249 results about "Synexpression" patented technology

The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 17th site, the 40th site and the 64th site of an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. Compared with a wild type enzyme, the carbonyl reductase mutant has high activity and high stereoselectivity, the enzyme activity can reach more than two times that of the wild type enzyme, the catalytic efficiency on a substrate precursor ketone is remarkably improved, the yield of a statin drug intermediate and chiral alcohol synthesized by an enzyme method of the carbonyl reductase mutant is remarkably improved, and the yield of a rosuvastatin intermediate (3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R) is remarkably improved. The highest yields of the (3R, 5R)-6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester and the atorvastatin intermediate (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester reach 99% and 97.6% respectively, e.e. Is larger than 99%, and the method has high industrial application value.

The invention discloses a thermostable conjugated polyketoreductase mutant, which is obtained by performing single-point mutation or multi-point combined mutation on the 25th site, the 92th site, the 214th site or the 256th site of an amino acid sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the thermostable conjugated polyketoreductase mutant. The initial enzyme activity of the mutant is high, and the residual enzyme activity is gt after incubation at 50 DEG C; the Tm value can be increased by more than 9 DEG C compared with that of an original strain, the 50 DEG C half-life period t1 / 2 is increased by more than 377.8 times, and the thermal stability is obviously improved. The invention also provides a coding gene, a recombinant vector containing the coding gene and a co-expression engineering bacterium. The thermostable conjugated polyketoreductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase are used for constructing a three-enzyme co-expression engineering bacterium as a biocatalyst, racemization pantolactone is catalyzed to synthesize DPL, for a 1M substrate, the yield of the DPL reaches 97.5% within 36 hours, KPL hydrolysis is avoided, the reaction time is shorter, the product concentration is higher, and the thermostable conjugated polyketoreductase mutant has great market application value.

The invention discloses a key gene identification method related to tobacco nitrogen response, which comprises the following steps: S1, setting four nitrogen fertilizer gradient treatments on the basis of same phosphorus and potassium fertilization by adopting a field experiment of a completely random block; s2, randomly taking the 6th to 8th leaves of the five plants in each area, and dividing a sample into two parts: quickly freezing one part with liquid nitrogen, and storing at-80 DEG C for RNA (Ribonucleic Acid) extraction and transcriptome sequencing; one part is used for measuring the nitrogen content, and after baking and drying treatment, a KjeltecTM8100 automatic nitrogen analyzer is used for measuring; s3, nitrogen content determination: determining the nitrogen content in the treated sample by using an automatic nitrogen analyzer KjeltecTM8100; according to the method, high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) are combined, so that not only can gene expression maps of flue-cured tobacco leaves treated by different nitrogen fertilizers be comprehensively captured, but also gene modules with similar expression modes can be mined from a global perspective.

The invention discloses an inference method and an inference system of a gene regulatory network. The inference method comprises the following steps: acquiring transcriptome data and prior gene network data of a single cell; extracting a gene sub-network related to the transcriptome data from the prior gene network data; obtaining a first view and a second view for the gene sub-network according to a first random deletion strategy and a second random deletion strategy; providing the first view and the second view to a neural network model, and performing comparative learning based on the neural network model to obtain incoming features and outgoing features of each node in the gene sub-network; and constructing a regulation score matrix based on the incoming features and the outgoing features, wherein the regulation score matrix displays the regulation association degree between genes in the transcriptome data of the single cell. According to the technical scheme, the direct causal relationship and the indirect association relationship can be effectively distinguished, so that the gene co-expression network more accurately reflects the real regulation relationship.

The invention discloses a recombinant plasmid, a recombinant strain and a method for synthesizing rebaudioside M by a one-step method, and belongs to the technical field of biological catalytic synthesis. The recombinant plasmid contains a coding gene of a glycosyl transferase UGT91C1 mutant, a coding gene of a glycosyl transferase UGT76G1 mutant and a coding gene of sucrose synthase. The mutant of the glycosyl transferase UGT91C1 is 2-12E, and the coding gene sequence of the glycosyl transferase UGT91C1 is as shown in SEQ ID NO. 1; the glycosyl transferase UGT76G1 mutant is 101H10, and the coding gene sequence of the glycosyl transferase UGT76G1 mutant is as shown in SEQ ID NO. 2; the sequence of the coding gene of the sucrose synthase is as shown in SEQ ID NO. 3. The rebaudioside M is synthesized by using a three-enzyme co-expression recombinant strain containing the plasmid through a one-step method, so that the loss of fermentation materials can be reduced, the utilization efficiency of equipment is improved, the energy consumption is reduced, and the large-scale production of the rebaudioside M is facilitated.

A method for recombinantly expressing feline serum albumin. A method for constructing an engineered strain for recombinantly expressing a feline serum albumin, wherein the method comprises: co-expressing a feline serum albumin, a protein disulfide isomerase, and regulatory factor HAC1 and / or vitreoscilla hemoglobin in a receptor yeast. The obtained strain is an engineered strain capable of expressing a recombinant feline serum albumin. The engineered strain can efficiently express rFSA. Compared with the direct and individual expression of rFSA, the expression level of rFSA in the co-expression strain is significantly increased, and the highest expression level can reach about 16 g / L. This lays a foundation for large-scale industrial production of rFSA.

The invention provides an application of a TGBp1 gene, or a TGBp1 gene coding protein, or a recombinant vector or host bacteria containing a TGBp1 gene coding region in down-regulation of the MeGRXC3 expression level. Researches find that TGBp1 and MeGRXC3 interact with each other, so that the growth ability of yeast in an SD / -Leu / -Trp / -His / -Ade auxotroph culture medium can be improved, the expression of MeGRXC3 can be down-regulated, the active oxygen content of tobacco leaves for co-expression of TGBp1 and MeGRXC3 is improved, and the growth ability of the yeast in the SD / -Leu / -Trp / -His / -Ade auxotroph culture medium is improved. Research results lay a theoretical foundation for breeding of common cassava mosaic virus disease resistance.

The invention belongs to the related technical field of gene regulation and control networks, and provides a gene regulation and control network construction method, system, equipment and medium in order to overcome the defects of the existing gene regulation and control network in the aspects of interpretability, authenticity and the like. Based on a graph neural network, efficient gene embedding representation learning is realized to capture a global dependency relationship; and then based on different types of regulation and control relationships, analyzing a linear causal relationship and a nonlinear causal relationship, integrating the two channels to obtain a confidence coefficient matrix of the gene, and further constructing a gene regulation and control network. The gene regulatory network constructed by the invention can reveal the regulatory relationship between genes, the interpretability, authenticity and credibility of the gene regulatory network are improved, and a more accurate and reliable tool is provided for analyzing a complex biological system subsequently.

The invention relates to the field of microbial metabolism engineering, and discloses a method for increasing the yield of 2 '-fucosyllactose (2'-FL) through the synergistic effect of various alpha-1, 2-fucosyltransferases. According to the method, two or more alpha-1, 2-fucosyltransferases with different sources are co-expressed in microbial cells, and the complementary enzymatic characteristics of the alpha-1, 2-fucosyltransferases are utilized, so that the synthesis efficiency of the 2 '-FL is remarkably improved, and the yield of the 2'-FL is effectively improved. The microbial cell is escherichia coli C43 (DE3) (E. coli C43 lacZ waaF with lacZ and waaF genes knocked out, the serial number is SL), a pETDuet-1 plasmid is constructed, and genes manB, manC, gmd, wcaG and zwf are overexpressed on a pETDuet-1 vector, so that the microbial cell can be used for preparing the microbial cell. In a 5L fermentation tank, after the final strain SL07 is used for final fermentation for 68 hours, the yield of 2 '-FL reaches 148g / L, and the production intensity reaches 2.18 g / L / h. The embodiment shows that the engineering strain for co-expressing the alpha-1, 2-fucosyltransferase from different sources can obviously improve the yield of the 2 '-FL.

The invention discloses an aspergillus niger recombinant strain for degrading vomitoxin and application thereof, and belongs to the field of gene engineering. In order to solve the problem that a method for efficiently degrading vomitoxin is lacked in the prior art, the invention provides an aspergillus niger recombinant strain for degrading vomitoxin, the aspergillus niger strain for co-expressing FsTRI12 is taken as a recipient bacterium, a vomitoxin acetyltransferase gene segment is selected, an intracellular gene expression vector is constructed, and a homozygous aspergillus niger recombinant strain is screened. The aspergillus niger recombinant strain provided by the invention can be applied to degradation of vomitoxin, provides a new choice for effectively solving the vomitoxin pollution problem in cereal raw materials and feeds, reducing grain loss and guaranteeing food and feed safety, and has potential industrial application prospects.

The invention provides a gene SbERF71 capable of improving waterlogging tolerance of sorghum, protein, application and a breeding method, and belongs to the technical field of crop breeding. On the basis of a sorghum plant with waterlogging tolerance, it is determined that SbERF71 is a key gene affecting the waterlogging tolerance of sorghum through RNA extraction and library building, RNA-Seg data analysis, weighted gene co-expression network analysis (WGCNA) and other methods. The SbERF71 gene is related to gene expression of various stress reaction pathways (such as energy metabolism and aero-tissue formation) in sorghum, is a central regulatory factor of sorghum waterlogging, and is beneficial to reaction of sorghum to flooding stress, expression height up-regulation of sorghum in a flooding stress environment and enhancement of flooding resistance of sorghum, so that sorghum can adapt to waterlogging stress. The invention reveals that sorghum can adapt to molecular and physiological mechanisms of waterlogging stress, provides a new direction for breeding of sorghum waterlogging-resistant varieties, and makes a contribution to food safety in waterlogging agricultural areas.

The invention discloses a prognosis evaluation method and system for diffuse large B-cell lymphoma. According to the method, firstly, a gene module closely related to lipid metabolism is screened from DLBCL transcriptome data through weighted gene co-expression network analysis (WGCNA), and then eight key prognosis genes including FNDC1, IL22RA2, C15orf48, OMD, MFAP2, BC017398, CXCL6 and TNFAIP6 are identified from the module by adopting multi-step regression analysis (single factor Cox, LASSO and multi-factor Cox). A risk scoring model is constructed based on the expression levels and regression coefficients of the genes, and DLBCL patients can be divided into a high-risk group and a low-risk group with significant survival differences. The risk score and the clinical pathological factors are further integrated to construct a column graph, and individualized survival probability prediction can be achieved. The invention further provides a corresponding prognosis evaluation system, electronic equipment and a storage medium. An independent data set verifies that the prognosis model has excellent prediction performance and clinical practical value.

The invention discloses a preparation method of recombinant mussel mucin rich in dopa, and belongs to the technical field of genetic engineering. When the recombinant mussel mucoprotein is co-expressed and modified, a certain amount of modifier is fed in the induction process according to the growth of thalli and the expression condition of protein, so that on one hand, the component concentration of a co-expression modification system is controlled, and a favorable environment is provided for the recombinant tyrosinase to oxidize tyrosine into DOPA; on the other hand, an environment is created for the synthesized recombinant mussel mucin containing dopa, the situation that dopa residues are oxidized into unnecessary substances such as dopaquinone and melanin due to long co-expression modification time is avoided, glutamic acid with a certain concentration in a modification auxiliary system serves as energy supply to promote modification, and the dopa oxidation rate and modification efficiency are improved.

The invention provides a pathogenic driver gene prediction method based on biological network fusion and related equipment. The method comprises the following steps: acquiring data of various driver genes for training; constructing an initial gene relationship map based on protein interaction, gene sequence similarity, KEGG pathway co-occurrence, a gene co-expression mode and semantic similarity of a gene ontology, and embedding various human driven gene data for training into each node in the initial gene relationship map to obtain various gene relationship maps; performing dynamic adjustment on each gene relationship map through edge discarding, feature discarding and difficult sample recognition enhancement to obtain an adjusted gene relationship map for training the constructed pathogenic driving gene prediction model to obtain a trained pathogenic driving gene prediction model; inputting the target driver gene data into the trained pathogenic driver gene prediction model for prediction to obtain a prediction result; and the accuracy and robustness of pathogenic driver gene prediction are improved.

The invention provides an application of a CP gene, or a protein encoded by the CP gene, or a recombinant vector or host bacteria containing a CP gene coding region in down-regulation of the MeGRXC3 expression level. Researches find that CP and MeGRXC3 interact with each other, so that the growth ability of yeast in an SD / -Leu / -Trp / -His / -Ade auxotrophic culture medium can be improved, cell autophagy can be activated, the expression of MeGRXC3 can be down-regulated, and the active oxygen content of plant tissues co-expressed with MeGRXC3 can be increased. Research results lay a theoretical foundation for breeding of common cassava mosaic virus disease resistance.

The invention discloses a construction method of immune-related key gene prognosis indexes of metastatic melanoma and related equipment, and belongs to the field of bioinformatics, the method comprises the following steps: obtaining an immune-related differential expression gene list, and constructing a weighted gene co-expression network to divide gene modules, two gene modules with the highest correlation degree with the matrix score and the immune score are screened out, and gene intersection is taken to recognize immune related key genes; and determining immune-related key genes significantly related to the lifetime, and sequentially screening through univariate Cox regression and LASSO regression to obtain genes of which the regression coefficients are not zero so as to construct and obtain immune-related key gene prognosis indexes of metastatic melanoma. The method can solve the problems that in the prior art, when a prognosis index is constructed, an immune-related gene pair is only determined by an LASSO regression coefficient, key disease treatment genes are difficult to recognize, too many immune-related genes are contained, and clinical application convenience is low.

The present disclosure discloses a Functional Genomic Imaging method. Genes are filtered based on the expression levels and other information first to improve the ratio of effective genes, and then a correlation matrix of gene co-expression is generated based on the filtered gene data to obtain a co-expression network; data conversion is performed based on the correlation matrix of gene co-expression to obtain a distance matrix; and finally, the co-expression network is subjected to dimensionality reduction and cluster analysis, thus obtaining the visualized gene expression according to the analysis result. The Functional Genomic Imaging method provided by the present disclosure achieves a better visualization effect of the genetic co-expression network and can obtain a co-expressed gene cluster more effectively.

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

The invention relates to the technical field of bioinformatics, in particular to an esophageal cancer RNA-protein interaction space prediction method and device based on a graph neural network, and can solve the problem that it is difficult to accurately distinguish the difference of cancer tissue and normal tissue on RPI in a traditional method to a certain extent. The method comprises the steps of obtaining multi-stage space transcriptome data related to esophageal cancer, and performing preprocessing to obtain a multi-modal feature tensor and a canceration stage tag; using the multi-modal feature tensor and the canceration stage tag, using RNA and protein as nodes, defining a static edge based on sequence complementarity and structure matching degree, defining a dynamic edge based on spatial co-expression correlation, and constructing a space-time dynamic graph updated along with the canceration stage; the space-time dynamic graph serves as input, interaction existence, site coordinates and binding energy are predicted through space-time double-branch coding and attention mechanism fusion features, and an interaction prediction result is output.

The invention discloses an adversity high-photosynthetic-efficiency transcription factor screening method based on deep learning, and relates to the technical field of biological information analys.The method comprises the steps that rice multi-modal stress response data is obtained and preprocessed, and preprocessed gene expression data is obtained; carrying out differential expression gene screening and co-expression network analysis on the preprocessed gene expression data, extracting multi-modal features, and fusing the multi-modal features to generate a multi-modal input feature matrix; constructing a double-layer deep learning model, training the double-layer deep learning model by using the multi-modal input feature matrix, and respectively outputting a regulation and control relationship matrix of transcription factors and target genes and a regulation and control relationship matrix of transcription factors and target pathways; and according to an output result, calculating a comprehensive score of each transcription factor through a multi-dimensional scoring system, and screening out the stress high-photosynthetic-efficiency transcription factor according to a predetermined screening standard.

The invention belongs to the technical field of biology, and discloses a method for screening plant interaction protein by combining big data with artificial intelligence and verifying protein interaction by using yeast two-hybrid. According to the method, AI is combined with big data, and co-expression protein of family protein is obtained through big data screening and analysis; aI interaction prediction and molecular docking are carried out on the proteins, and interaction information is further analyzed and extracted. And further analyzing the condition of the screened interaction protein by using a yeast two-hybridization or fluorescence co-localization experiment. According to the method, aiming at the characteristics of complex genomes and abundant sequencing data of species such as wheat, public sequencing data is creatively utilized to carry out an interaction protein screening process of subgenomes. The problems of long protein screening period, high cost, large workload, false positive protein screening, no co-expression and the like are solved. The interaction relationship between important gene families can be quickly and conveniently obtained, the interaction structural domain can be accurately and clearly found, and technical support is provided for research on analysis of a wheat key gene functional mechanism.

The present application relates to a kind of sulfide monooxygenase mutant and its application in esomeprazole synthesis.The sulfide monooxygenase mutant, its encoding gene, the preparation method of the recombinant expression vector containing the gene sequence, co-expression recombinant vector and recombinant expression transformant, and the application of the recombinant sulfide monooxygenase mutant catalyst in esomeprazole synthesis are specifically disclosed.Compared with other sulfide monooxygenases, the sulfide monooxygenase mutant obtained by the present application can efficiently utilize the coenzyme NADH with lower cost and higher stability to catalyze the asymmetric oxidation reaction of omeprazole sulfide, prepare esomeprazole, has the advantages of high substrate concentration, mild reaction condition, environment-friendly, simple operation, high yield, low production cost and good industrial application prospect.

The invention discloses a recombinant 2.1 d subtype swine fever E2 protein, a subunit vaccine and application of the subunit vaccine, and belongs to the technical field of genetic engineering. According to the 2.1 d subtype swine fever E2 protein, 344-375 amino acids of the 2.1 d subtype swine fever E2 protein are removed, and 1-343 amino acids of the 2.1 d subtype swine fever E2 protein are reserved, so that the most important antigenic epitope with a protective effect of the 2.1 d subtype swine fever E2 protein can be reserved, the space structure of the recombinant 2.1 d subtype swine fever E2 protein is basically not influenced, and the immunogenicity of the 2.1 d subtype swine fever E2 protein can be reserved to the greatest extent; in addition, the recombinant 2.1 d subtype swine fever E2 protein can be efficiently expressed in a prokaryotic expression system; furthermore, the soluble expression level of the recombinant 2.1 d subtype swine fever E2 protein can be remarkably improved through co-expression of the molecular chaperone, and after the recombinant 2.1 d subtype swine fever E2 protein is prepared into a subunit vaccine, the subunit vaccine has a good protection effect on 2.1 d subtype swine fever virus infection.

The invention relates to an expression cassette and a method for producing humanized triple helix collagen by using the expression cassette, and belongs to the technical field of genetic engineering. The expression cassette sequentially comprises a promoter sequence, a fusion gene and a terminator sequence from upstream to downstream, the fusion gene comprises nucleotide fragments A, B and C, each fragment comprises an immutable region and a variable region, and the variable region is selected from I, II, III, IV and XVII type human collagen. A kex2 protease recognition site KR is introduced into an expression box, so that an expression product can be subjected to trisection cutting, and three equal-length peptide chains with a triple-helix basic structure are formed. The invention also provides a recombinant humanized collagen with a self-assembled triple helix structure and a production method thereof, and the method comprises the following steps: preparing a recombinant expression vector containing the expression cassette and a proline hydroxylase expression vector, and transferring the two expression vectors into competent cells for co-expression. The method can be used for producing the recombinant humanized collagen with the self-assembled triple-helix structure, and has a good application prospect.

The invention belongs to the technical field of biological detection, and discloses a screening method of respiratory tract infection related biomarkers based on metatranscriptomics. Respiratory tract infection samples with different clinical phenotypes are subjected to metatranscriptome sequencing, data quality control, comparison and transcript quantification, pathogen and host information is reserved, then genes which are stably expressed or remarkably changed in different groups are identified by combining differential expression analysis and co-expression analysis, and potential biomarkers are obtained. And taking an intersection gene of three machine learning algorithms including an LASSO algorithm, a random forest model and an SVM model to obtain the biomarker. The invention provides a biomarker screening method for rapid and accurate identification of respiratory tract infection.

The application discloses application of SbWOX gene and SbBBM gene in corn breeding and a corn genetic transformation method, and relates to a bioengineering technology.The application provides a nucleic acid molecule with co-expression of SbWOX protein gene and SbBBM protein gene, and corresponding gene co-expression vectors and microbial transformants.The application also provides application of the co-expression of the SbWOX gene and the SbBBM gene in the corn genetic transformation method.The application obtains corresponding genes and mutant proteins based on the SbWOX gene and the SbBBM gene of sorghum, the two genes are co-expressed in the corn genetic transformation process, even without adding plant growth hormones in the antibiotic screening medium in the tissue culture process, the plant genetic transformation process can be normally completed, and the transformation efficiency and the regeneration efficiency of the corn can be remarkably improved, the transformation rate reaches 10.42%, and the differentiation rate reaches 32.89%.

The application provides a construction of a fluorescent protein labeled microtubulin and a microtubule binding protein universal bivalent carrier, and the process is as follows: a first stage is to construct a GFP-alpha tubulin carrier, and a second stage is to construct a GFP-alpha tubulin-mCherry universal bivalent co-expression carrier; in the second stage, 35S, mCherry and NOS sequences are inserted into a KpnI enzyme cutting site of a multiple cloning site of the GFP-alpha tubulin carrier constructed in the first stage, and a single nucleic acid enzyme cutting site is inserted into the 5' end and the 3' end of the mCherry sequence, respectively, and the single nucleic acid enzyme cutting site is XbaI, KpnI / Acc65I and AscI sequences, respectively, so as to obtain the universal bivalent carrier. The universal bivalent carrier provided by the application can express two target genes simultaneously, has the fluorescent signals of GFP and mCherry, can quickly and accurately identify a transgenic plant, is convenient for positive seedling screening, can be used for a tobacco transient expression experiment, can shorten an experimental period, and saves time and effort.

The invention relates to the technical field of biology, in particular to a gene co-expression network-based sepsis marker screening method and application. The screening method comprises the following steps: screening differential expression genes; performing hierarchical clustering analysis on each differential expression gene to combine each differential expression gene into a plurality of target gene modules; and screening out hub genes from each target gene module. According to the method, gene expression data and clinical feature data are combined, the accuracy of screening sepsis marker genes is improved, possible abnormal genes irrelevant to the pathophysiological process of sepsis are avoided, gene modules are divided more finely through primary clustering analysis and secondary clustering analysis, GO function analysis is combined, and the accuracy of screening the sepsis marker genes is improved. According to an analysis result, selection of a GO term subset is optimized, and a step length set by primary clustering is corrected, so that the fine granularity and accuracy of analysis are improved, division of gene modules is optimized, and more refined treatment and screening of markers of sepsis are realized.

The invention discloses an AAV2 recombinant gene system for double expression of sFLT-1 and shVEGF as well as a preparation method and application of the AAV2 recombinant gene system, and co-expression of the sFLT-1 and the shVEGF is realized. A target gene (sFLT-1: NM001159920.2, shVEGF: NM001025366.3) is cloned to a pAAV-CMV vector through BamHI, HindIII, EcoRI and MluI sites, a pAAV-CMV-sFLT-1-shVEGF plasmid is constructed, and the plasmid is subjected to PCR (Polymerase Chain Reaction), enzyme digestion and sequencing verification, so that the recombinant plasmid is obtained. And then, co-transfecting HEK293 cells with pAAV-RC2 and pHelper plasmids to produce rAAV particles, carrying out gradient ultracentrifugation purification on the rAAV particles by virtue of iodo-pentose, and storing the rAAV particles at the temperature of 80 DEG C below zero. The AAV2 recombinant gene system has the following advantages that the sFLT-1 and the shVEGF are stably expressed, the immunoreactivity is low, and the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF plays a role for a long time, so that the AAV2 recombinant gene system for double expression of the sFLT-1 and the shVEGF can be applied to various neovascularization diseases.