total) by the reaction product b, a gene fragment I solution and a solution I (1. Reaction product c is obtained.
(2) The expression gene in the helicobacter pylori is connected with a carrier
The reaction product c was digested with Xba I at 37 ℃ for 5min, and the reaction system is shown in Table 7 below. Reaction product d is obtained.
TABLE 7 Xba I reaction System
The reaction product d was digested with Not I at 37 ℃ for 5min, and the reaction system is shown in Table 8 below. Reaction product e is obtained.
TABLE 8 Not I reaction System
Agarose gel electrophoresis, cutting gel and recovering, preparing a reaction system (10 mu L in total) by the reaction product e, the gene fragment VI solution and the solution I (1. Reaction product f is obtained.
2. Post ligation vector purification
And (3) carrying out agarose gel electrophoresis on the reaction product f, cutting the gel, recovering to obtain a recombinant vector pHP-EC-shuttle, and carrying out sequence identification (Shandong Qibang Viukang biotechnology, co., ltd.). Plasmids identified as having the correct sequence were purified using the Plasmid Mini Kit I (OMEGA bio-tek).
(VI) detecting the expression of the Green fluorescent protein gene
1. Transferring into colibacillus to detect green fluorescent protein gene expression and observe
Adding 20 μ L of the ligation product into the subpackaged 200mL DH5 alpha competent cells, uniformly mixing on ice, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, and rapidly carrying out ice bath cooling for 2min; 890. Mu.L of LB medium was added to the tube, and the mixture was cultured with shaking at 37 ℃ for 45 to 60min. 10. Mu.L of 1mol/L IPTG was pipetted and uniformly spread on LB medium containing 50. Mu.g/mL ampicillin and streptomycin, after drying IPTG, 10. Mu.L of the bacterial solution was pipetted and uniformly spread on the induction medium, and cultured at 37 ℃ for 12 hours. Picking single colony, mixing well in 500. Mu.L physiological saline, sucking 20. Mu.L tabletting. Excited with blue light and observed under a fluorescence microscope (objective 40x, eyepiece 10 x). The positive strain was named Ec-shuttle.
2. Detection of Gene expression in helicobacter pylori
Helicobacter pylori was spread evenly on Horse Blood Agar (HBA) plates, placed in an incubator, under microaerophilic conditions (5% 2 、10％CO 2 、85％N 2 ) Culturing at 37 deg.C for 48h. The bacteria on the plate were gently scraped off, collected at 5000 Xg for 5min, washed 3 times with TBS buffer (0.01M TBS, pH 7.5) and resuspended. The plasmid was electrotransformed (voltage 1 800V, capacitance 25. Mu.F, resistance 200. Omega.) into H.pylori, then spread evenly on Horse Blood Agar (HBA) plates, placed in an incubator, and subjected to microaerophilic conditions (5% O) 2 、10％CO 2 、85％N 2 ) Culturing at 37 deg.C for 48h. Picking single colony, mixing the single colony in 500 μ L normal saline, sucking 20 μ L tablet. Excited with blue light and observed under a fluorescent microscope (objective 40x, eyepiece 10 x). The positive strain was named Hp-shuttle.
(VII) measurement of Carrier stability
1. Determination of stability in E.coli
The strain Ec-shuttle is cultured in an induction medium for 12h to obtain a 1 st generation culture. Sucking 10. Mu.L of 1mol/L IPTG, uniformly coating the mixture on LB culture medium containing 50. Mu.g/mL ampicillin and streptomycin, after IPTG is dried, picking out single colony obtained by culture by using an inoculating loop, and carrying out streak culture on the induction culture medium for 12h to obtain a culture of the 2 nd generation. The above steps are repeated to obtain 3 rd and 4 th generation cultures. If the 4 th culture has colony growth, the carrier is stable in Escherichia coli.
2. Determination of stability in helicobacter pylori
Strain Hp-shuttle was cultured overnight in liquid medium supplemented with 50. Mu.g/ml amp. The resulting day 0 culture was used to inoculate a day 1 culture in liquid medium without amp. Day 1 cultures were grown overnight and subcultured in fresh liquid medium without amp at 1. This re-inoculation cycle was repeated at the beginning of the liquid culture on day 5. Culture samples from days 0, 1, 3 and 5 were plated on HBA plates to obtain single colonies. Four days later, colonies were replica plated onto HBA plates and 50. Mu.g/ml amp was added to HBA plates. Two days after replica plating, the HBA plates supplemented with amp were compared to the unsupplemented HBA plates to identify any amp-sensitive colonies and to assess the stability of the plasmid when repeatedly passaged in liquid medium without selection. Collecting thallus, extracting plasmid, and detecting by electrophoresis. If a band is evident, it is shown that the newly constructed plasmid does remain free in H.pylori cells.
2. As a result, the
(I) construction of E.coli-H.pylori shuttle expression vector
At present, the number of tool vectors for researching helicobacter pylori is relatively small, so that the shuttle expression vector which is simple in structure, stable in passage and convenient to operate is designed. The vector takes a pCDFDuet-1 vector as a skeleton, is connected with elements such as a helicobacter pylori replication origin, a promoter and the like which are optimized based on the usage preference of helicobacter pylori codons, and is designed with a penicillin and streptomycin dual-resistance box which can be used as a marker gene. The vector also contains a highlighted green fluorescent protein gene, and when helicobacter pylori parasitizes in gastric mucosa cells, an expression product of the highlighted green fluorescent protein can be used as a marker for helicobacter pylori localization.
An Escherichia coli-helicobacter pylori shuttle expression vector pHP-EC-shuttle is constructed, and the structure is shown in the figure. First, the optimized gene fragments (containing the restriction enzyme sites at both ends) were subjected to whole gene synthesis, and then the fragments obtained by whole gene synthesis were ligated to pCDFDuet-1 vector using Bgl II, kpn I, xba I and Not I to generate plasmid pHP-EC-shuttle (4889 bp in length). The expression vector pHP-EC-shuttle was verified by restriction digestion and DNA sequencing.
In the process of vector construction, penicillin and streptomycin are used as markers for successful transformation of escherichia coli and helicobacter pylori, and the escherichia coli and the helicobacter pylori grow on a culture medium containing penicillin and streptomycin, which shows that the expression vector pHP-EC-shuttle is successfully expressed in thalli.
(II) detection of Green fluorescent protein Gene expression
The 4 th generation product obtained by repeated streaking of DH 5. Alpha. E.coli containing shuttle expression vector on induction medium is shown in the figure. DH 5. Alpha. E.coli smears in the green under a fluorescent microscope.
Helicobacter pylori containing the shuttle expression vector is cultured on the culture medium for 12h, and the colony is shown in the figure. Helicobacter pylori smears, which appear green under a fluorescence microscope.
(III) stability detection of expression vector pHP-EC-shuttle
And (4) repeatedly carrying out streak inoculation culture on the DH5 alpha escherichia coli containing the shuttle expression vector on an induction culture medium to obtain a 4 th generation product. As shown in fig. 4.
Helicobacter pylori containing the shuttle expression vector was repeatedly streaked on horse blood medium (HBA) to obtain the 4 th generation product. As shown in fig. 5.
In conclusion, the invention develops a tool vector pHP-EC-shuttle for helicobacter pylori design, the vector is transformed into Escherichia coli and helicobacter pylori and is induced and expressed in an induction culture medium, a green fluorescent protein gene carried on the vector pHP-EC-shuttle is expressed, after streak culture and passage, the plasmid pHP-EC-shuttle can still stably exist in thalli, and the plasmid is proved to have certain stability and can be used as a tool plasmid of the helicobacter pylori.
The invention selects the pCDFDuet-1 empty vector as the skeleton of the vector pHP-EC-shuttle, because the pCDFDuet-1 empty vector contains an escherichia coli replication origin (CloDF 13 ori) and a streptomycin resistance gene (expressed in escherichia coli), the pCDFDuet-1 empty vector can be directly utilized, and the construction of the vector is convenient. Meanwhile, the replication origin in the helicobacter pylori selected by the invention is the replication origin of the natural plasmid pHP666, the natural plasmid pHP666 is a strict plasmid, the copy number is low, and the replication origin of the natural plasmid pHP666 can be used for effectively controlling the copy number of the plasmid pHP-EC-shuttle in the helicobacter pylori to be maintained at a low level.
The plasmid pHP-EC-shuttle also comprises a lactose operon, so that the expression of a green fluorescent protein gene (AausFP 1) in Escherichia coli can be induced, both ends of the green fluorescent protein gene (AausFP 1) are designed with flat end enzyme cutting sites (5 ': bgl II,3': kpn I), and the green fluorescent protein gene can be cut off and replaced into a gene sequence with the same enzyme cutting sites at both ends, and the replaced gene sequence can be induced to express a large amount under the action of the lactose operon, thereby providing convenience for researching the action of the gene expression in helicobacter pylori. Helicobacter pylori depends on the colonization factor to colonize the gastric mucosa surface, and gastrointestinal diseases are caused by virulence factor [5] The plasmid pHP-EC-shuttle provides a good tool vector for researching the related genes in the aspects of helicobacter pylori pathogenicity, immune escape mechanism and the like, and has stronger practical significance and development prospect.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered in the protection scope of the present invention.
SEQUENCE LISTING
<110> Weihai city hospital
<120> helicobacter pylori-escherichia coli shuttle expression vector, and construction method and application thereof
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 4860
<212> DNA
<213> Artificial sequence
<400> 1
gcgctgcgga cacatacaaa gttacccaca gattccgtgg ataagcaggg gactaacatg 60
tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca taggctccgc cctcctgcca 120
gagttcacat aaacagacgc ttttccggtg catctgtggg agccgtgagg ctcaaccatg 180
aatctgacag tacgggcgaa acccgacagg acttaaagat ccccaccgtt tccggcgggt 240
cgctccctct tgcgctctcc tgttccgacc ctgccgttta ccggatacct gttccgcctt 300
tctcccttac gggaagtgtg gcgctttctc atagctcaca cactggtatc tcggctcggt 360
gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc cccgttcagc ccgactgctg 420
cgccttatcc ggtaactgtt cacttgagtc caacccggaa aagcacggta aaacgccact 480
ggcagcagcc attggtaact gggagttcgc agaggatttg tttagctaaa cacgcggttg 540
ctcttgaagt gtgcgccaaa gtccggctac actggaagga cagatttggt tgctgtgctc 600
tgcgaaagcc agttaccacg gttaagcagt tccccaactg acttaacctt cgatcaaacc 660
acctccccag gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa aaaaaggatc 720
tcaagaagat cctttgatct tttctactga accgctctag attaccaatg cttaatcagt 780
gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 840
gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 900
cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 960
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1020
gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca 1080
ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1140
tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1200
ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1260
cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1320
accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1380
cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1440
tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1500
cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1560
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1620
atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1680
tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcggtc gacaagaact 1740
acacctagtg ttggcaagaa ctacacctag tgttggcaag aactacacct agtgttggca 1800
agaactacac ctagtgttgg caagaactac acctagtgtt ggcaagaact acacctagtg 1860
ttggccccgg ggatatctgt atttttaaac taatgaaact tgatacaaat agacttaata 1920
atccttatag ttatattatt agctttgttt ttatggcttg acttatccct aaaaatgcgc 1980
tatagttatg tcgcttaata acaataagcg ctaaatttct attttattta tcaaaactta 2040
ggagaactaa tatggatatc agctatgggg ctttattatt tagggaaaaa attccttatg 2100
tggtggaaat ggaaggggat gtggaaggga tgaaatttag cgtgaggggg aaagggcatg 2160
gggatgctaa taccgggaaa attgaagcta gctttatttg caccaccggg gaattacctg 2220
tgccttggag cagcatttta accaccgtga cctatggggc tcaatgcttt gctaaatatc 2280
ctaatgatat taaagattat cctaaaagcg ctatgcctga agggtatgtg caagaaagga 2340
ccattacctt tgaaaatgat ggggtgtata aaaccagggc tgaagtgacc tatgaaaaag 2400
ggagcgtgta taatagggtg accttaaatg ggagcgggtt taaaaaaggg gggaatattt 2460
tagggaaaaa attagaattt aattataatc ctcattgcat ttatgtgtta cctgatgtgc 2520
aaaataatgg gattaaatgc tatattaata ttgtgcatga tgtgattggg ggggggcaaa 2580
ttattgctgc tcatcaacaa ttaaataccc ctttaggggg ggggcctgtg gatattcctc 2640
attatcatca tattcaagct cataccattt taagcaaaga tcctaaagaa accagggatc 2700
atatgaatgt ggtggaagtg tttagggcta ttgattgcaa aaccgcttat gctcatcatc 2760
atcatcatca ttaatgagcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg 2820
tacacggccg cataatcgaa attaatacga ctcactatag gggaattgtg agcggataac 2880
aattccccat cttagtatat tagttaagta taagaaggag atatacatat ggcagatctc 2940
tcttacggtg ctctgctgtt ccgtgaaaaa atcccgtacg ttgttgaaat ggaaggtgac 3000
gttgaaggta tgaaattctc tgttcgtggt aaaggtcacg gtgacgctaa caccggtaaa 3060
atcgaagctt ctttcatctg caccaccggt gaactgccgg ttccgtggtc ttctatcctg 3120
accaccgtta cctacggtgc tcagtgcttc gctaaatacc cgaacgacat caaagactac 3180
ccgaaatctg ctatgccgga aggttacgtt caggaacgta ccatcacctt cgaaaacgac 3240
ggtgtttaca aaacccgtgc tgaagttacc tacgaaaaag gttctgttta caaccgtgtt 3300
accctgaacg gttctggttt caaaaaaggt ggtaacatcc tgggtaaaaa actggaattc 3360
aactacaacc cgcactgcat ctacgttctg ccggacgttc agaacaacgg tatcaaatgc 3420
tacatcaaca tcgttcacga cgttatcggt ggtggtcaga tcatcgctgc tcaccagcag 3480
ctgaacaccc cgctgggtgg tggtccggtt gacatcccgc actaccacca catccaggct 3540
cacaccatcc tgtctaaaga cccgaaagaa acccgtgacc acatgaacgt tgttgaagtt 3600
ttccgtgcta tcgactgcaa aaccgcttac gctcatcacc atcatcacca cggtaccctc 3660
gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact 3720
agcgcagctt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt 3780
ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac 3840
acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct 3900
atttaacgac cctgccctga accgacgact tatttgccga ctaccttggt gatctcgcct 3960
ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg aggccaagcg atcttcttct 4020
tgtccaagat aagcctgtct agcttcaagt atgacgggct gatactgggc cggcaggcgc 4080
tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac 4140
caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag 4200
ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca 4260
aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc 4320
aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg 4380
cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg 4440
tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc 4500
gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacggtc 4560
accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg 4620
tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct 4680
gatagttgag tcgatacttc ggcgatcacc gcttccctca tactcttcct ttttcaatat 4740
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 4800
aaaaataaac aaatagctag ctcactcggt cgctacgctc cgggcgtgag actgcggcgg 4860
<210> 2
<211> 752
<212> DNA
<213> Artificial sequence
<400> 2
gatatacata tggcagatct ctcttacggt gctctgctgt tccgtgaaaa aatcccgtac 60
gttgttgaaa tggaaggtga cgttgaaggt atgaaattct ctgttcgtgg taaaggtcac 120
ggtgacgcta acaccggtaa aatcgaagct tctttcatct gcaccaccgg tgaactgccg 180
gttccgtggt cttctatcct gaccaccgtt acctacggtg ctcagtgctt cgctaaatac 240
ccgaacgaca tcaaagacta cccgaaatct gctatgccgg aaggttacgt tcaggaacgt 300
accatcacct tcgaaaacga cggtgtttac aaaacccgtg ctgaagttac ctacgaaaaa 360
ggttctgttt acaaccgtgt taccctgaac ggttctggtt tcaaaaaagg tggtaacatc 420
ctgggtaaaa aactggaatt caactacaac ccgcactgca tctacgttct gccggacgtt 480
cagaacaacg gtatcaaatg ctacatcaac atcgttcacg acgttatcgg tggtggtcag 540
atcatcgctg ctcaccagca gctgaacacc ccgctgggtg gtggtccggt tgacatcccg 600
cactaccacc acatccaggc tcacaccatc ctgtctaaag acccgaaaga aacccgtgac 660
cacatgaacg ttgttgaagt tttccgtgct atcgactgca aaaccgctta cgctcatcac 720
catcatcacc acggtaccct cgagtctggt aa 752
<210> 3
<211> 978
<212> DNA
<213> Artificial sequence
<400> 3
tctagattac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 60
atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 120
tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 180
aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 240
catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 300
gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc 360
ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 420
aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 480
atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 540
cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 600
gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 660
agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 720
gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 780
caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 840
ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 900
tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 960
aggggttccg cggtcgac 978
<210> 4
<211> 113
<212> DNA
<213> Artificial sequence
<400> 4
cccgggaaga actacaccta gtgttggcaa gaactacacc tagtgttggc aagaactaca 60
cctagtgttg gcaagaacta cacctagtgt tggcaagaac tacacctccc ggg 113
<210> 5
<211> 183
<212> DNA
<213> Artificial sequence
<400> 5
gatatctgta tttttaaact aatgaaactt gatacaaata gacttaataa tccttatagt 60
tatattatta gctttgtttt tatggcttga cttatcccta aaaatgcgct atagttatgt 120
cgcttaataa caataagcgc taaatttcta ttttatttat caaaacttag gagaactaat 180
atg 183
<210> 6
<211> 731
<212> DNA
<213> Artificial sequence
<400> 6
gatatcagct atggggcttt attatttagg gaaaaaattc cttatgtggt ggaaatggaa 60
ggggatgtgg aagggatgaa atttagcgtg agggggaaag ggcatgggga tgctaatacc 120
gggaaaattg aagctagctt tatttgcacc accggggaat tacctgtgcc ttggagcagc 180
attttaacca ccgtgaccta tggggctcaa tgctttgcta aatatcctaa tgatattaaa 240
gattatccta aaagcgctat gcctgaaggg tatgtgcaag aaaggaccat tacctttgaa 300
aatgatgggg tgtataaaac cagggctgaa gtgacctatg aaaaagggag cgtgtataat 360
agggtgacct taaatgggag cgggtttaaa aaagggggga atattttagg gaaaaaatta 420
gaatttaatt ataatcctca ttgcatttat gtgttacctg atgtgcaaaa taatgggatt 480
aaatgctata ttaatattgt gcatgatgtg attggggggg ggcaaattat tgctgctcat 540
caacaattaa ataccccttt aggggggggg cctgtggata ttcctcatta tcatcatatt 600
caagctcata ccattttaag caaagatcct aaagaaacca gggatcatat gaatgtggtg 660
gaagtgttta gggctattga ttgcaaaacc gcttatgctc atcatcatca tcatcattaa 720
tgagcggccg c 731
Claims (6)
1. A helicobacter pylori-escherichia coli shuttle expression vector is characterized in that the nucleotide sequence of the shuttle expression vector is shown in SEQ ID NO. 1.
2. The method for constructing a helicobacter pylori-escherichia coli shuttle expression vector according to claim 1, wherein the method comprises:
s1, a green fluorescent protein AausFP1 optimized based on the codon preference of escherichia coli and used for expression in the escherichia coli; after the whole gene is synthesized, two enzyme cutting sites of bgl II and Kpn I are connected to a pCDFDuet-1 vector; the sequence is shown in SEQ ID NO. 2;
s2, constructing an ampicillin resistance expression cassette; the restriction enzyme sites are Xba I and Sal I respectively; the sequence is shown in SEQ ID NO. 3;
s3, a replication origin in helicobacter pylori, wherein both enzyme cutting sites are Smal; the sequence is shown in SEQ ID NO. 4;
s4, a promoter, a transcription initiation site and a ribosome binding site in helicobacter pylori, wherein the double enzyme cutting sites are all EcoR V; the sequence is shown as SEQ ID NO. 5;
s5, a green fluorescent protein AausFP1 optimized based on the codon preference of helicobacter pylori and used for expressing in the helicobacter pylori; the restriction enzyme sites are EcoR V and Not I; the sequence is shown in SEQ ID NO. 6;
the fragments obtained in steps S2 to S5 were integrated into one fragment, and then ligated to the pCDFDuet-1 vector containing the green fluorescent protein gene obtained in step S1 using Xba I, not I.
3. The use of the helicobacter pylori-escherichia coli shuttle expression vector of claim 1 for the expression of foreign genes in helicobacter pylori.
4. The use of the helicobacter pylori-E.coli shuttle expression vector of claim 1 for the expression of a foreign gene in E.coli.
5. The use of the H.pylori-E.coli shuttle expression vector of claim 1 in genetic regulation and control studies of H.pylori and/or E.coli.
6. The use of the helicobacter pylori-escherichia coli shuttle expression vector of claim 1 in the development of helicobacter pylori and/or escherichia coli related drugs.
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