CN113130009A - Application of regulating EIF4A3 expression to regulating apoptosis, migration and invasion capacity of liver cancer cells - Google Patents
Application of regulating EIF4A3 expression to regulating apoptosis, migration and invasion capacity of liver cancer cells Download PDFInfo
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Abstract
The invention relates to an application of regulating EIF4A3 expression to regulate and control apoptosis, migration and invasion capacity of liver cancer cells, which analyzes the effect of EIF4A3 on liver cancer and explores the path and function of gene enrichment related to EIF4A3 through bioinformatics, and then predicts and constructs a comprehensive regulation and control network of EIF4A3 participating in apoptosis, migration and invasion of liver cancer cells. On the basis, a cell line interfering the expression of EIF4A3 is constructed by using 97H and HepG2 cell lines as sources, the influence of interfering the expression of EIF4A3 on the apoptosis, migration and invasion of liver cancer cells is further clarified, a new insight is provided for the effect in the progress of liver cancer, a potential treatment target of the disease is determined, and a reliable theoretical basis is provided for the application of EIF4A3 in genetic engineering, oncology and clinical diagnosis indexes and treatment targets of liver cancer.
Description
Technical Field
The invention relates to the technical field of bioinformatics and genetic engineering, discloses application of regulating EIF4A3 expression to regulate and control apoptosis, migration and invasion capabilities of liver cancer cells, and particularly relates to a molecular mechanism and application of regulating EIF4A3 expression to influence apoptosis, migration and invasion of liver cancer cells.
Background
Primary liver Cancer is the second leading cause of Cancer-related death worldwide (Sia D, Villanueva A, Friedman S L, et al. liver Cancer Cell of Origin, Molecular Class, and Effects on Patient diagnosis [ J ]. Gastroenterology, 2016, 152 (4): 745.). Hepatocellular carcinoma (HCC, hereinafter referred to as liver Cancer) is the digestive system tumor with the highest mortality in China, and the first-line treatment of patients with advanced liver Cancer is mainly molecular targeted drugs (Liu Z, Lin Y, Zhang J, et al. molecular targeted and immune therapy for advanced hepatolar carcinoma carcinosoma [ J ]. Journal of Experimental, Clinical Cancer Research: CR, 2019, 38.). The first liver cancer-targeting drug, sorafenib, was published in 2007, but its clinical manifestations were poor (Zhu Y J, Zheng B, Wang H Y, et al. New knowledge of the mechanisms of sorafenib resistance in liver cancer [ J ]. Acta Pharmacological Sinica, 2017, 38 (005): 614-622.). Late-stage treatment of liver cancer is very limited, and a second targeted drug, ranvatinib, is not on the market until 2018, but the high price is prohibitive for many patients. The metastatic property of the liver cancer is one of the main reasons for difficult cure of the liver cancer, so that the dilemma of independently researching and developing innovative drugs is urgently needed to break through the dilemma and searching for mechanisms influencing apoptosis, migration and invasion of the liver cancer, which has great significance for breaking the monopoly of importing targeted drugs for the liver cancer, reducing the medical cost and meeting the clinical requirements.
EIF4a3 is an ATP-dependent RNA helicase,is also a member of the EIF4A family of translation initiation factors, is a novel component of the Exon Junction Complex (EJC) (Chan C,
DOSTIE，Diem M D，et al.eIF4A3 is a novel component of the exon junction complex[J]rna-a Publication of the Rna Society, 2004, 10 (2): 200-9.). EJC is a protein complex that assembles at the exon-exon junction of mRNA by splicing. EJC proteins play important roles in post-splicing events, including mRNA export, cytoplasmic localization, and nonsense-mediated decay. EJC may be involved in increasing the efficiency of translation of spliced mRNAs (Hl.W, Sh.L, Br.C. Exon junction complexes mediate of splicing mRNA expression [ J.].Proceedings of the National Academy of ences，2003，100(20)：11327-11332.；Dreyfuss G，Kim V N，Kataoka N.Messenger-RNA-binding proteins and the messages they carry.[J]Nature Reviews Molecular Cell Biology, 2002, 3 (3): 195.). EIF4A3 was shown to promote glioblastoma migration and invasion (Wang R, Zhang S, Chen x, et al, EIF4A3-induced circular RNA MMP9 (circular MMP9) acts as a space of miR-124 and proteins cytology multiforma cell genetics [ J]Molecular Cancer, 2018, 17 (1)), also EIF4a3 can play a key role in regulating apoptosis as a bridging protein through multiple tumor-associated signaling pathways, promoting tumor cell migration, invasion and drug resistance (Lin Y, Liang R, Mao Y, et al]Journal of Cellular Biochemistry, 2020) and is one of the important phosphoproteins for liver cancer metastasis (phosphogenomic Analysis of the high-grade-Metastic Hepatocellular Carcinoma Cell Line, MHCC97-H [ J]International Journal of Molecular Sciences, 2015, 16 (2): 4209-4225.). However, the role of EIF4a3 in apoptosis, migration and invasion of liver cancer cells is not clear.
The theoretical basis for treating liver cancer can be provided by analyzing the mechanism of EIF4A3 for promoting apoptosis, migration and invasion of liver cancer cells.
Disclosure of Invention
In order to solve the problems in the prior art, the invention analyzes the effect of EIF4A3 on liver cancer through bioinformatics, explores the path and function of gene enrichment related to EIF4A3, and then predicts and constructs a comprehensive regulation and control network of EIF4A3 participating in apoptosis, migration and invasion of liver cancer cells. On the basis, the liver cancer cell lines 97H and HepG2 are used as cell sources, an own experimental technology platform and method are established, the method for designing and constructing the lentiviral plasmid by the EIF4A3 sequence is further optimized, and the influence of the lentiviral plasmid on the apoptosis, migration and invasion of the liver cancer cells is identified. Exploring the mechanism of EIF4A3 participating in apoptosis, migration and invasion of liver cancer cells. Contributes to the role in the progression of liver cancer, provides new insights and helps to determine potential therapeutic targets for the disease. Provides reliable theoretical basis for applying EIF4A3 in gene engineering, oncology, and clinical diagnosis indexes and treatment targets of glioma.
The invention is realized by the following technical scheme:
on one hand, the invention provides a method for constructing a comprehensive regulation and control network for regulating EIF4A3 expression to influence apoptosis, migration and invasion of liver cancer cells, which comprises the following steps:
(1) data processing: downloading liver cancer data from a cancer genome atlas database, and preprocessing the liver cancer data; the liver cancer data comprises a gene expression profile of RNA _ Seq, a methylated gene expression profile, SNP data and CNV data based on the Affymetrix human genome U133a array platform;
(2) differential expression analysis: performing differential expression analysis on the condition that the gene expression profiles of RNA _ Seq in liver cancer data are respectively in an EIF4A3 low-expression sample and an EIF4A3 high-expression sample to find genes related to EIF4A3 expression; exploring multiple groups of scientific conditions of EIF4A3 in the liver cancer sample as assistance;
(3) weighted gene co-expression network analysis: extracting low-expression and high-expression mRNAs in the EIF4A3 low-expression sample and the EIF4A3 high-expression sample respectively, and executing WGCNA to search a module related to liver cancer;
(4) constructing a comprehensive regulation and control network: and performing functional enrichment analysis on the modular gene with the maximum phenotype correlation, and constructing a comprehensive regulation network for regulating the expression of EIF4A3 so as to influence the apoptosis, migration and invasion of the liver cancer cells.
Further, the data processing steps are as follows:
in the liver cancer data, 375 liver cancer samples and 391 control samples exist in CNV data, gene annotation is carried out on the CNV data, and copy number sites are converted into gene symbols for subsequent analysis;
380 liver cancer samples and 50 control samples exist in a liver cancer methylation spectrum, methylation array analysis is carried out by using an R software package ChAMP, data are filtered, then the data are standardized, and finally differential methylation is calculated;
the total 387 samples of SNP data comprise 345 liver cancer samples and 42 control samples, the Pvalue value of the SNP locus is calculated by using MATLAB2018b software, and the significance of the gene mutation is checked;
there were 387 samples in total in the gene expression profile data of RNA-seq, including 345 liver cancer samples and 42 control samples.
Further, the step of differential expression analysis is as follows:
performing difference analysis of a normal sample and a disease sample on the CNV data by using perl language;
performing differential expression analysis on RNA expression profile data by using a limma package of an R language; further, mRNA and lncRNA expression profiles were extracted based on the background set Homo _ sapiens.grch38.98.chr.gtf and differential expression analysis was performed on liver cancer samples compared to healthy controls; further, the normalizeBetweenArrays function in the limma package was used to normalize the expression profiles; further, when a certain RNA corresponds to a plurality of probes, the average expression value of the probes is selected as the expression value of the RNA.
Further, the weighted gene co-expression network analysis comprises the following steps:
performing hierarchical clustering analysis on 7574 extracted mRNA which are significantly related to EIF4A3 and are subjected to up-regulation and down-regulation by using an hclust function; screening a Soft Threshold power value in the module construction process by using a pick Soft Threshold function; testing the average connectivity and the independence of different modules by using the candidate power; the WGCNA R packet is used for constructing a co-expression network; the minimum module size is set to 30 and each module is assigned a unique color; based on string database, a protein interaction network (PPI network) is constructed, and only the interaction with the joint score more than 500 is reserved; the ClusterONE plug-in of the cytoscape software is used for conducting modular analysis on the PPI network. Further, the candidate power is 1-30. Further, when the degree of independence is > 0.9, an appropriate power value is selected.
Further, the construction of the comprehensive regulation and control network comprises the following steps:
performing function enrichment analysis on the 3 1619 mRNAs with the maximum positive correlation with EIF4A3 phenotype by using a clusterProfiler package to explore functions and signal paths involved by module genes;
gene Set Enrichment Analysis (GSEA) is applied to explore the functions and the paths of enrichment in liver cancer samples and EIF4A3 high-expression samples;
an lncRNA capable of regulating EIF4A3 is extracted based on an RNA Inter database, and a comprehensive regulation network which is mediated by lncRNA targeting EIF4A3 and influences apoptosis, migration and invasion of liver cancer cells is constructed by combining a passage obtained in a function enrichment process.
On the other hand, the invention provides an application of regulating EIF4A3 expression to regulate and control apoptosis, migration and invasion capacity of liver cancer cells, which comprises the following steps:
step A: constructing a stably infected knockout EIF4A3 cell line;
and B: constructing a cell line stably infected with the over-expression EIF4A3 gene;
and C: and detecting the influence of interfering EIF4A3 gene expression on the apoptosis, migration and invasion capacity of the liver cancer cells.
Further, the process of step a is as follows: designing shRNA according to an EIF4A3 sequence, constructing an EIF4A3 interference lentivirus plasmid and packaging lentivirus; preparing a recombinant EIF4A3-shRNA lentivirus; and infecting a lentiviral vector for knocking out the EIF4A3 gene in 97H cells to obtain a stably infected knocked-out EIF4A3 cell line.
Further, the stably infected knockout EIF4A3 cell line was prepared as follows
(1) Nucleotide sequences as described by SED ID NO: 1 to the sequence shown as SED ID NO: 2 to obtain an EIF4A3 interference lentivirus vector;
(2) interference with lentiviral vector packaging using EIF4a3 preparation sequences such as SED ID NO: EIF4a3 shown in fig. 3 interferes with lentiviruses; the EIF4A3 interferes the lentivirus to infect the hepatoma cell strain, the infected cell is cultured and screened by using antibiotic, and the cell strain with the EIF4A3 gene stably knocked down is obtained.
Further, the conditions of the packaging are: mixing at room temperature for 20min, transfecting for 20 hr, and collecting.
Further, the connection conditions are as follows: ligation was performed at 22 ℃ for 1 h.
Further, the lentiviral vector is hU 6-MCS-CMV-zsGreenl-PGK-puromycin; the antibiotic is puromycin.
Further, the liver cancer cell strain is selected from 97H cells.
Further, the method also comprises the step of verifying the expression level of EIF4A3 in the cell strain with the stable reduced EIF4A3 gene by using qRT-PCR and western blot methods.
Further, the process of step B is as follows: an expression plasmid marked with EIF4A3 is constructed by using a CMV-MCS-3XFLAg-PGK-Puro vector, and the plasmid is transfected into 293T cells; the recombinant retroviruses of the cells are used for multiple infection of the liver cancer cell line HepG2, and the cells are infected by a lentiviral vector over-expressing EIF4A3 gene, so as to obtain a cell line stably infected with the over-expressing EIF4A3 gene.
Further, the preparation steps of the cell line which is stably infected and over-expresses the EIF4A3 gene are as follows:
(a) the sequences were cut with restriction enzymes XhoI and EcoR, respectively, as SED ID NO: 4, electrophoresing to obtain the sequence shown as SED ID NO: 5, a linearized support;
(b) extracting total RNA from fresh tissue specimen, reverse transcribing to cDNA, PCR amplifying to obtain sequence such as SED ID NO: 6 or a fragment of the EIF4A3 gene;
(c) connecting the linearized vector with an EIF4A3 gene fragment by using DNA ligase to obtain an EIF4A3 overexpression lentiviral vector;
(d) the use of EIF4a3 to overexpress lentiviral vector packaging to prepare sequences such as SED ID NO: 7, infecting a hepatoma cell strain by using the EIF4A3 over-expressed lentivirus, and performing drug screening by using antibiotics to obtain a cell strain stably over-expressing EIF4A3 gene.
Further, the conditions of the packaging are: mixing at room temperature for 20min, transfecting for 20 hr, and collecting.
Further, the electrophoresis conditions are as follows: 1% agarose gel, 230V, 30 min.
Further, the PCT amplification system is as follows:
the sequence of primer 1 here: CTACCGGACTCAGATCTCGAGGCCACCATGGCGACCACGGCCACGAT, respectively;
the sequence of primer 2 is: GTCATCCTTGTAATCGAATTCGATAAGATCAGCAACGTTCATCGG, respectively;
further, the connection conditions are as follows: water bath at 37 deg.c for 30min for conversion.
Further, the lentiviral vector is CMV-MCS-3 XFlag-PGK-Puro; the antibiotic is puromycin.
Further, the liver cancer cell line is selected from HepG2 cells.
Further, the method also comprises the step of verifying the expression level of EIF4A3 in the cell strain which stably over-expresses the EIF4A3 gene by utilizing qRT-PCR and western blot method.
According to the invention, the liver cancer cells are directly infected by the EIF4A3 overexpression lentivirus, so that a cell strain for stably overexpressing the EIF4A3 gene is constructed; and verifying the expression quantity of the EIF4A3 gene in the cell strain by utilizing a qRT-PCR and western blot method, and showing that the expression quantity of the constructed cell strain is higher than that of a control cell strain. Verification shows that the EIF4A3 gene can be efficiently and stably over-expressed in the constructed cell strain.
Further, the process of step C is as follows: respectively exploring the influence of knocking out or over-expressing EIF4A3 on the apoptosis of the liver cancer cell by a cell line stably infected with an over-expressed EIF4A3 gene and a knocked-out EIF4A3 cell line stably infected with the gene through an apoptosis test; respectively carrying out Transwell and Matrigel-Transwell experiments on a cell line which is stably infected and overexpresses EIF4A3 gene and a stably infected knockout EIF4A3 cell line to explore the influence of knocking out or overexpressing EIF4A3 on migration and invasion of the liver cancer cells; the effect of knocking out EIF4A3 on liver cancer is observed at the animal level.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
according to the invention, by constructing a comprehensive regulation and control network of the EIF4A3 participating in apoptosis, migration and invasion of the liver cancer cells, the EIF4A3 is screened out to inhibit the apoptosis of the liver cancer cells through a mediated gap junction pathway, and the EIF4A3 promotes the migration and invasion of the liver cancer cells through an ErbB signal pathway, so that the action mechanism of the EIF4A3 participating in the apoptosis, migration and invasion of the liver cancer cells is further demonstrated. The comprehensive regulation and control network constructed by the invention is further verified by constructing a cell line over-expressing or knocking out the EIF4A3 gene, so that a theoretical basis is provided for the subsequent exploration of the molecular mechanism of EIF4A3 participating in apoptosis, migration and invasion of the liver cancer cell, and a reliable theoretical basis is also provided for the diagnosis index and the treatment target of the liver cancer.
In the process of constructing the comprehensive regulation and control network, the biological information data of a large sample is mined and effective information is extracted, mRNA with consistent expression is extracted by adopting differential expression analysis, a module related to liver cancer is searched by applying weighted gene co-expression network analysis, functions and paths of module genes influenced by EIF4A3 are analyzed, a regulation and control network for regulating apoptosis, migration and invasion of liver cancer cells by EIF4A3 is constructed, the requirements of standard analysis can be met, meanwhile, personalized analysis requirements are met, meaningful exploration and basis are provided for research of liver cancer marker screening and molecular mechanisms, detection markers and new treatment paths are provided for diagnosis of liver cancer, and reliable scientific basis is provided for prevention and treatment of diseases.
According to the invention, by successfully constructing a cell line for knocking out or over-expressing EIF4A3, the ELF4A3 protein can be stably and lowly expressed or highly expressed, significant exploration and basis are provided for research on liver cancer marker screening and molecular mechanism, a new idea for liver cancer treatment is provided, and a new way is provided for research on liver cancer drug treatment targets.
Drawings
FIG. 1 is a flow chart of the molecular mechanism and application of EIF4A3 expression regulation to influence apoptosis, migration and invasion of hepatoma cells
FIG. 2 shows the expression of EIF4A3 in different liver cancer samples, and the multiple groups of SNPs, CNVs and methylation of EIF4A3 in liver cancer samples.
FIG. 3 analysis of a weighted gene co-expression network explores the molecular expression co-dysregulation landscape associated with EIF4A 3.
FIG. 4 is a comprehensive network landscape of EIF4A 3-mediated molecular mechanisms affecting apoptosis, migration and invasion of hepatoma carcinoma cells.
FIG. 5 graph of transfection efficiency following knockdown or overexpression of EIF4A 3.
FIG. 6 is a graph of the results of modulating EIF4A3 expression to affect hepatoma apoptosis.
FIG. 7 is a graph of results of modulating EIF4A3 expression to affect hepatoma cell migration and invasion.
FIG. 8 is a graph of tumor growth after knock-out of EIF4A3 at the animal level.
Detailed Description
The following examples are intended to illustrate the invention without further limiting its scope.
Example 1
As shown in fig. 1, the present invention provides a flow chart of a molecular mechanism and an application of the molecular mechanism for regulating EIF4a3 expression to affect apoptosis, migration and invasion of liver cancer cells. Four types of liver cancer data are obtained from a TCGA database, after differential expression analysis, genes related to EIF4A3 expression are identified, modules related to liver cancer are explored through weighted gene co-expression network analysis, and a comprehensive regulation and control network of EIF4A3 participating in apoptosis, migration and invasion of liver cancer cells is constructed through function enrichment analysis.
As shown in fig. 1, the invention provides a method for constructing a comprehensive regulation network for regulating EIF4a3 expression to influence apoptosis, migration and invasion of liver cancer cells, comprising the following steps:
(1) data processing:
downloading data of liver cancer from a cancer genome atlas (TCGA) database to obtain a gene expression profile comprising RNA _ Seq, Methylation 2 omics and clinical information thereof, and data of liver cancer SNP and CNV; wherein the gene expression profiles are based on the Affymetrix human genome U133a array platform (Affymetrix; Thermo Fisher ScientiIFC Inc., Waltham, Mass., USA).
Carrying out gene annotation on 375 liver cancer samples and 391 control samples in CNV data, and converting copy number sites into gene symbol for subsequent analysis;
380 liver cancer samples and 50 control samples exist in a liver cancer methylation spectrum, methylation array analysis is carried out by using an R software package ChAMP, data are filtered, then the data are subjected to standardization processing, and finally differential methylation is calculated;
the total 387 samples of SNP data comprise 345 liver cancer samples and 42 control samples, the Pvalue value of the SNP locus is calculated by using MATLAB2018b software, and the significance of the gene mutation is checked;
there were 387 samples in total in the RNA-seq data, including 345 liver cancer samples and 42 control samples.
(2) Differential expression analysis, as follows:
differential expression analysis was performed on RNA expression profile data using limma in R language, mRNA and lncRNA expression profiles were extracted based on the background set Homo sapiens grch38.98 chr.gtf, and on liver cancer samples compared to healthy controls, and the normalizebetweenrays function in the limma package was used to normalize the expression profiles. Differential expression analysis was performed on samples with high expression of EIF4A3 to look for differentially expressed genes, compared to samples with low expression of EIF4 A3. Differential expression analysis of normal and disease samples was performed on CNV data using perl language.
When a gene corresponds to a plurality of probes, the average expression value of the probes is selected as the expression value of the gene.
The multi-cohort of EIF4a3 in liver cancer samples was subsequently explored.
As shown in fig. 2, the expression of EIF4A3 in different liver cancer samples, and the multiple mathematical conditions of SNP, CNV and methylation of EIF4A3 in liver cancer samples; wherein, FIG. 2A is the expression situation of EIF4A3 differential expression and each phenotype in the RNA _ Seq data downloaded from TCGA for liver cancer, and FIG. 2B is the expression situation of SNP-CNV-methyl-RNAexp four omics of EIF4A3 in the liver cancer sample-health sample differential group.
(3) The weighted gene co-expression network analysis specifically comprises the following steps: mRNA with high and low expression in both groups was extracted and WGCNA was performed to explore modules associated with liver cancer.
First, a hierarchical clustering analysis was performed on 7574 up-and down-regulated mrnas significantly correlated to EIF4a3 using the hclust function, and then Soft Threshold power values in the module construction process were screened using the pick Soft Threshold function. And (3) testing the average connectivity and the independence of different modules by using the candidate power (1-30). When the degree of independence is greater than 0.9, a suitable power value is selected. The WGCNA R package is used for constructing co-expression networks (modules); the minimum module size is set to 30 and each module is assigned a unique color. To obtain interactive genes significantly associated with EIF4A3 expression, a protein interaction (PPI) network was constructed for 7574 up-and down-regulated mrnas significantly associated with EIF4A3 based on string databases, only the interactions with a joint score > 500 were retained, and a total of 315 interactive genes significantly associated with EIF4A3 expression were obtained.
cytoscape software is used to visualize the network. The ClusterONE plug-in of cytoscape software was used for modular analysis of PPI networks (minimum 30).
As shown in FIG. 3, the analysis of the weighted gene co-expression network in the present example explores the molecular expression co-dysregulation landscape associated with EIF4A 3. Where fig. 3A determines that the weighted gene co-expression network analysis has a weight of 7, fig. 3B results in 19 modules significantly associated with EIF4A3 and each module is assigned a unique color, fig. 3C is a constructed PPI network, and cytoscape software shows 315 of the 16 modules significantly associated with EIF4A3 expression and maps the module colors.
(4) Enrichment analysis and construction of a network for regulating EIF4A3 expression to influence apoptosis, migration and invasion of liver cancer cells are as follows:
the clusterProfiler package is used to perform functional enrichment analysis on 1619 mRNAs in total of 3 modules with the maximum positive correlation with EIF4A3 phenotype so as to explore functions and signal paths involved by module genes. P < 0.05 was considered to be significantly different.
Gene Set Enrichment Analysis (GSEA) was applied to explore the functions and pathways of enrichment in liver cancer samples and EIF4A3 high expression samples. GSEA was done using JAVA software (software index http:// jsp/jsp /) found on official websites. Gene sets c5.bp. v6.2.symbols.gmt and c2.cp.kegg. v6.2.symbols.gmt in the MSigDB database serve as reference gene sets.
And finally, extracting lncRNA capable of regulating EIF4A3 based on an RNAInter database, and constructing an lncRNA-targeted EIF4A 3-mediated liver cancer global imbalance comprehensive network. The result shows that 5 lncRNA and EIF4A3 interact with each other and control 43 KEGG channels through 289 modular genes to further mediate the comprehensive network of the global disorder of the liver cancer, based on literature reference, the fact that EIF4A3 can inhibit the apoptosis of the liver cancer through a gap junction channel can be found, and EIF4A3 mediates an ErbB signal channel to promote the migration and invasion of the liver cancer.
As shown in fig. 4, the comprehensive network landscape of the molecular mechanism that affects apoptosis, migration, and invasion of the liver cancer cell mediated by EIF4A3 in the embodiment of the present invention indicates that EIF4A3 can inhibit apoptosis of the liver cancer cell through a gap junction pathway, and EIF4A3 mediates ErbB signaling pathway to promote migration and invasion of the liver cancer. Wherein fig. 4A is a biological function in which 1619 genes are involved in 3 modules with the largest positive correlation among EIF4A3_ Case _ Control phenotypes, fig. 4B is a biological function in which 1619 genes are involved in the group EIF4A3_ Case _ Control, the right is a biological function in which 1619 genes are involved in the group EIF4A3_ high _ low, fig. 4C is a KEGG pathway in which 1619 genes are involved in the group EIF4A3_ Case _ Control, the positive correlation among the 3 modules with the largest positive correlation among EIF4A3_ Case _ Control, the left is a KEGG pathway in which KEGG is enriched in the group EIF4A3_ Case _ Control, the right is a KEGG pathway in which KEGG is enriched in the group EIF4A3_ high _ low, fig. 4E is a kencrna interaction EIF4A3 inhibits apoptosis through gap junon pathway, and fig. 4F is an ErbB pathway mediated by einc rna invasion promoting signal 3 and liver cancer migration through gap junon.
Example 2
(1) Constructing a cell line with EIF4A3 knockout:
preparing a recombinant EIF4A3-shRNA lentivirus and a Negative Control (NC) lentivirus; and infecting a slow virus vector (EIF4A3-KD) for knocking out EIF4A3 gene in LM3 cells to obtain stably infected knocked-out EIF4A3 cell lines and control cell lines. Transfection efficiency after knocking out EIF4A3 as shown in FIG. 5A indicates that the hepatoma cell line LM3 did knock out EIF4A 3. The specific process is as follows:
and (3) detecting by adopting qRT-PCR, and comparing and analyzing a sequencing result and an EIF4A3 sequence. Landing on Public TRC Portal website, searching EIF4A3 and selecting the verified and high-scoring shRNA interference sequence 5'-GGATGAAGCTGATGAAATG-3' of EIF4A3 from the results, and comparing through BLAST homology. The synthesized shRNA template (nucleotide sequence is shown as SED ID NO: 1, specifically GGATGAAGCTGATGAAATG; the specific synthesis process aims at the target gene sequence of a target gene, multiple RNA interference target point sequences are designed by using the RNA interference sequence design principle provided in a public website, evaluation and determination are carried out according to the design experience and design software, the target point with the best kinetic parameter is selected to enter the subsequent experimental process) and connected with a lentiviral vector (connected for 1h at 22 ℃) to obtain the EIF4A3 interference lentiviral vector.
The obtained EIF4A3 interference lentivirus vector is packaged to prepare EIF4A3 interference lentivirus (the conditions are that the mixture is evenly mixed for 20min at room temperature, the transfection is carried out for 20h, and the virus is collected), and the EIF4A3 interference lentivirus infects LM3 cells.
Positive clones (2. mu.g/mL for 97H cells) were selected with antibiotics and a 14-day stable cell line 97H-EIF4A3-KD was established.
The efficiency of EIF4A3 knockdown was examined by qRT-PCR and western blot, and the conclusion is shown in FIG. 5A.
The lentivirus vector is hU6-MCS-CMV-zsGreen 1-PGK-puromycin; the antibiotic is puromycin.
Here lentiviral vector sequences such as SED ID NO: 2, specifically:
TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAGGATCCGGACAAGCTTCGAATTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGTCGCCACCATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTCCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGCCCTAACTCGAGTAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAACGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
here EIF4a3 interferes with lentiviral sequences such as SED ID NO: 3, specifically:
TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAGGATCCGGATGAAGCTGATGAAATGTTCAAGAGACATTTCATCAGCTTCATCCTTTTTTGAATTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGTCGCCACCATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGTGGAGGGCGGCCCCTTGCCCTTCGCCGAGGACATCTTGTCCGCCGCCTTCATGTACGGCAACCGCGTGTTCACCGAGTACCCCCAGGACATCGTCGACTACTTCAAGAACTCCTGCCCCGCCGGCTACACCTGGGACCGCTCCTTCCTGTTCGAGGACGGCGCCGTGTGCATCTGCAACGCCGACATCACCGTGAGCGTGGAGGAGAACTGCATGTACCACGAGTCCAAGTTCTACGGCGTGAACTTCCCCGCCGACGGCCCCGTGATGAAGAAGATGACCGACAACTGGGAGCCCTCCTGCGAGAAGATCATCCCCGTGCCCAAGCAGGGCATCTTGAAGGGCGACGTGAGCATGTACCTGCTGCTGAAGGACGGTGGCCGCTTGCGCTGCCAGTTCGACACCGTGTACAAGGCCAAGTCCGTGCCCCGCAAGATGCCCGACTGGCACTTCATCCAGCACAAGCTGACCCGCGAGGACCGCAGCGACGCCAAGAACCAGAAGTGGCACCTGACCGAGCACGCCATCGCCTCCGGCTCCGCCTTGCCCTAACTCGAGTAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAACGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGTTTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
constructing a cell line over expressing EIF4A3, and concretely comprising the following steps:
the lentiviral vector was digested with restriction enzymes XhoI and EcoRI, respectively, the vector digestion products were subjected to agarose gel electrophoresis (1% agarose gel, 230V, 30min), and the target band was recovered to obtain a linearized vector (plasmid).
Here the sequences of the lentiviral vectors are as SED ID NO: 4, the concrete steps are as follows:
TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGAGGATCGCTAGCGCTACCGGACTCAGATCTCGAGACAACGTTCGAACGCGTACAAGCGCGGCCGCACAACGTCTAGAGAATTCGATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACAAAGATGATGACGATAAATGAGGATCCACCGGATCTAGATAACTGATCATAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGACCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
the sequences of the vectors linearized herein are as SED ID NO: 5, the concrete steps are as follows:
TGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGAGGATCGCTAGCGCTACCGGACTCAGATCTCGAGGATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACAAAGATGATGACGATAAATGAGGATCCACCGGATCTAGATAACTGATCATAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGACCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
extracting total RNA of tissues from a cell specimen, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification on the reverse transcribed cDNA to obtain a purified EIF4A3 gene fragment, wherein the system is as follows: 1-2 mu g of template; primer 12 uL; 22 mu L of primer; PCR mix 25. mu.L; ddH2O made up to 50. mu.L.
The sequence of primer 1 here is: CTACCGGACTCAGATCTCGAGGCCACCATGGCGACCACGGCCACGAT, respectively;
the sequence of primer 2 is: GTCATCCTTGTAATCGAATTCGATAAGATCAGCAACGTTCATCGG
The materials are added into a thin-wall tube, mixed evenly and placed into a PCR instrument after point separation, and proper annealing temperature and extension temperature are selected, so that PCR amplification can be started.
Here, the sequence of the EIF4a3 gene fragment is as defined by SED ID NO: 6, the concrete steps are as follows:
ATGGCGACCACGGCCACGATGGCGACCTCGGGCTCGGCGCGAAAGCGGCTGCTCAAAGAGGAAGACATGACTAAAGTGGAATTCGAGACCAGCGAGGAGGTGGATGTGACCCCCACGTTCGACACCATGGGCCTGCGGGAGGACCTGCTGCGGGGCATCTACGCTTACGGTTTTGAAAAACCATCAGCAATCCAGCAACGAGCAATCAAGCAGATCATCAAAGGGAGAGATGTCATCGCACAGTCTCAGTCCGGCACAGGAAAAACAGCCACCTTCAGTATCTCAGTCCTCCAGTGTTTGGATATTCAGGTTCGTGAAACTCAAGCTTTGATCTTGGCTCCCACAAGAGAGTTGGCTGTGCAGATCCAGAAGGGGCTGCTTGCTCTCGGTGACTACATGAATGTCCAGTGCCATGCCTGCATTGGAGGCACCAATGTTGGCGAGGACATCAGGAAGCTGGATTACGGACAGCATGTTGTCGCGGGCACTCCAGGGCGTGTTTTTGATATGATTCGTCGCAGAAGCCTAAGGACACGTGCTATCAAAATGTTGGTTTTGGATGAAGCTGATGAAATGTTGAATAAAGGTTTCAAAGAGCAGATTTACGATGTATACAGGTACCTGCCTCCAGCCACACAGGTGGTTCTCATCAGTGCCACGCTGCCACACGAGATTCTGGAGATGACCAACAAGTTCATGACCGACCCAATCCGCATCTTGGTGAAACGTGATGAATTGACTCTGGAAGGCATCAAGCAATTTTTCGTGGCAGTGGAGAGGGAAGAGTGGAAATTTGACACTCTGTGTGACCTCTACGACACACTGACCATCACTCAGGCGGTCATCTTCTGCAACACCAAAAGAAAGGTGGACTGGCTGACGGAGAAAATGAGGGAAGCCAACTTCACTGTATCCTCAATGCATGGAGACATGCCCCAGAAAGAGCGGGAGTCCATCATGAAGGAGTTCCGGTCGGGCGCCAGCCGAGTGCTTATTTCTACAGATGTCTGGGCCAGGGGGTTGGATGTCCCTCAGGTGTCCCTCATCATTAACTATGATCTCCCTAATAACAGAGAATTGTACATACACAGAATTGGGAGATCAGGTCGATACGGCCGGAAGGGTGTGGCCATTAACTTTGTAAAGAATGACGACATCCGCATCCTCAGAGATATCGAGCAGTACTATTCCACTCAGATTGATGAGATGCCGATGAACGTTGCTGATCTTATC
connecting the EIF4A3 gene fragment amplified and purified by PCR with a linearized vector (plasmid) after enzyme digestion by using DNA ligase (the conditions are: water bath at 37 ℃ for 30min, transformation, plate coating and single clone extraction of the plasmid) to obtain an EIF4A3 overexpression lentiviral vector, packaging the EIF4A3 overexpression lentiviral vector into lentivirus (the conditions are: uniformly mixing at room temperature for 20min, transfecting for 20h, collecting the virus), and obtaining the EIF4A3 overexpression lentivirus.
Here EIF4a3 overexpresses lentiviral sequences such as SED ID NO: 7, specifically:
TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGCCTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTACTAGAGGATCGCTAGCGCTACCGGACTCAGATCTCGAGGCCACCATGGCGACCACGGCCACGATGGCGACCTCGGGCTCGGCGCGAAAGCGGCTGCTCAAAGAGGAAGACATGACTAAAGTGGAATTCGAGACCAGCGAGGAGGTGGATGTGACCCCCACGTTCGACACCATGGGCCTGCGGGAGGACCTGCTGCGGGGCATCTACGCTTACGGTTTTGAAAAACCATCAGCAATCCAGCAACGAGCAATCAAGCAGATCATCAAAGGGAGAGATGTCATCGCACAGTCTCAGTCCGGCACAGGAAAAACAGCCACCTTCAGTATCTCAGTCCTCCAGTGTTTGGATATTCAGGTTCGTGAAACTCAAGCTTTGATCTTGGCTCCCACAAGAGAGTTGGCTGTGCAGATCCAGAAGGGGCTGCTTGCTCTCGGTGACTACATGAATGTCCAGTGCCATGCCTGCATTGGAGGCACCAATGTTGGCGAGGACATCAGGAAGCTGGATTACGGACAGCATGTTGTCGCGGGCACTCCAGGGCGTGTTTTTGATATGATTCGTCGCAGAAGCCTAAGGACACGTGCTATCAAAATGTTGGTTTTGGATGAAGCTGATGAAATGTTGAATAAAGGTTTCAAAGAGCAGATTTACGATGTATACAGGTACCTGCCTCCAGCCACACAGGTGGTTCTCATCAGTGCCACGCTGCCACACGAGATTCTGGAGATGACCAACAAGTTCATGACCGACCCAATCCGCATCTTGGTGAAACGTGATGAATTGACTCTGGAAGGCATCAAGCAATTTTTCGTGGCAGTGGAGAGGGAAGAGTGGAAATTTGACACTCTGTGTGACCTCTACGACACACTGACCATCACTCAGGCGGTCATCTTCTGCAACACCAAAAGAAAGGTGGACTGGCTGACGGAGAAAATGAGGGAAGCCAACTTCACTGTATCCTCAATGCATGGAGACATGCCCCAGAAAGAGCGGGAGTCCATCATGAAGGAGTTCCGGTCGGGCGCCAGCCGAGTGCTTATTTCTACAGATGTCTGGGCCAGGGGGTTGGATGTCCCTCAGGTGTCCCTCATCATTAACTATGATCTCCCTAATAACAGAGAATTGTACATACACAGAATTGGGAGATCAGGTCGATACGGCCGGAAGGGTGTGGCCATTAACTTTGTAAAGAATGACGACATCCGCATCCTCAGAGATATCGAGCAGTACTATTCCACTCAGATTGATGAGATGCCGATGAACGTTGCTGATCTTATCGAATTCGATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACAAAGATGATGACGATAAATGAGGATCCACCGGATCTAGATAACTGATCATAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGACCGCGTCTGGAACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAATTAATTCTGCAGTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGAGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTGATATCGAGCTTGCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGCCTTGACATTGCTAGCGTTTTACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAATATGTACTACAAACTTAGTAGT
the obtained EIF4a3 overexpressing lentivirus was infected into cells.
Positive clones (2. mu.g/mL of HepG2 cells) were selected with antibiotics and a 14-day stable cell line HepG2-EIF4A3-OE cell was established.
The efficiency of EIF4A3 overexpression was examined by qRT-PCR and western blot, and the conclusion is shown in FIG. 5B.
Wherein the lentiviral vector is CMV-MCS-3 xFlag-PGK-Puro; the antibiotic is puromycin.
Establishing an EIF4A3 control cell line: hep3B-EIF4A3-NC and LM3-EIF4A3-NC cells were constructed in a similar manner to that described in 1 above.
(2) The interference efficiency of EIF4A3 was verified by RT-qPCR and Western blot.
The RT-qPCR method is as follows: liver cancer cell lines with EIF4a3 knocked out or overexpressed were collected, total RNA of the tissue was extracted using Trizol (TaKaRa, a-79061) reagent, and the total RNA was reverse-transcribed into cDNA according to the kit instructions. Reaction conditions are as follows: pre-denaturation at 95 ℃, enzyme activation (30s), denaturation at 95 ℃ (5s), 60 ℃ (30s), 40 cycles. Beta actin is used as internal reference and 2-ΔΔCtThe method calculates the relative expression quantity of the target gene EIF4A3 mRNA. The EIF4A3 upstream primer is 5'-GGCACAGGAAAAACAGCCACCT-3', and the downstream primer is 5'-TGTAGTCACCGAGAGCAAGCAG-3'; the upstream primer of the beta-actin is 5' -CACCATTGGCAATGAGCGGTTC-3 ', and the downstream primer is 5'-AGGTCTTTGCGGATGTCCACGT-3'. The experiment was repeated 3 times. Finally, the interference efficiency of the EIF4A3 is tested.
The Western blot method comprises the following steps: total protein in cells was extracted with cell lysate and protein concentration was measured using BCA kit (Biyun day) following the protocol strictly. Adding 30-50 μ g of standardized protein sample into each well, performing constant-pressure electrophoresis of concentrated gel at 80V in a vertical electrophoresis tank filled with Tris-Glycine electrophoresis solution to separation gel interface, and adjusting to 120V to continue electrophoresis to the bottom of bromocyan separation gel. Transfer to poly PVDF membrane, designated primary antibody room temperature incubation for 2h or 4 degrees C were incubated overnight. TBST was washed for 3 times for 10min, and a secondary antibody was added and incubated for 1h at room temperature. Taking out the PVDF membrane, washing for 10min by TBST for 3 times; sucking the liquid on the film, adding ECL working solution on the clean preservative film, and reacting for 2-5 min; tabletting and developing in a dark room. Detecting the gray value of each band by Image J software, representing the expression level of the EIF4A3 protein in each group of cells by the ratio of the gray value of the target band to the gray value of the beta-actin band,
(3) apoptosis experiments explore the influence of knocking out or over-expressing EIF4A3 on the apoptosis of liver cancer cells.
At 37 ℃ 5% CO2The 97H cell line of the EIF4A3 is knocked out and the HepG2 cell line of the over-expression EIF4A3 and the control thereof are cultured in an incubator, when the cell density reaches 70%, cephalotaxin is added to induce apoptosis, after the cell is continuously cultured for 2.5 hours, the cell sap is taken out and added with Ho33342 mother liquor, PI staining is carried out for 15 minutes, a glass slide is taken to surround a small chamber, the stained cell suspension is taken from a centrifuge tube and added into the small chamber, then the glass slide is covered, and the cell apoptosis condition is observed by a fluorescence microscope through ultraviolet excitation light or a flow cytometer.
As shown in FIG. 6, in the present example, after EIF4A3 is knocked out at the cellular level, the apoptosis of hepatoma carcinoma cells 97H is increased. In the Hep3B liver cancer cell line over-expressing EIF4A3, the liver cancer cell HepG2 has no obvious apoptosis.
(4) Transwell and Matrigel-Transwell explored the effect of knocking out or overexpressing EIF4a3 on migration and invasion of hepatoma cells, as follows: the cells of each group were collected, cell suspensions were prepared, and the cell concentration was adjusted to 1X106 cells/ml. Matrigel was diluted 1: 8 and added to the upper chamber of the Transwell. Fibronectin was added to the bottom of the chamber. Cell suspensions were prepared and added to the upper chamber and DMEM containing FBS was added to the lower chamber. Methanol fixation, crystal violet staining, random selection of 5-8 fields under the microscope, counting the number of cells passing through each field, and calculating cell mobility.
As shown in FIG. 7, in the present example, after EIF4A3 is knocked out at the cellular level, the cell migration and invasion of hepatoma carcinoma cells 97H are inhibited. In the Hep3B liver cancer cell line over-expressing EIF4A3, the cell migration and invasion of the liver cancer cell HepG2 are not obviously inhibited.
(5) The influence of knocking-out EIF4A3 on liver cancer is observed at the animal level, and the specific steps are as follows: EIF4A3-KD or a control cell LM3 is transplanted subcutaneously in BALC nude mice by utilizing a mouse stereotaxic technology, and the size of a tumor is measured by a vernier caliper every week after the cells are inoculated. The longest and shortest tumor sites were measured with a vernier caliper, and when the subcutaneous tumor volume of the nude mice in the experimental group was grown to an appropriate volume (less than 1cm ^ 3), the nude mice were sacrificed, the final tumor volume of the nude mice was measured, and the tumor weight was weighed.
As shown in fig. 8, after EIF4a3 knock-out at the animal level, the tumor diameter was smaller compared to the control group.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> forest swallow
<120> application of regulating EIF4A3 expression to regulating liver cancer cell apoptosis, migration and invasion capacity
<130> 2021
<141> 2021-03-19
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 1
ggatgaagct gatgaaatg 19
<210> 2
<211> 8997
<212> DNA
<213> Artificial Sequence
<400> 2
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatctgggc aggaagaggg cctatttccc atgattcctt 2220
catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa 2280
acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 2340
cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 2400
tcgatttctt ggctttatat atcttgtgga aaggacgagg atccggacaa gcttcgaatt 2460
ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 2520
gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 2580
tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 2640
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 2700
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 2760
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 2820
ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 2880
gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 2940
gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 3000
tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct 3060
accggtcgcc accatggccc agtccaagca cggcctgacc aaggagatga ccatgaagta 3120
ccgcatggag ggctgcgtgg acggccacaa gttcgtgatc accggcgagg gcatcggcta 3180
ccccttcaag ggcaagcagg ccatcaacct gtgcgtggtg gagggcggcc ccttgccctt 3240
cgccgaggac atcttgtccg ccgccttcat gtacggcaac cgcgtgttca ccgagtaccc 3300
ccaggacatc gtcgactact tcaagaactc ctgccccgcc ggctacacct gggaccgctc 3360
cttcctgttc gaggacggcg ccgtgtgcat ctgcaacgcc gacatcaccg tgagcgtgga 3420
ggagaactgc atgtaccacg agtccaagtt ctacggcgtg aacttccccg ccgacggccc 3480
cgtgatgaag aagatgaccg acaactggga gccctcctgc gagaagatca tccccgtgcc 3540
caagcagggc atcttgaagg gcgacgtgag catgtacctg ctgctgaagg acggtggccg 3600
cttgcgctgc cagttcgaca ccgtgtacaa ggccaagtcc gtgccccgca agatgcccga 3660
ctggcacttc atccagcaca agctgacccg cgaggaccgc agcgacgcca agaaccagaa 3720
gtggcacctg accgagcacg ccatcgcctc cggctccgcc ttgccctaac tcgagtaatt 3780
ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 3840
gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 3900
taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 3960
agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 4020
agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 4080
cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 4140
aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 4200
gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 4260
ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccatgac cgagtacaag 4320
cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 4380
gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 4440
gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 4500
gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 4560
gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 4620
cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 4680
gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 4740
cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 4800
cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 4860
gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg tctggaacaa 4920
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 4980
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 5040
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 5100
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 5160
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 5220
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 5280
gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc 5340
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 5400
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 5460
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattaattc 5520
tgcagtcgag acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat 5580
gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct 5640
caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa 5700
gaaaagaggg gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg 5760
tggatctacc acacacaagg ctacttccct gattagcaga actacacacc agggccaggg 5820
gtcagatatc cactgacctt tggatggtgc tacaagctag taccagttga gccagataag 5880
gtagaagagg ccaataaagg agagaacacc agcttgttac accctgtgag cctgcatggg 5940
atggatgacc cggagagaga agtgttagag tggaggtttg acagccgcct agcatttcat 6000
cacgtggccc gagagctgca tccggagtac ttcaagaact gctgatatcg agcttgctac 6060
aagggacttt ccgctgggga ctttccaggg aggcgtggcc tgggcgggac tggggagtgg 6120
cgagccctca gatcctgcat ataagcagct gctttttgcc tgtactgggt ctctctggtt 6180
agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 6240
ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 6300
ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg 6360
tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc 6420
ttgacattgc tagcgtttac cgtcgacctc tagctagagc ttggcgtaat catggtcata 6480
gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 6540
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 6600
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 6660
acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 6720
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 6780
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 6840
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 6900
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 6960
ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 7020
taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 7080
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 7140
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 7200
aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 7260
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 7320
agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 7380
ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 7440
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 7500
tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 7560
cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 7620
aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 7680
atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 7740
cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 7800
tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 7860
atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 7920
taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 7980
tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 8040
gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 8100
cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 8160
cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 8220
gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 8280
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 8340
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 8400
ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 8460
gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 8520
aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 8580
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc 8640
gggagatcaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 8700
atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 8760
atgtatctta tcatgtctgg atcaactgga taactcaagc taaccaaaat catcccaaac 8820
ttcccacccc ataccctatt accactgcca attacctgtg gtttcattta ctctaaacct 8880
gtgattcctc tgaattattt tcattttaaa gaaattgtat ttgttaaata tgtactacaa 8940
acttagtagt ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagt 8997
<210> 3
<211> 9039
<212> DNA
<213> Artificial Sequence
<400> 3
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgatctgggc aggaagaggg cctatttccc atgattcctt 2220
catatttgca tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa 2280
acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 2340
cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 2400
tcgatttctt ggctttatat atcttgtgga aaggacgagg atccggatga agctgatgaa 2460
atgttcaaga gacatttcat cagcttcatc cttttttgaa ttctagttat taatagtaat 2520
caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 2580
taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 2640
atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 2700
ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 2760
acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 2820
ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 2880
ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 2940
ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 3000
gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 3060
taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggc 3120
ccagtccaag cacggcctga ccaaggagat gaccatgaag taccgcatgg agggctgcgt 3180
ggacggccac aagttcgtga tcaccggcga gggcatcggc taccccttca agggcaagca 3240
ggccatcaac ctgtgcgtgg tggagggcgg ccccttgccc ttcgccgagg acatcttgtc 3300
cgccgccttc atgtacggca accgcgtgtt caccgagtac ccccaggaca tcgtcgacta 3360
cttcaagaac tcctgccccg ccggctacac ctgggaccgc tccttcctgt tcgaggacgg 3420
cgccgtgtgc atctgcaacg ccgacatcac cgtgagcgtg gaggagaact gcatgtacca 3480
cgagtccaag ttctacggcg tgaacttccc cgccgacggc cccgtgatga agaagatgac 3540
cgacaactgg gagccctcct gcgagaagat catccccgtg cccaagcagg gcatcttgaa 3600
gggcgacgtg agcatgtacc tgctgctgaa ggacggtggc cgcttgcgct gccagttcga 3660
caccgtgtac aaggccaagt ccgtgccccg caagatgccc gactggcact tcatccagca 3720
caagctgacc cgcgaggacc gcagcgacgc caagaaccag aagtggcacc tgaccgagca 3780
cgccatcgcc tccggctccg ccttgcccta actcgagtaa ttctaccggg taggggaggc 3840
gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta 3900
cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc 3960
gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc 4020
cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct 4080
cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca 4140
atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg 4200
cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc 4260
attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg 4320
cctttcgacc tgcagcccaa gcttaccatg accgagtaca agcccacggt gcgcctcgcc 4380
acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 4440
gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 4500
ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 4560
gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 4620
ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 4680
ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 4740
cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 4800
gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac 4860
gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 4920
tgcatgaccc gcaagcccgg tgcctgaacg cgtctggaac aatcaacctc tggattacaa 4980
aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 5040
cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 5100
cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 5160
tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 5220
ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 5280
cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 5340
ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat 5400
tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 5460
ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 5520
tcggatctcc ctttgggccg cctccccgcc tggaattaat tctgcagtcg agacctagaa 5580
aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta 5640
gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca 5700
atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaagag gggactggaa 5760
gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa 5820
ggctacttcc ctgattagca gaactacaca ccagggccag gggtcagata tccactgacc 5880
tttggatggt gctacaagct agtaccagtt gagccagata aggtagaaga ggccaataaa 5940
ggagagaaca ccagcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga 6000
gaagtgttag agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg 6060
catccggagt acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg 6120
gactttccag ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc 6180
atataagcag ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg 6240
ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt 6300
gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc 6360
cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta 6420
tttataactt gcaaagaaat gaatatcaga gagtgagagg ccttgacatt gctagcgttt 6480
accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 6540
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 6600
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 6660
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 6720
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 6780
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 6840
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 6900
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 6960
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 7020
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 7080
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 7140
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 7200
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 7260
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 7320
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 7380
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 7440
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 7500
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 7560
acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 7620
ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 7680
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 7740
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 7800
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 7860
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 7920
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 7980
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 8040
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 8100
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 8160
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 8220
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 8280
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 8340
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 8400
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 8460
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 8520
gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 8580
ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 8640
gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc aacttgttta 8700
ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 8760
ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 8820
ggatcaactg gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta 8880
ttaccactgc caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat 8940
tttcatttta aagaaattgt atttgttaaa tatgtactac aaacttagta gtttttaaag 9000
aaattgtatt tgttaaatat gtactacaaa cttagtagt 9039
<210> 4
<211> 8148
<212> DNA
<213> Artificial Sequence
<400> 4
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag 2820
acaacgttcg aacgcgtaca agcgcggccg cacaacgtct agagaattcg attacaagga 2880
tgacgacgat aaggactata aggacgatga tgacaaggac tacaaagatg atgacgataa 2940
atgaggatcc accggatcta gataactgat cataattcta ccgggtaggg gaggcgcttt 3000
tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa 3060
gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct 3120
ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc 3180
cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc 3240
agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc 3300
agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc 3360
tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct 3420
gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt 3480
cgacctgcag cccaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg 3540
cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 3600
gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 3660
cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 3720
ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 3780
gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 3840
ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 3900
ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 3960
cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg 4020
gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 4080
gacccgcaag cccggtgcct gaccgcgtct ggaacaatca acctctggat tacaaaattt 4140
gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 4200
ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 4260
ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 4320
tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 4380
agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 4440
cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 4500
tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc 4560
gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 4620
gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 4680
tctccctttg ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc tagaaaaaca 4740
tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc 4800
acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac 4860
ttacaaggca gctgtagatc ttagccactt tttaaaagaa aagaggggac tggaagggct 4920
aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta 4980
cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg 5040
atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga 5100
gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt 5160
gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc 5220
ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt 5280
tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata 5340
agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc 5400
tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc 5460
aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt 5520
agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat 5580
aacttgcaaa gaaatgaata tcagagagtg agaggccttg acattgctag cgttttaccg 5640
tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 5700
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 5760
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 5820
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 5880
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 5940
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 6000
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 6060
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 6120
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 6180
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 6240
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 6300
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 6360
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 6420
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 6480
ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 6540
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 6600
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 6660
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 6720
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 6780
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 6840
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 6900
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 6960
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 7020
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 7080
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 7140
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 7200
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 7260
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 7320
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 7380
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 7440
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 7500
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 7560
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 7620
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 7680
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 7740
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 7800
acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcaact tgtttattgc 7860
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 7920
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat 7980
caactggata actcaagcta accaaaatca tcccaaactt cccaccccat accctattac 8040
cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg aattattttc 8100
attttaaaga aattgtattt gttaaatatg tactacaaac ttagtagt 8148
<210> 5
<211> 6899
<212> DNA
<213> Artificial Sequence
<400> 5
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 60
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 120
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 180
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 240
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 300
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 360
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 420
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 480
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 540
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 600
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 660
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 720
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 780
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 840
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 900
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 960
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 1020
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 1080
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 1140
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 1200
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 1260
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 1320
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 1380
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 1440
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 1500
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 1560
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag 1620
gattacaagg atgacgacga taaggactat aaggacgatg atgacaagga ctacaaagat 1680
gatgacgata aatgaggatc caccggatct agataactga tcataattct accgggtagg 1740
ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg 1800
gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc 1860
ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt 1920
cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac 1980
tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag 2040
cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc 2100
cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg 2160
cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc 2220
tccgggcctt tcgacctgca gcccaagctt accatgaccg agtacaagcc cacggtgcgc 2280
ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 2340
taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 2400
caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 2460
ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 2520
gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 2580
gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 2640
gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 2700
gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 2760
ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 2820
acctggtgca tgacccgcaa gcccggtgcc tgaccgcgtc tggaacaatc aacctctgga 2880
ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 2940
tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 3000
ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 3060
gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 3120
caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 3180
actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 3240
ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac 3300
ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 3360
tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 3420
gacgagtcgg atctcccttt gggccgcctc cccgcctgga attaattctg cagtcgagac 3480
ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc 3540
tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta 3600
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagagggga 3660
ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac 3720
acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt cagatatcca 3780
ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt agaagaggcc 3840
aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat ggatgacccg 3900
gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca cgtggcccga 3960
gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa gggactttcc 4020
gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg agccctcaga 4080
tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag accagatctg 4140
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 4200
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 4260
cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat 4320
tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt gacattgcta 4380
gcgttttacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 4440
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 4500
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 4560
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 4620
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4680
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4740
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4800
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4860
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4920
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4980
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 5040
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 5100
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 5160
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 5220
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 5280
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 5340
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 5400
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 5460
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 5520
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 5580
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 5640
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5700
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5760
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5820
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5880
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5940
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6000
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6060
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6120
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6180
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6240
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 6300
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 6360
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 6420
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 6480
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6540
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcaac 6600
ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 6660
aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 6720
catgtctgga tcaactggat aactcaagct aaccaaaatc atcccaaact tcccacccca 6780
taccctatta ccactgccaa ttacctgtgg tttcatttac tctaaacctg tgattcctct 6840
gaattatttt cattttaaag aaattgtatt tgttaaatat gtactacaaa cttagtagt 6899
<210> 6
<211> 1233
<212> DNA
<213> Artificial Sequence
<400> 6
atggcgacca cggccacgat ggcgacctcg ggctcggcgc gaaagcggct gctcaaagag 60
gaagacatga ctaaagtgga attcgagacc agcgaggagg tggatgtgac ccccacgttc 120
gacaccatgg gcctgcggga ggacctgctg cggggcatct acgcttacgg ttttgaaaaa 180
ccatcagcaa tccagcaacg agcaatcaag cagatcatca aagggagaga tgtcatcgca 240
cagtctcagt ccggcacagg aaaaacagcc accttcagta tctcagtcct ccagtgtttg 300
gatattcagg ttcgtgaaac tcaagctttg atcttggctc ccacaagaga gttggctgtg 360
cagatccaga aggggctgct tgctctcggt gactacatga atgtccagtg ccatgcctgc 420
attggaggca ccaatgttgg cgaggacatc aggaagctgg attacggaca gcatgttgtc 480
gcgggcactc cagggcgtgt ttttgatatg attcgtcgca gaagcctaag gacacgtgct 540
atcaaaatgt tggttttgga tgaagctgat gaaatgttga ataaaggttt caaagagcag 600
atttacgatg tatacaggta cctgcctcca gccacacagg tggttctcat cagtgccacg 660
ctgccacacg agattctgga gatgaccaac aagttcatga ccgacccaat ccgcatcttg 720
gtgaaacgtg atgaattgac tctggaaggc atcaagcaat ttttcgtggc agtggagagg 780
gaagagtgga aatttgacac tctgtgtgac ctctacgaca cactgaccat cactcaggcg 840
gtcatcttct gcaacaccaa aagaaaggtg gactggctga cggagaaaat gagggaagcc 900
aacttcactg tatcctcaat gcatggagac atgccccaga aagagcggga gtccatcatg 960
aaggagttcc ggtcgggcgc cagccgagtg cttatttcta cagatgtctg ggccaggggg 1020
ttggatgtcc ctcaggtgtc cctcatcatt aactatgatc tccctaataa cagagaattg 1080
tacatacaca gaattgggag atcaggtcga tacggccgga agggtgtggc cattaacttt 1140
gtaaagaatg acgacatccg catcctcaga gatatcgagc agtactattc cactcagatt 1200
gatgagatgc cgatgaacgt tgctgatctt atc 1233
<210> 7
<211> 9344
<212> DNA
<213> Artificial Sequence
<400> 7
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag 2820
gccaccatgg cgaccacggc cacgatggcg acctcgggct cggcgcgaaa gcggctgctc 2880
aaagaggaag acatgactaa agtggaattc gagaccagcg aggaggtgga tgtgaccccc 2940
acgttcgaca ccatgggcct gcgggaggac ctgctgcggg gcatctacgc ttacggtttt 3000
gaaaaaccat cagcaatcca gcaacgagca atcaagcaga tcatcaaagg gagagatgtc 3060
atcgcacagt ctcagtccgg cacaggaaaa acagccacct tcagtatctc agtcctccag 3120
tgtttggata ttcaggttcg tgaaactcaa gctttgatct tggctcccac aagagagttg 3180
gctgtgcaga tccagaaggg gctgcttgct ctcggtgact acatgaatgt ccagtgccat 3240
gcctgcattg gaggcaccaa tgttggcgag gacatcagga agctggatta cggacagcat 3300
gttgtcgcgg gcactccagg gcgtgttttt gatatgattc gtcgcagaag cctaaggaca 3360
cgtgctatca aaatgttggt tttggatgaa gctgatgaaa tgttgaataa aggtttcaaa 3420
gagcagattt acgatgtata caggtacctg cctccagcca cacaggtggt tctcatcagt 3480
gccacgctgc cacacgagat tctggagatg accaacaagt tcatgaccga cccaatccgc 3540
atcttggtga aacgtgatga attgactctg gaaggcatca agcaattttt cgtggcagtg 3600
gagagggaag agtggaaatt tgacactctg tgtgacctct acgacacact gaccatcact 3660
caggcggtca tcttctgcaa caccaaaaga aaggtggact ggctgacgga gaaaatgagg 3720
gaagccaact tcactgtatc ctcaatgcat ggagacatgc cccagaaaga gcgggagtcc 3780
atcatgaagg agttccggtc gggcgccagc cgagtgctta tttctacaga tgtctgggcc 3840
agggggttgg atgtccctca ggtgtccctc atcattaact atgatctccc taataacaga 3900
gaattgtaca tacacagaat tgggagatca ggtcgatacg gccggaaggg tgtggccatt 3960
aactttgtaa agaatgacga catccgcatc ctcagagata tcgagcagta ctattccact 4020
cagattgatg agatgccgat gaacgttgct gatcttatcg aattcgatta caaggatgac 4080
gacgataagg actataagga cgatgatgac aaggactaca aagatgatga cgataaatga 4140
ggatccaccg gatctagata actgatcata attctaccgg gtaggggagg cgcttttccc 4200
aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 4260
cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 4320
tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 4380
gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 4440
ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 4500
ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 4560
gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 4620
gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac 4680
ctgcagccca agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 4740
gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 4800
cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 4860
acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 4920
gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 4980
atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 5040
ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 5100
cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 5160
ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 5220
ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 5280
cgcaagcccg gtgcctgacc gcgtctggaa caatcaacct ctggattaca aaatttgtga 5340
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 5400
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 5460
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 5520
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 5580
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 5640
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 5700
ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg 5760
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 5820
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 5880
cctttgggcc gcctccccgc ctggaattaa ttctgcagtc gagacctaga aaaacatgga 5940
gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa 6000
gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac 6060
aaggcagctg tagatcttag ccacttttta aaagaaaaga ggggactgga agggctaatt 6120
cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc 6180
cctgattagc agaactacac accagggcca ggggtcagat atccactgac ctttggatgg 6240
tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac 6300
accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta 6360
gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag 6420
tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca 6480
gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca 6540
gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc 6600
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 6660
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 6720
agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact 6780
tgcaaagaaa tgaatatcag agagtgagag gccttgacat tgctagcgtt ttaccgtcga 6840
cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 6900
cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 6960
aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 7020
acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 7080
ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 7140
gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 7200
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 7260
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 7320
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 7380
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 7440
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 7500
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 7560
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 7620
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 7680
agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 7740
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 7800
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 7860
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 7920
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 7980
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 8040
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 8100
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 8160
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 8220
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 8280
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 8340
ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 8400
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 8460
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 8520
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 8580
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 8640
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 8700
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 8760
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 8820
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 8880
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 8940
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 9000
ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcaacttgtt tattgcagct 9060
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 9120
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac 9180
tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact 9240
gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt 9300
taaagaaatt gtatttgtta aatatgtact acaaacttag tagt 9344
Claims (10)
1. A construction method of a comprehensive regulation and control network for regulating EIF4A3 expression to influence apoptosis, migration and invasion of liver cancer cells is characterized by comprising the following steps:
(1) data processing: downloading liver cancer data from a cancer genome atlas database, and preprocessing the liver cancer data; the liver cancer data comprises a gene expression profile of RNA _ Seq, a methylated gene expression profile, SNP data and CNV data based on the Affymetrix human genome U133a array platform;
(2) differential expression analysis: performing differential expression analysis on the condition that the gene expression profiles of RNA _ Seq in liver cancer data are respectively in an EIF4A3 low-expression sample and an EIF4A3 high-expression sample to find genes related to EIF4A3 expression; exploring multiple groups of scientific conditions of EIF4A3 in the liver cancer sample as assistance;
(3) weighted gene co-expression network analysis: extracting low-expression and high-expression mRNAs in the EIF4A3 low-expression sample and the EIF4A3 high-expression sample respectively, and executing WGCNA to search a module related to liver cancer;
(4) constructing a comprehensive regulation and control network: and performing functional enrichment analysis on the modular gene with the maximum phenotype correlation, and constructing a comprehensive regulation network for regulating the expression of EIF4A3 so as to influence the apoptosis, migration and invasion of the liver cancer cells.
2. The method for constructing the comprehensive regulation and control network for regulating the expression of EIF4A3 to influence the apoptosis, migration and invasion of the hepatoma cells according to claim 1, wherein the data processing comprises the following steps:
in the liver cancer data, 375 liver cancer samples and 391 control samples exist in CNV data, gene annotation is carried out on the CNV data, and copy number sites are converted into gene symbols for subsequent analysis;
380 liver cancer samples and 50 control samples are adopted in a liver cancer methylation gene expression profile, methylation array analysis is carried out by using an R software package ChAMP, data are filtered, then the data are standardized, and finally differential methylation is calculated;
the total 387 samples of SNP data comprise 345 liver cancer samples and 42 control samples, the Pvalue value of the SNP locus is calculated by using MATLAB2018b software, and the significance of the gene mutation is checked;
there were 387 samples in total in the gene expression profile data of RNA-seq, including 345 liver cancer samples and 42 control samples.
3. The method for constructing the comprehensive regulation network for regulating the expression of EIF4A3 to influence the apoptosis, migration and invasion of the hepatoma cells according to claim 1, wherein the differential expression analysis comprises the following steps:
performing differential expression analysis on the normal sample compared with the liver cancer sample on the CNV data of the liver cancer by using perl language;
performing differential expression analysis on RNA expression profile data by using a limma package of an R language;
extracting mRNA and lncRNA expression profiles based on a background set Homo _ sapiens.GRCh38.98.chr.gtf, and performing differential expression analysis on the liver cancer sample compared with a healthy control; the normalizeBetwenArrays function in the limma package was used to normalize the expression profiles; when a certain RNA corresponds to a plurality of probes, the average expression value of the probes is selected as the expression value of the RNA.
4. The method for constructing the comprehensive regulation network for regulating the expression of EIF4A3 to influence the apoptosis, migration and invasion of liver cancer cells according to claim 1, wherein the weighted gene co-expression network analysis comprises the following steps:
performing hierarchical clustering analysis on the extracted 7574 mRNAs which are significantly related to EIF4A3 and are down-regulated by using an hclust function; screening a Soft Threshold power value in the module construction process by using a pick Soft Threshold function; testing the average connectivity and the independence of different modules by using the candidate power; the WGCNA R packet is used for constructing a co-expression network; the minimum module size is set to 30 and each module is assigned a unique color; based on the string database, a protein interaction network is constructed, and only the interaction with the joint score > 500 is reserved.
5. The method for constructing the comprehensive regulation network for regulating the expression of EIF4A3 to influence the apoptosis, migration and invasion of liver cancer cells according to claim 1, wherein the weighted gene co-expression network analysis comprises the following steps: the ClusterONE plug-in of the cytoscape software is used for conducting modular analysis on the PPI network.
6. The method for constructing the comprehensive regulation network for regulating the expression of EIF4A3 to influence the apoptosis, migration and invasion of the hepatoma cells according to claim 1, wherein the method for constructing the comprehensive regulation network comprises the following steps:
performing functional enrichment analysis on 1619 mRNAs with the maximum expression correlation with EIF4A3 by using a clusterProfiler package to explore functions and signal paths involved by module genes;
the gene set enrichment analysis is applied to exploring the functions and the channels of the enrichment in liver cancer samples and EIF4A3 high-expression samples;
an lncRNA capable of regulating EIF4A3 is extracted based on an RNA Inter database, and a comprehensive regulation network which is mediated by lncRNA targeting EIF4A3 and influences apoptosis, migration and invasion of liver cancer cells is constructed by combining a passage obtained in a function enrichment process.
7. An application of regulating EIF4A3 expression to regulate apoptosis, migration and invasion capacity of liver cancer cells is characterized by comprising the following steps:
step A: constructing a stably infected cell line with the EIF4A3 gene knocked out;
and B: constructing a cell line stably infected with the over-expression EIF4A3 gene;
and C: and detecting the influence of interfering EIF4A3 gene expression on the apoptosis, migration and invasion capacity of the liver cancer cells.
8. The use of the modulation of EIF4a3 expression to modulate the ability of liver cancer cells to undergo apoptosis, migration and invasion as claimed in claim 7, wherein the process of step a is as follows: designing shRNA according to an EIF4A3 sequence, constructing an EIF4A3 interference lentivirus plasmid and packaging lentivirus; preparing a recombinant EIF4A3-shRNA lentivirus; and infecting a lentiviral vector for knocking out the EIF4A3 gene in 97H cells to obtain a stably infected knocked-out EIF4A3 cell line.
9. The use of the modulation of EIF4a3 expression to modulate the ability of liver cancer cells to undergo apoptosis, migration and invasion as claimed in claim 7, wherein the process of step B is as follows: constructing an expression plasmid marked by EIF4A3 by using a pMSCV-IRES-GFP vector, and transfecting 293T cells with the plasmid; the recombinant retroviruses of the cells are used for multiple infection of the liver cancer cell line HepG2, and the cells are infected by a lentiviral vector over-expressing EIF4A3 gene, so as to obtain a cell line stably infected with the over-expressing EIF4A3 gene.
10. The use of the modulation of EIF4a3 expression to modulate the ability of liver cancer cells to undergo apoptosis, migration and invasion as claimed in claim 7, wherein the process of step C is as follows: respectively exploring the influence of knocking out or over-expressing EIF4A3 on the apoptosis of the liver cancer cell by a cell line stably infected with an over-expressed EIF4A3 gene and a knocked-out EIF4A3 cell line stably infected with the gene through an apoptosis test; respectively carrying out Transwell and Matrigel-Transwell experiments on a cell line which is stably infected and overexpresses EIF4A3 gene and a stably infected knockout EIF4A3 cell line to explore the influence of knocking out or overexpressing EIF4A3 on migration and invasion of the liver cancer cells; the effect of knocking out EIF4A3 on liver cancer is observed at the animal level.
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