CN109837301A - Humanization helicobacter pylori cagA construction of eukaryotic expression vector method - Google Patents
Humanization helicobacter pylori cagA construction of eukaryotic expression vector method Download PDFInfo
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Abstract
The invention belongs to biomedicine fields, and in particular to a kind of humanization helicobacter pylori cagA construction of eukaryotic expression vector method.The construction method includes codon optimization step, humanization cagA gene is synthesized in the codon optimization step, the nucleotide sequence of the humanization cagA gene is as shown in the SEQ ID NO:1 in sequence table.This law constructs to obtain humanization helicobacter pylori cagA carrier for expression of eukaryon and great expression cagA albumen, the carcinogenic pathogenic mechanism for the albumen of research cagA gene coding can create condition in stomach cancer cell.The technical program can be applied in the practice of pathogenic mechanism research of helicobacter pylori.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to humanization helicobacter pylori cagA construction of eukaryotic expression vector
Method.
Background technique
Helicobacter pylori (Helicobacter pylori, H.pylori) is a kind of negative micro- need of spiral helicine Gram
Oxygen bacterium infects the population of global half, is the Etiological for causing gastritis, peptic ulcer, gastric cancer.In infection population, greatly
Majority cause asymptomatic gastritis, and about 20-25% development is chronic gastritis, and 10% development is peptic ulcer, there is 1-
3% development is gastric cancer.Gastric cancer is multifactor functioning as a result, main includes Helicobacter pylori infection, host
Inherent cause and environmental factor.Wherein to be that gastric cancer is most important originate factor to Helicobacter pylori infection, by world health group
Knit the I class carcinogen for being classified as Human Gastric carcinoma.Cytotoxin-associated gene A (cytotoxin associated gene A,
CagA) the albumen encoded is known today uniquely by helicobacter pylori injection gastric epithelial cell and to simulate intracellular egg
White " cancer protein " to play a role, the albumen of cagA gene coding activate many A signal pathways after being phosphorylated in the cell, join
With the occurrence and development of gastric cancer, but its carcinogenic mechanism is not fully understood.
To study the influence that associated signal paths occur for further research cagA gene pairs gastric cancer, the present inventor's PCR amplification
Cloning vector pMD18-T/cagA is constructed after helicobacter pylori cagA full length gene segment out, after restricted digestion processing,
It will be connected on pcDNA3.1/ZEO (-) carrier for expression of eukaryon after cagA gene digestion on prokaryotic vector, construct eukaryotic expression
Carrier pcDNA3.1ZEO (-)/cagA.By the eukaryotic expression vector transfection stomach cancer cell line, the eukaryon table of cagA gene is realized
It reaches, and detects the albumen of cagA gene or cagA gene coding to the regulating and controlling effect of downstream gene.But above-mentioned technical proposal
It has the following problems: although prokaryotic gene cagA is building up to carrier for expression of eukaryon and realizes the eukaryon table of the prokaryotic gene
It reaches, but heterogenous expression is lower there are expression quantity in eukaryocyte and expression is unstable for protokaryon cagA gene, or even
It replaces condition of culture or when stomach cancer cell line, the case where gene is not expressed often occurs, be not able to satisfy further grinding at this stage
Study carefully the requirement of cagA regulatory pathway.Because cagA gene expression amount is too low or does not express, the GAP-associated protein GAP amount of generation is less or nothing
GAP-associated protein GAP generates, and can not activate or cannot activate all cagA gene effect albumen, effector or downstream passages completely,
So that causing the further investigation of the molecular mechanism of gastric cancer to receive certain limitation cagA gene.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of humanization helicobacter pylori cagA carrier for expression of eukaryon
Construction method, this method realize the humanization of cagA, benefit by carrying out codon optimization to helicobacter pylori cagA gene
Carrier for expression of eukaryon is constructed with the cagA of humanization, the high expression in gastric carcinoma cells of humanization cagA gene can be made.
In order to solve the above technical problems, the invention proposes technical solutions once:
Humanization helicobacter pylori cagA construction of eukaryotic expression vector method, comprising:
(1) codon optimization step: synthesizing ribonucleotide sequence is the humanization cagA gene of SEQ ID NO:1；
(2) humanization cagA construction of eukaryotic expression vector step.
In the above-mentioned technical solutions, inventor to helicobacter pylori cagA gene (NCBI sequence ID:
KR154737 codon optimization) is carried out, the sequence after optimization is known as humanization cagA.Humanization cagA and helicobacter pylori
CagA gene order comparison result is shown in that Fig. 2, humanization cagA nucleotide sequence are shown in SEQ ID NO:1.
The technical principle of the technical solution are as follows: remove methionine and tryptophan, other amino acid all correspond to 2-6 password
Son.The codon for wherein encoding same amino acid is known as synonym, in different biologies or cell, synonymous code
The frequency that son is used also has very big difference, and there are codon preferences.Inventor compares the Preference of human codon,
Processing is optimized by the codon to helicobacter pylori cagA gene.According to codon preference to helicobacter pylori
After the nucleotide sequence of cagA gene is transformed, inventor, which has found that cagA gene is appointed in eukaryocyte, cannot so stablize and greatly
The expression of amount.Inventor is on the basis of the optimization processing carried out according to codon preference, to helicobacter pylori cagA base
3 ' ends of the template strand of cause are transformed, and increase one section of nucleotide sequence (5 '-at 3 ' ends
GACTACAAGGACGACGATGACAAG-3 '), the increased nucleotide sequence is to humanization cagA gene in eukaryocyte
Expression plays facilitation.Inventor is the study found that region and 855-1048 amino acids between 629-817 amino acids
Between region be helicobacter pylori cagA gene coding albumen functional area (endotoxin function, that is, cause a disease function),
And the terminal region of the albumen does not work to the realization of protein endotoxins function, so to helicobacter pylori cagA gene 3 '
Although the transformation of end can increase several amino acid on the albumen that cagA is encoded, but not influence protein function, increased widow
Polynucleotide plays adjustment effect to gene expression.
What the technical program reached has the beneficial effect that the table that humanization cagA gene can be a large amount of and stable in eukaryocyte
It reaches, overcomes the defect of helicobacter pylori cagA expression quantity in eukaryocyte small (or even not expressing) and unstable expression,
The research of gastric cancer molecular mechanism is caused to create condition for helicobacter pylori pathogenic mechanism and cagA.
Further, I restriction enzyme of Nhe is equipped at 5 ' ends of the template strand of the humanization cagA gene identify sequence
Column are equipped with I restriction endonuclease recognition sequence of Xba at 3 ' ends of the template strand of the humanization cagA gene.
By adopting the above technical scheme, restriction enzyme site is added in humanization cagA gene, can by digestion and
Humanization cagA gene is connected in all kinds of carriers by the operation such as connection.It is not contained in humanization cagA gene open reading frame
I restriction enzyme site of I restriction enzyme site of Nhe and Xba, to ensure that digestion operation will not be humanization cagA gene
Open reading frame cutting, ensure that the integrality of humanization cagA gene.
Further, Kozak sequence, the Kozak sequence are equipped at 5 ' ends of the template strand of the humanization cagA gene
Between I restriction endonuclease recognition sequence of Nhe and the initiation codon ATG of humanization cagA gene.
By adopting the above technical scheme, technical principle is as follows: Kozak sequence (Kozak consensus sequence) is
According to the sequence that Kozak rule designs, Kozak rule is that the base distribution of the flanking sequence of gene start codon ATG is expired
The statistical law of foot.Kozak sequence is applied in the building of carrier for expression of eukaryon, when gene is transcribed into mRNA, ribose physical efficiency
This section of sequence on mRNA is enough identified, and using it as translation initiation site, to enhance gene in eukaryotic expression system
Translation efficiency, to increase the expressing quantity of gene coding.If being not provided with Kozak sequence, ribosomes may be with
The site of the upper mistake of mRNA combines, to the case where translation error or translation omission occur.
Further, the Kozak sequence is 5 '-GCCACC-3 '.
By adopting the above technical scheme, sequence 5 '-is added after restriction enzyme site and before initiation codon ATG
GCCACC-3 ' meets the requirement of Kozak rule, and the accuracy of protein translation can be improved, to improve the expression of gene
Amount.
Further, in the codon optimization step, the humanization cagA base is synthesized by polymerase chain reaction
Cause.
By adopting the above technical scheme, it can be achieved to synthesize outside humanization cagA genosome by polymerase chain reaction (PCR).
Further, the cagA construction of eukaryotic expression vector step are as follows: I site Nhe of pcDNA3.1 (+) and Xba I
Between point, it is inserted into the humanization cagA gene, obtains humanization cagA eukaryotic expression vector pcDNA3.1 (+)-cagA.
By adopting the above technical scheme, pcDNA3.1 (+) is common one of carrier for expression of eukaryon, can be in the large intestines bar such as DH5 α
Duplication is cloned in bacterium, and albumen can also be expressed in mammalian cell.Contain CMV promoter in pcDNA3.1 (+), which can open
Dynamic eukaryotic gene expression, starting activity with higher.Utilize the nucleotides sequence of pcDNA3.1 (+) connection humanization cagA gene
Column, building source cagA carrier for expression of eukaryon is, it can be achieved that great expression of the albumen in eukaryocyte that cagA gene encodes.?
Humanization cagA carrier for expression of eukaryon obtained in this step is pcDNA3.1 (+)-cagA.
Further, there are also transfection procedure, the transfections after the humanization cagA construction of eukaryotic expression vector step
Step are as follows: with humanization cagA eukaryotic expression vector pcDNA3.1 (+)-cagA transfection host cell.
By adopting the above technical scheme, the eukaryotic expression may be implemented after pcDNA3.1 (+)-cagA Successful transfection host cell
The clone's duplication or the expression of target gene of carrier.
Further, the host cell is gastric carcinoma cells.
By adopting the above technical scheme, the main existence position of helicobacter pylori is people's antrum portion, helicobacter pylori
The site of action of the albumen of cagA coded by said gene is people's stomach cell.By humanization cagA eukaryotic expression vector pcDNA3.1
(+)-cagA transfects gastric carcinoma cells, it can be achieved that pathogenic mechanism and cagA gene and stomach to the albumen of cagA coded by said gene
The research of relationship between cancer.
Further, the gastric carcinoma cells derive from ags cell strain.
By adopting the above technical scheme, ags cell belongs to low noble cells, and ags cell is isolated from poorly differentiated adenocarcinoma cancer cell,
The grade malignancy of poorly differentiated adenocarcinoma cancer is relatively high, treats relative difficulty.So humanization cagA eukaryon table prepared by this law
Up to carrier pcDNA3.1 (+)-cagA transfection ags cell strain, it can be achieved that grinding to the relationship between cagA gene and pernicious gastric cancer
Study carefully.
Further, between the codon optimization step and the humanization cagA construction of eukaryotic expression vector step also
There is nucleotide sequence authentication step.
By adopting the above technical scheme, nucleotide sequence is carried out to the nucleotide sequence synthesized in codon optimization step
Identification, reuse by evaluation and screening nucleotide sequence carry out humanization cagA carrier for expression of eukaryon building, can avoid by
The nucleotide sequence mistake caused by resultant fault or gene mutation, and then avoid the nucleotide sequence of mistake to subsequent step
It influences, ensure that and finally give expression to correct albumen in host cell.Conventional nucleotide sequence identification method is sequencing mirror
It is fixed.
Detailed description of the invention
Fig. 1 is the schematic diagram of empty carrier pcDNA3.1 (+).
Fig. 2 is the nucleotide sequence comparison result of helicobacter pylori cagA gene and humanization cagA gene.
Fig. 3 is the electrophoresis result of pcr amplification product.
Fig. 4 is that WB detects protein expression level (after pcDNA3.1 (+)-cagA transfects ags cell).
Specific embodiment
It is further described below by specific embodiment:
The acquisition of humanization cagA gene and its building of carrier for expression of eukaryon
1. the acquisition of humanization cagA gene nucleotide series
According to helicobacter pylori cagA gene order, its codon is optimized, obtains humanization cagA gene
Nucleotide sequence (see SEQ ID NO:1).Helicobacter pylori cagA gene (hpcagA；NCBI sequence ID:
) and the nucleotide sequence comparison of humanization cagA gene (containing restriction endonuclease recognition sequence and Kozak sequence) KR154737
As a result see Fig. 2.
2. artificial full genome synthesis humanization cagA gene and identification
(1) experimental material
Restriction endonuclease (Fermentas)；Primer Oligo (Invitrogen synthesis)；T4DNA ligase(1U/μl)
(EP0061,Fermentas)；Gel reclaims kit (AP-GX_250, Axygen)；GeneRuler DNA Ladder
(SM0332, Fermentas), competent cell E.coli DH5 α (promise hundred) etc., the small pumping kit of plasmid (AP-MN-P-250,
Ayxgen), kit (AP-MD-P-25, Ayxgen) is taken out in plasmid, the big pumping kit (AP-MX-P-25, Ayxgen) of plasmid.
(2) laboratory apparatus
Tabletop refrigerated centrifuge (Neofuge13R, Heal Force), Biohazard Safety Equipment (HFSafe-1800, Heal
Force), PCR instrument (T100TM, Bio-Rad), electrophoretic image system (Tanon1600, day energy), dry type thermostat (TU-100C,
One permanent science and technology) temperature control shaking table (THZ-98, granary) etc..
(3) full genome synthesis and identification
According to the nucleotide sequence of the humanization cagA gene in SEQ ID NO:1, humanization cagA is manually directly synthesized
Gene (commission biotech company synthesizes according to the nucleotide sequence of SEQ ID NO:1), obtains artificial synthesized humanization cagA
Gene, humanization cagA gene are connected in carrier T (T-cagA).To the humanization cagA gene being connected on T-cagA into
Row sequencing is to verify nucleotide sequence as correct sequence.
(4) building of carrier for expression of eukaryon
Artificial synthesized humanization cagA gene is connected to eukaryotic expression vector pcDNA3.1 (+), and (pcDNA3.1 (+) is empty
Carrier structure is as shown in Figure 1) on, must being connected with the pcDNA3.1 (+) of gene, (whether the gene to be detected connected is correct mesh
Gene).Specific carrier connection procedure are as follows: to artificial synthesized humanization cagA gene and eukaryotic expression vector pcDNA3.1
(+) carries out double digestion (digestion system and condition are shown in Table 1, table 2) respectively, then by the artificial synthesized humanization cagA base after digestion
Cause is connected to (linked system and condition are shown in Table 3) on the pcDNA3.1 (+) after digestion.Humanization cagA eukaryotic expression of the invention
The restriction enzyme site that the building of carrier is selected is I restriction enzyme site of I restriction enzyme site of Nhe and Xba.
Table 1: artificial synthesized humanization cagA gene double digestion
Table 2: eukaryotic expression vector pcDNA3.1 (+) double digestion
Table 3: target gene is connect with carrier
Using the connection product transformed competence colibacillus bacterium E.coli DH5 α in table 3, plated overnight culture, from plate with
Machine is selected falls single bacterium colony (monoclonal colonies) completely, is detected with bacterial examination PCR method, the positive gram obtained in picking bacterial examination PCR
It is grand that sequencing company is sent to carry out sequence verification, it chooses the Escherichia coli positive colony that sequence verification is correct sequence and carries out expanding bacterium training
It supports, that is to say, that sequence verification is in the Escherichia coli positive colony of correct sequence containing the upper humanization cagA gene of connection
pcDNA3.1(+).The pcDNA3.1 (+) for connecting upper humanization cagA gene is named as pcDNA3.1 (+)-cagA, i.e. source of people
Change cagA carrier for expression of eukaryon.
After the bacterium solution amplification culture that the E.coli DH5 α of pcDNA3.1 (+)-cagA will be contained, Axygen plasmid extraction is utilized
Kit and method extract pcDNA3.1 (+)-cagA, to the humanization cagA gene in pcDNA3.1 (+)-cagA extracted
PCR detection is carried out, electrophoresis result is as shown in Figure 3.From the figure 3, it may be seen that the length of humanization cagA gene is in 3700bp or so.It is right
PcDNA3.1 (+)-cagA carries out PCR detection, and the Loaded samples of pcr amplification product electrophoresis are as follows: the 1/2/3rd swimming lane loading is
The pcr amplification product of pcDNA3.1 (+)-cagA, the 4th swimming lane loading are that DNA molecular amount marks (DNA marker15000).
(4) stomach cancer cell is transfected with pcDNA3.1 (+)-cagA
(4.1) stomach cancer cell culture
Ags cell strain (it is purchased from ATCC,CRL-1739TM), with RPMI-1640 complete medium (10%FBS), set
In 37 DEG C of 5%CO2Incubator carries out routine culture, and cell culture processes are as follows:
The recovery of ags cell: after the cell strain that -80 DEG C of ultra low temperature freezers take out preservation, being put into rapidly 37 DEG C of water-baths,
Until completely dissolved, 800rpm is centrifuged 5min.It discards supernatant, after being mixed with culture solution piping and druming, is added in Tissue Culture Dish, uniformly
It rocks, is put into CO2In incubator.
Ags cell passage and counting: growth, which has, is added 0.5ml pancreatin in the culture bottle of cell, be added after digesting 2~3min
1ml culture medium, culture solution is transferred in centrifuge tube, and 800rpm is centrifuged 5min, abandons supernatant.2~3ml culture solution is added, piping and druming is mixed
It is even, it is transferred in culture dish.The 75 μ l of cell of cancellationization is counted on cell counter, and calculates 1ml cell quantity.
The preservation of ags cell: by medium centrifugal, 800rpm is centrifuged 5min, abandons supernatant.Addition frozen stock solution (fetal calf serum:
DMSO=9:1), piping and druming mixes.It is distributed into cryopreservation tube and is put into liquid nitrogen chamber.
(4.2) stomach cancer cell is transfected
Cultured ags cell is laid in 6 orifice plates, transfection experiment is carried out.Experimental group is as shown in table 4:
Table 4: transfection experiment design
| Experimental group | Number | Expression vector | Transfection reagent |
| Blank control group | T0 | N/A | lipofectamine2000 |
| Negative control group | T1 | pcDNA3.1(+) | lipofectamine2000 |
| Experimental group | T2 | pcDNA3.1(+)-cagA | lipofectamine2000 |
| Prior art group | T3 | pcDNA3.1(+)-hpcagA* | lipofectamine2000 |
* annotate: the preparation method of pcDNA3.1 (+)-hpcagA is that will not carry out the helicobacter pylori of codon optimization
CagA gene (hpcagA, NCBI number KR154737) is directly connected to pcDNA3.1 (+).
In experimental group, pcDNA3.1 (+)-cagA is mixed with lipofectamine2000 with the ratio of 3 μ g:5 μ l,
Above-mentioned 5 μ l of mixed liquor addition is covered in the 6 orifice plates of ags cell and carries out cell transfecting.In blank control group, 5 μ are added
Llipofectamine2000 carries out cell transfecting to the 6 orifice plates for being covered with ags cell are interior.It, will in negative control group
PcDNA3.1 (+) is mixed with lipofectamine2000 with the ratio of 3 μ g:5 μ l, and above-mentioned 5 μ l of mixed liquor addition is covered with AGS
Cell transfecting is carried out in the 6 orifice plates of cell.In prior art group, by pcDNA3.1 (+)-hpcagA with
Lipofectamine2000 is mixed with the ratio of 3 μ g:5 μ l, and above-mentioned 5 μ l of mixed liquor addition is covered in the 6 orifice plates of ags cell
Carry out cell transfecting.In above-mentioned each group, transfection stops and replaces culture medium after handling lasting 6h, continues after cultivating cell 48h
Terminate cell culture and receives cell.
(4.3) albumen and WB detection are extracted after transfecting
The total protein that the ags cell after the transfection harvested in (4.2) is extracted using BCA protein quantification kit, using WB
Method detects the expressing quantity of cagA gene, and experimental result is as shown in Figure 4.It is blank control that each swimming lane, which is respectively as follows: T0, and T1 is real
Group is tested, T2 is negative control group, and T3 is prior art group, and rightmost swimming lane is protein markers (marker).Experiment knot
Fruit shows: the humanization cagA gene on humanization cagA eukaryotic expression vector pcDNA3.1 (+)-cagA is in stomach cancer cell line
Great expression in AGS, the molecular weight of albumen of humanization cagA gene coding is in 135KD or so.Helicobacter pylori cagA gene
Codon optimization processing was not carried out, and the gene is in stomach cancer cell line AGS without expression.
What has been described above is only an embodiment of the present invention, and the common sense such as well known specific structure and characteristic are not made herein in scheme
Excessive description.It, without departing from the structure of the invention, can be with it should be pointed out that for those skilled in the art
Several modifications and improvements are made, these also should be considered as protection scope of the present invention, these all will not influence what the present invention was implemented
Effect and patent practicability.The scope of protection required by this application should be based on the content of the claims, in specification
The records such as specific embodiment can be used for explaining the content of claim.
SEQUENCE LISTING
<110>Guizhou medical university
<120>humanization helicobacter pylori cagA construction of eukaryotic expression vector method
<130> 2019.3.21
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 3552
<212> DNA
<213>artificial sequence
<400> 1
atgacaaatg aaactatcaa ccagacgact accccagatc agactcccaa tcagaccgac 60
ttcgtaccgc agaggtttat taacaatctg caggttgcct ttattaaggt tgatagcgca 120
gtcgccagtt tcgacccaga ccaaaagccc attgtggata agaatgatag ggacaaccgg 180
caggcctttg agaagatctc acagctgagg gaggagtacg ccaacaaggc catcaaaaat 240
cccgcgaaaa agaaccaata tttttccgac ttcatcaaca agtcaaacga tctaattaat 300
aaggataacc tgattgcagt cgactctagt gtggactctt ttaagaaatt cggcgaccag 360
cgctaccaga tttttacaag ttgggtctct cttcagaagg acccttcaca aataaacact 420
caaaagattc gggactttat ggagaacatc atccagccac ccatttcaga cgataaggag 480
aaagctgaat ttctgaggtc cgcgaagcag agtttcgcag gcatcatcat cgggaatcag 540
atcagatctg atgagaagtt catgggtgtc ttcgatgaat cactgaaggc ccgacaggaa 600
gctgaaaaga acgcagagcc cgccggcgga gactggcttg atatttttct gagcttcgtt 660
ttcaacaaga agcagtcctc tgaccttaaa gaaactttga accaggaacc acgacctgac 720
tttgagcaga accttgcgac caccacaacc gatattcagg ggctgccacc cgaggcccgc 780
gacttgttgg atgaacgcgg caacttctcc aaattcacac tgggcgacat ggagatgctg 840
gatgtcgagg gggtggctga caaggaccca aattataagt tcaatcaact cctgatccac 900
aataacgctc tgtcatcagt gctgatggga ggacactcca acatcgaacc cgaaaaggtc 960
agcctgttgt acggtgataa cgggggccca gaagcaaggc acgactggaa cgctactgtt 1020
gggtataaag accagcaagg gaacaacgtg gctacactga tcaacgcgca cctgcacaat 1080
ggaagcggct tgatcatcgc cggcaacgaa aacggaatca agaatccttc attctacttg 1140
cacaaagaag accagctaac tggcttgaag caagccctga gccaggaaga gatccagaac 1200
aacgtggact ttatggagtt cttggctcag aataatgcta aattagataa cctctcggaa 1260
aaggagaaag agaaattcca gaccgagatt gagaactttc agaaagatag gaaagcatac 1320
ctggacgctc tcggcaatga tcacatagcc ttcgtgagta agaaggatcc gaagcatctg 1380
gccttagtga cggaattcgg caatggggaa gtgtcttata cgttgaaaga ttatggaaag 1440
aaacaggaca aagccttgga tggtgagatc aaaacaacac tacaggggtc tctcaaatac 1500
gatggtgtca tgttcgttaa ttactcaaac tttaaatata caaatgcatc caagtcccca 1560
gacaaaggtg tggggacaac gaatggagtg tctcatcttg aggccaattt tagtaaagtc 1620
gctgtcttca atctgcctaa cctcaacaat ctcgcgatca ccagttatat tagacgagac 1680
ctcgaggata agttatgggc aaaaggctta tccccccagg aggccaacaa gttaatcaaa 1740
gattttctga acagcaataa agagatggtg gaaaaggtga gtaatttcaa caaggcagtc 1800
gccgaggcta agaataccgg gaattacgac gaggtgaaga aggcccagaa ggatctggag 1860
aaatccctcc ggaaaagaga acatctggag aaagaggttg ctaagaagct ggaaagtaga 1920
aacgataata agaaccgcat ggaggccaag gcacaagcga atagtcaaaa ggataaaatc 1980
tttgctctga tcaaccaaga agcatccaag gaagccagag ctgccgcctt cgacccaaac 2040
gtaaagggtg tacgctccga actgtctgac aaactcgaga acatcaataa gaatctcaag 2100
gacttcggga aatcgttcga cgagctaaaa aatggaaaaa acaaggactt tagcaaggca 2160
gaagagacct taaaggccct taaggatagc gtgaaagatc tggggataaa ccccgagtgg 2220
ataagcaaaa tagagaacct gaacgccgcc ctcaacgatt ttaaaaacgg gaagaacaag 2280
gacttttcca aagtgaccca ggcaaagagt gaccttgaaa actccattaa ggatgttatc 2340
ataaatcaga agattaccga taaggtagat aacctcaacc aggccgtgtc tgagacaaag 2400
ctcactgggg acttttctaa ggtggaacag gccctcgctg agctgaagaa cctctctcta 2460
gacctcggta aaaacagcga tctgcagtcg gtgaaaaatt ctgtaaatgg aacccttgtc 2520
gggaatggac tttcgaaaac tgaggccacc acacttacaa agaacttctc cgatatacgg 2580
aaagagttga acgagaaact tttcggaaat tctaataaca ataacaatgg cctggagaac 2640
aataccgagc ctatctacgc taaagttaac aagaagaaga cgggacaggt cgcatctcct 2700
gaggagccaa tctacgccca agtggcaaag aaagtaagcg ctaaaattga tcaactgaac 2760
gaggcgacgt cagccataaa tcgcaagatt gatcgcatta acaaaatcgc gtccgccggg 2820
aaaggcgtgg gcgcgtttag cggtgctcgt cagtcagcct cccccgaacc tatctatgcc 2880
accatagatt tcgacgaagc aaatcaagct ggatttccgc tgcgtagatc gacagcggtc 2940
ggcgatctga gcaaagtggg gttgtccagg gaggaagagc tgacacggcg aatcggtgac 3000
ctgaaccagg ctgtcagcga ggccaaaacc ggacgcttcg acaagctcca gcagaagatc 3060
gacgaactga aagactccac aaagaaaaat gccctgaagc tatgggtgga gtccgctaaa 3120
caggtgccca cgggactcca agccaaactg gacaattacg caactaattc ccacactaga 3180
attaacagca acgttcgaaa tggagcaatt aatgaaaagg caaccggaat gctgacccag 3240
aagaatcccg agtggctcaa gttggtgaac gataagattg tagcccataa catcggcagc 3300
actcacctga gcgaatatga taagataggc ttcaatcaga agaatatgaa agactatagc 3360
gacagcttca agttcagtac aaagctcaac aatgctgtta aggacataaa gagttctttt 3420
gtgcagtttc ttaccaacgc tttctctacc ggttcttaca gcctgatgaa agctaacgtg 3480
gaacatggcg tgaaaaatac caccaagagc ggattccaaa aatcagacta caaggacgac 3540
gatgacaagt aa 3552
Claims (10)
1. humanization helicobacter pylori cagA construction of eukaryotic expression vector method characterized by comprising
(1) codon optimization step: synthesizing ribonucleotide sequence is the humanization cagA gene of SEQ ID NO:1；
(2) humanization cagA construction of eukaryotic expression vector step.
2. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 1, feature exist
In 5 ' in the template strand of the humanization cagA gene hold equipped with I restriction endonuclease recognition sequence of Nhe, in the source of people
3 ' the ends for changing the template strand of cagA gene are equipped with I restriction endonuclease recognition sequence of Xba.
3. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 2, feature exist
In, the humanization cagA gene template strand 5 ' end be equipped with Kozak sequence, the Kozak sequence be located at Nhe I limit
Between property endonuclease recognition sequence and the initiation codon ATG of humanization cagA gene.
4. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 3, feature exist
In the Kozak sequence is 5 '-GCCACC-3 '.
5. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 4, feature exist
In synthesizing the humanization cagA gene by polymerase chain reaction in the codon optimization step.
6. according to humanization helicobacter pylori cagA carrier for expression of eukaryon described in any one of claim 2-5 claim
Construction method, which is characterized in that the cagA construction of eukaryotic expression vector step are as follows: pcDNA3.1 (+) I site Nhe and
Between I site Xba, it is inserted into the humanization cagA gene, obtains humanization cagA eukaryotic expression vector pcDNA3.1 (+)-cagA.
7. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 6, feature exist
In there are also transfection procedure, the transfection procedures are as follows: described in use after the humanization cagA construction of eukaryotic expression vector step
Humanization cagA eukaryotic expression vector pcDNA3.1 (+)-cagA transfection host cell.
8. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 7, feature exist
In the host cell is gastric carcinoma cells.
9. humanization helicobacter pylori cagA construction of eukaryotic expression vector method according to claim 8, feature exist
In the gastric carcinoma cells derive from ags cell strain.
10. according to claim 1-5, humanization helicobacter pylori cagA eukaryon table described in any one of 7-9 claim
Up to carrier construction method, which is characterized in that in the codon optimization step and the humanization cagA carrier for expression of eukaryon structure
Building between step further includes nucleotide sequence authentication step.
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