CN109837301B

CN109837301B - Construction method of humanized helicobacter pylori cagA eukaryotic expression vector

Info

Publication number: CN109837301B
Application number: CN201910252127.5A
Authority: CN
Inventors: 谢渊; 周建奖
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2019-03-29
Filing date: 2019-03-29
Publication date: 2021-01-26
Anticipated expiration: 2039-03-29
Also published as: CN109837301A

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a construction method of a humanized helicobacter pylori cagA eukaryotic expression vector. The construction method comprises a codon optimization step, wherein a humanized cagA gene is synthesized in the codon optimization step, and the nucleotide sequence of the humanized cagA gene is shown as SEQ ID NO. 1 in a sequence table. The humanized helicobacter pylori cagA eukaryotic expression vector constructed by the method can express a large amount of cagA protein in gastric cancer cells, and creates conditions for researching the carcinogenic pathogenic mechanism of the protein coded by the cagA gene. The technical scheme can be applied to the practice of researching the pathogenic mechanism of the helicobacter pylori.

Description

Construction method of humanized helicobacter pylori cagA eukaryotic expression vector

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a construction method of a humanized helicobacter pylori cagA eukaryotic expression vector.

Background

Helicobacter pylori (Helicobacter pylori, H.pylori) Is a spiral gram-type negative microaerophilic bacterium, infects half of the global population, and is the main cause of gastritis, peptic ulcer and gastric cancer. Among the infected people, most people cause asymptomatic gastritis, with about 20-25% developing into chronic gastritis, 10% developing into peptic ulcer, and 1-3% developing into gastric cancer. The occurrence of gastric cancer is the result of a combination of multiple factors, mainly including helicobacter pylori infection, host genetic factors and environmental factors. Among them, helicobacter pylori infection is the most important initiating factor of gastric cancer, and has been classified as a class I carcinogenic factor of human gastric cancer by the world health organization. The protein coded by the cytotoxin associated gene A (cagA) is the only known 'oncoprotein' which is injected into gastric epithelial cells by helicobacter pylori and can simulate intracellular proteins to play a role, the protein coded by the cagA gene is phosphorylated in cells to activate various signal paths and participate in the generation and development of gastric cancer, but the carcinogenic mechanism is not completely clear.

In order to further research the influence of the cagA gene on a gastric cancer generation related signal path, the inventor constructs a cloning vector pMD18-T/cagA after amplifying a helicobacter pylori cagA gene full-length fragment by PCR, and after the restriction enzyme treatment, the cagA gene on a prokaryotic vector is connected to a pcDNA3.1/ZEO (-) eukaryotic expression vector after the restriction enzyme digestion, thereby constructing the eukaryotic expression vector pcDNA3.1ZEO (-)/cagA. The eukaryotic expression vector is transfected into gastric cancer cell strains, so that eukaryotic expression of the cagA gene is realized, and the regulation and control effect of the cagA gene or protein encoded by the cagA gene on downstream genes is detected. However, the above technical solutions have the following problems: although the prokaryotic gene cagA is constructed to an eukaryotic expression vector and eukaryotic expression of the prokaryotic gene is realized, the prokaryotic gene cagA has lower expression level and unstable expression level in heterologous expression in eukaryotic cells, and even when culture conditions or gastric cancer cell strains are changed, the situation that the gene is not expressed frequently occurs, and the requirement of further researching the cagA regulation and control pathway at the present stage can not be met. Because the expression level of the cagA gene is too low or is not expressed, the produced related protein is less or no related protein is produced, and all the effector proteins, effector genes or downstream channels of the cagA gene cannot be completely activated or activated, so that the deep research on the molecular mechanism of gastric cancer caused by the cagA gene is limited to a certain extent.

Disclosure of Invention

The invention mainly solves the technical problem of providing a method for constructing a humanized helicobacter pylori cagA eukaryotic expression vector, the method realizes the humanization of the cagA by carrying out codon optimization on the helicobacter pylori cagA gene, and the humanized cagA is used for constructing the eukaryotic expression vector, so that the humanized cagA gene can be highly expressed in human gastric cancer cells.

In order to solve the technical problems, the invention provides the following technical scheme:

the construction method of the humanized helicobacter pylori cagA eukaryotic expression vector comprises the following steps:

(1) a codon optimization step: synthesizing a humanized cagA gene with a nucleotide sequence of SEQ ID NO. 1;

(2) constructing a humanized cagA eukaryotic expression vector.

In the technical scheme, the inventor carries out codon optimization on a helicobacter pylori cagA gene (NCBI sequence ID: KR 154737), and the optimized sequence is called humanized cagA. The result of the alignment of the humanized cagA gene sequence and the helicobacter pylori cagA gene sequence is shown in figure 2, and the humanized cagA nucleotide sequence is shown in SEQ ID NO: 1.

The technical principle of the technical scheme is as follows: except methionine and tryptophan, the other amino acids correspond to 2-6 codons. Wherein codons encoding the same amino acid are called synonymous codons, the synonymous codons are used at frequencies which are also greatly different in different organisms or cells, and there is a codon preference. The inventors controlled codon preference in humans by optimizing the codons of the helicobacter pylori cagA gene. After modifying the nucleotide sequence of the helicobacter pylori cagA gene according to codon preference, the inventors found that the cagA gene can not be stably and massively expressed in eukaryotic cells at all. The inventor modifies the 3 'end of the template chain of the helicobacter pylori cagA gene on the basis of optimization treatment according to codon preference, adds a nucleotide sequence (5'-GACTACAAGGACGACGATGACAAG-3') at the 3' end, and the added nucleotide sequence plays a role in promoting the expression of the humanized cagA gene in eukaryotic cells. The inventor researches and discovers that the region between 629-817 amino acids and 855-1048 amino acids is the functional region (endotoxin function, namely pathogenic function) of the protein encoded by the helicobacter pylori cagA gene, while the terminal region of the protein does not play a role in realizing the endotoxin function of the protein, so that the modification of the 3' terminal of the helicobacter pylori cagA gene can add a plurality of amino acids on the protein encoded by the cagA, but does not influence the protein function, and the added oligonucleotide plays a role in regulating the gene expression.

The technical scheme has the following beneficial effects: the humanized cagA gene can be expressed in a large amount and stably in eukaryotic cells, overcomes the defects of small expression amount (even no expression) and unstable expression of the helicobacter pylori cagA in the eukaryotic cells, and creates conditions for researching pathogenic mechanisms of the helicobacter pylori and molecular mechanisms of gastric cancer caused by the cagA.

Furthermore, a Nhe I restriction endonuclease recognition sequence is arranged at the 5 'end of the template chain of the humanized cagA gene, and an Xba I restriction endonuclease recognition sequence is arranged at the 3' end of the template chain of the humanized cagA gene.

By adopting the technical scheme, restriction enzyme sites are added into the humanized cagA gene, and the humanized cagA gene can be connected to various vectors through operations such as enzyme digestion, connection and the like. The humanized cagA gene open reading frame does not contain a Nhe I restriction site and an Xba I restriction site, so that the open reading frame of the humanized cagA gene cannot be cut off by enzyme cutting operation, and the integrity of the humanized cagA gene is ensured.

Further, a Kozak sequence is provided at the 5' end of the template strand of the humanized cagA gene, and the Kozak sequence is located between the recognition sequence of the Nhe I restriction endonuclease and the ATG initiation codon of the humanized cagA gene.

By adopting the technical scheme, the technical principle is as follows: kozak sequence (Kozak consensus sequence), is a sequence designed according to the Kozak rule, which is a statistical rule satisfied by the base distribution of sequences flanking the ATG of the gene start codon. When the Kozak sequence is applied to the construction of a eukaryotic expression vector, when a gene is transcribed into mRNA, ribosome can recognize the sequence on the mRNA and use the sequence as a translation starting site, so that the translation efficiency of the gene in a eukaryotic expression system is enhanced, and the expression amount of protein coded by the gene is increased. If the Kozak sequence is not provided, the ribosome may bind to the wrong site on the mRNA, resulting in erroneous translation or missing translation.

Further, the Kozak sequence is 5 '-GCCACC-3'.

By adopting the technical scheme, the sequence 5 '-GCCACC-3' is added after the restriction enzyme site and before the initiation codon ATG, the requirement of a Kozak rule is met, the accuracy of protein translation can be improved, and the expression quantity of the gene is improved.

Further, in the codon optimization step, the humanized cagA gene is synthesized by polymerase chain reaction.

By adopting the technical scheme, the humanized cagA gene can be synthesized in vitro by Polymerase Chain Reaction (PCR).

Further, the construction steps of the cagA eukaryotic expression vector are as follows: the humanized cagA gene is inserted between the Nhe I site and the Xba I site of pcDNA3.1(+) to obtain the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA.

By adopting the technical scheme, pcDNA3.1(+) is one of common eukaryotic expression vectors, can be cloned and replicated in Escherichia coli such as DH5 alpha and the like, and can also express protein in mammalian cells. The pcDNA3.1(+) contains CMV promotor, which can start eukaryotic gene expression and has higher start activity. The nucleotide sequence of the humanized cagA gene is connected with pcDNA3.1(+), so that a humanized cagA eukaryotic expression vector is constructed, and the large-scale expression of the protein coded by the cagA gene in eukaryotic cells can be realized. The humanized cagA eukaryotic expression vector obtained in this step is pcDNA3.1(+) -cagA.

Further, the construction step of the humanized cagA eukaryotic expression vector is followed by a transfection step which comprises the following steps: the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA is used for transfecting a host cell.

By adopting the technical scheme, the cloning and replication of the eukaryotic expression vector or the expression of the target gene can be realized after the pcDNA3.1(+) -cagA successfully transfects the host cell.

Further, the host cell is a human gastric cancer cell.

By adopting the technical scheme, the main living part of the helicobacter pylori is the antrum of the human stomach, and the action part of the protein coded by the cagA gene of the helicobacter pylori is the human stomach cell. The humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA is transfected into human gastric cancer cells, so that the research on the pathogenic mechanism of the protein coded by the cagA gene and the relation between the cagA gene and gastric cancer can be realized.

Further, the human gastric cancer cell is derived from an AGS cell line.

By adopting the technical scheme, the AGS cells belong to poorly differentiated cells, the AGS cells are separated from gastric poorly differentiated adenocarcinoma cells, the malignancy degree of the gastric poorly differentiated adenocarcinoma is relatively high, and the treatment is relatively difficult. Therefore, the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA prepared by the method is used for transfecting an AGS cell strain, and the research on the relation between the cagA gene and malignant gastric cancer can be realized.

Further, there is a nucleotide sequence identification step between the codon optimization step and the humanized cagA eukaryotic expression vector construction step.

By adopting the technical scheme, the nucleotide sequence synthesized in the codon optimization step is identified, and the humanized cagA eukaryotic expression vector is constructed by using the identified and screened nucleotide sequence, so that the nucleotide sequence error caused by synthesis error or gene mutation can be avoided, the influence of the wrong nucleotide sequence on the subsequent steps is further avoided, and the correct protein is finally expressed in the host cell. The conventional method for identifying nucleotide sequences is sequencing identification.

Drawings

FIG. 1 is a schematic diagram of the empty vector pcDNA3.1 (+).

FIG. 2 shows the result of alignment of the nucleotide sequences of the helicobacter pylori cagA gene and the humanized cagA gene.

FIG. 3 shows the result of electrophoresis of the PCR amplification product.

FIG. 4 shows the expression level of WB detection protein (pcDNA3.1 (+) -cagA transfected AGS cells).

Detailed Description

The following is further detailed by way of specific embodiments:

acquisition of humanized cagA gene and construction of eukaryotic expression vector thereof

1. Acquisition of nucleotide sequence of humanized cagA Gene

The codon of the helicobacter pylori cagA gene sequence is optimized according to the helicobacter pylori cagA gene sequence to obtain the nucleotide sequence of the humanized cagA gene (shown as SEQ ID NO: 1). The nucleotide sequence alignment of the helicobacter pylori cagA gene (hpcagA; NCBI sequence ID: KR 154737) and the humanized cagA gene (containing restriction enzyme recognition sequence and Kozak sequence) is shown in FIG. 2.

2. Artificial total gene synthesis humanized cagA gene and identification

(1) Experimental Material

Endonucleases (Fermentas); primer Oligo (synthesized by Invitrogen); t4 DNA ligase (1U/. mu.l) (EP 0061, Fermentas); gel recovery kit (AP-GX-250, Axygen); GeneRuler DNA Ladder (SM 0332, Fermentas), competent cell E. coli DH5 alpha (Nobel), etc., plasmid minipump kit (AP-MN-P-250, Ayxgen), plasmid medium pump kit (AP-MD-P-25, Ayxgen), plasmid large pump kit (AP-MX-P-25, Ayxgen).

(2) Laboratory apparatus

A table refrigerated centrifuge (Neoflux 13R, Heal Force), a biosafety cabinet (HFSafe-1800, Heal Force), a PCR instrument (T100 TM, Bio-Rad), an electrophoresis imaging system (Tanon 1600, Nature), a dry thermostat (TU-100C, a constant technology) temperature-controlled shaking table (THZ-98, Taicang), and the like.

(3) Whole gene synthesis and identification

The humanized cagA gene was artificially ligated directly to the humanized cagA gene (synthesized by Biotech Co., Ltd. according to the nucleotide sequence of SEQ ID NO: 1) according to the nucleotide sequence of the humanized cagA gene in SEQ ID NO:1 to obtain an artificially synthesized humanized cagA gene, and the humanized cagA gene was ligated to a T vector (T-cagA). The humanized cagA gene linked to T-cagA was sequenced to verify that the nucleotide sequence was the correct sequence.

(4) Construction of eukaryotic expression vectors

The artificially synthesized humanized cagA gene is connected to a eukaryotic expression vector pcDNA3.1(+) (the structure of the empty vector pcDNA3.1(+) is shown in figure 1) to obtain the gene-connected pcDNA3.1(+) (whether the connected gene is a correct target gene is to be detected). The specific carrier connection process is as follows: the artificially synthesized humanized cagA gene and the eukaryotic expression vector pcDNA3.1(+) are subjected to double enzyme digestion respectively (the enzyme digestion system and conditions are shown in tables 1 and 2), and the artificially synthesized humanized cagA gene after enzyme digestion is connected to the enzyme digested pcDNA3.1(+) (the connection system and conditions are shown in table 3). The construction of the humanized cagA eukaryotic expression vector selects Nhe I restriction enzyme site and Xba I restriction enzyme site.

Table 1: artificially synthesized humanized cagA gene double enzyme digestion

Table 2: eukaryotic expression vector pcDNA3.1(+) double enzyme digestion

Table 3: the target gene is linked to a vector

Transforming competent bacteria E, coli DH5 alpha by using the ligation products in Table 3, plating for overnight culture, randomly selecting completely single-falling colonies (monoclonal colonies) from the plate, detecting by using a bacteria detection PCR method, selecting positive clones obtained in the bacteria detection PCR to send to a sequencing company for sequencing verification, and selecting Escherichia coli positive clones with a sequencing verified to be a correct sequence for carrying out amplification culture, namely, the Escherichia coli positive clones with the sequencing verified to be the correct sequence contain pcDNA3.1(+) with the humanized cagA gene connected thereto. The pcDNA3.1(+) linked with the humanized cagA gene is named pcDNA3.1(+) -cagA, namely the humanized cagA eukaryotic expression vector.

After the bacterial liquid of E.coli DH5 alpha containing pcDNA3.1(+) -cagA is amplified and cultured, the Axygen plasmid extraction kit and the method are utilized to extract the pcDNA3.1(+) -cagA, PCR detection is carried out on the humanized cagA gene in the extracted pcDNA3.1(+) -cagA, and the electrophoresis result is shown in figure 3. As is clear from FIG. 3, the length of the product of the humanized cagA gene amplified by PCR was about 3700 bp. PCR detection is carried out on pcDNA3.1(+) -cagA, and the loading condition of electrophoresis of PCR amplification products is as follows: the PCR amplification product of pcDNA3.1(+) -cagA is loaded in lane 1/2/3, and the DNA molecular weight marker (DNA marker 15000) is loaded in lane 4.

(4) Transfection of gastric cancer cells with pcDNA3.1(+) -cagA

(4.1) gastric cancer cell culture

AGS cell line (purchased from ATCC, ATCC ^ CRL-1739;) is cultured in RPMI-1640 complete medium (10% FBS) at 37 ℃ in 5% CO₂The incubator is used for conventional culture, and the cell culture method is as follows:

recovery of AGS cells: the cell line preserved is taken out from an ultra-low temperature refrigerator at minus 80 ℃, and then is quickly put into a water bath at 37 ℃, and after complete dissolution, the cell line is centrifuged at 800rpm for 5 min. Discarding supernatant, beating with culture solution, mixing, adding into cell culture dish, shaking, and adding CO₂An incubator.

Passage and counting of AGS cells: adding 0.5ml of pancreatin into a culture bottle with cells growing, digesting for 2-3 min, adding 1ml of culture medium, transferring the culture solution into a centrifuge tube, centrifuging for 5min at 800rpm, and removing the supernatant. Adding 2-3 ml of culture solution, blowing, beating and uniformly mixing, and transferring to a culture dish. 75 μ l of the digested cells were counted in a cell counter and the number of cells in 1ml was counted.

Preservation of AGS cells: the culture medium was centrifuged at 800rpm for 5min, and the supernatant was discarded. The frozen stock solution (fetal bovine serum: DMSO = 9: 1) was added, blown and mixed well. And respectively filling into freezing tubes and putting into a liquid nitrogen chamber.

(4.2) transfection of gastric cancer cells

Cultured AGS cells were plated in 6-well plates for transfection experiments. Experimental groups are shown in table 4:

table 4: design of transfection experiment

Experiment grouping	Numbering	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

Note: the pcDNA3.1(+) -hpcagA is prepared by directly linking the non-codon optimized helicobacter pylori cagA gene (hpcagA, NCBI number KR 154737) to pcDNA3.1 (+).

In the experimental group, pcDNA3.1(+) -cagA and lipofectamine2000 were mixed at a ratio of 3. mu.g: 5. mu.l, and 5. mu.l of the above mixture was added to a 6-well plate plated with AGS cells to perform cell transfection. In the blank control group, 5. mu.l lipofectamine2000 was added to 6-well plates plated with AGS cells for cell transfection. In the negative control group, pcDNA3.1(+) and lipofectamine2000 were mixed at a ratio of 3. mu.g: 5. mu.l, and 5. mu.l of the above mixture was added to a 6-well plate plated with AGS cells to perform cell transfection. In the prior art group, pcDNA3.1(+) -hpcagA and lipofectamine2000 were mixed at a ratio of 3. mu.g: 5. mu.l, and 5. mu.l of the above mixture was added to 6-well plates plated with AGS cells to perform cell transfection. In each of the above groups, the transfection treatment was stopped after 6 hours, the medium was changed, and the cells were cultured for 48 hours, whereupon the cell culture was terminated and the cells were harvested.

(4.3) protein extraction after transfection and WB detection

Total proteins of transfected AGS cells harvested in (4.2) were extracted using a BCA protein quantification kit, and the protein expression level of the cagA gene was measured by WB method, and the results are shown in FIG. 4. Lanes are: t0 is blank control, T1 is experimental group, T2 is negative control group, T3 is prior art group, and the rightmost lane is protein molecular weight marker (marker). The experimental results show that: the humanized cagA gene on the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA is expressed in a large amount in a gastric cancer cell line AGS, and the molecular weight of the protein coded by the humanized cagA gene is about 135 KD. The helicobacter pylori cagA gene is not subjected to codon optimization treatment, and the gene is not expressed in a gastric cancer cell strain AGS.

The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Guizhou medical university

<120> construction method of humanized helicobacter pylori cagA eukaryotic expression vector

<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

1. The construction method of the humanized helicobacter pylori cagA eukaryotic expression vector is characterized by comprising the following steps:

(1) codon optimization, adding an additional fragment at the 3' end, and finally synthesizing a humanized cagA gene of which the nucleotide sequence is SEQ ID NO. 1; the nucleotide sequence of the additional fragment is:

5’-GACTACAAGGACGACGATGACAAG-3’；

(2) constructing a humanized cagA eukaryotic expression vector, wherein a host cell of the humanized cagA eukaryotic expression vector is a human gastric cancer AGS cell;

an Nhe I restriction endonuclease recognition sequence is arranged at the 5 'end of the template chain of the humanized cagA gene, and an Xba I restriction endonuclease recognition sequence is arranged at the 3' end of the template chain of the humanized cagA gene; a Kozak sequence is arranged at the 5' end of the template chain of the humanized cagA gene and is positioned between the recognition sequence of the Nhe I restriction endonuclease and the initiation codon ATG of the humanized cagA gene; the Kozak sequence is 5 '-GCCACC-3'.

2. The method for constructing a humanized helicobacter pylori cagA eukaryotic expression vector according to claim 1, wherein in the codon optimization step, the humanized cagA gene is synthesized by polymerase chain reaction.

3. The method for constructing the humanized helicobacter pylori cagA eukaryotic expression vector as claimed in claim 2, wherein the construction steps of the cagA eukaryotic expression vector are as follows: the humanized cagA gene is inserted between the Nhe I site and the Xba I site of pcDNA3.1(+) to obtain the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA.

4. The method for constructing the humanized helicobacter pylori cagA eukaryotic expression vector as claimed in claim 3, wherein the construction step of the humanized cagA eukaryotic expression vector is followed by a transfection step, and the transfection step is as follows: the humanized cagA eukaryotic expression vector pcDNA3.1(+) -cagA is used for transfecting a host cell.

5. The method for constructing the humanized helicobacter pylori cagA eukaryotic expression vector according to claim 4, further comprising a nucleotide sequence identification step between the codon optimization step and the humanized cagA eukaryotic expression vector construction step.