CN112746076B

CN112746076B - Codon-optimized COL7A1 gene, slow virus and application

Info

Publication number: CN112746076B
Application number: CN202011584157.5A
Authority: CN
Inventors: 董文吉; 张艳君; 刘子瑾; 程谟斌; 赵忠亮
Original assignee: Zhongji Zhiyao Nanjing Biotechnology Co ltd
Current assignee: Zhongji Zhiyao Nanjing Biotechnology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2023-05-12
Anticipated expiration: 2040-12-28
Also published as: CN112746076A

Abstract

The invention relates to a codon-optimized COL7A1 gene sequence, which is a nucleotide sequence with at least 80% homology with the sequence shown in SEQ ID NO. 1. The Western Blot detection results show that: the expression level of the codon optimized COL7A1 gene is obviously higher than that of the wild type COL7A1 gene. After the human epidermal stem cells are infected by the slow virus carrying the codon optimized COL7A1 gene, the slow virus can be successfully cultured in vitro to form an epidermal sheet. Therefore, lentiviruses carrying the codon optimized COL7A1 gene can be used as RDEB gene therapy drugs for establishing RDEB clinical therapy products that bind to the advantages of both LV and epidermal stem cells.

Description

Codon-optimized COL7A1 gene, slow virus and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a codon-optimized COL7A1 gene, slow virus and application.

Background

Human skin is composed of epidermis (epidermis), dermis (dermis), subcutaneous tissue and skin appendages. The basal membrane strip (basement membrane zone, BMZ) is located between the epidermis and the dermis, and is a connection structure between the epidermis and the dermis. Hereditary epidermolysis bullosa (epidermolysis bullosa, EB) is a monogenic genetic disorder characterized by the appearance of blisters and bloodblisters between the epidermis and dermis due to structural abnormalities of BMZ, and can be divided into four major categories: epidermolysis bullosa simplex (epidermolysis bullosa simplex, EBS); junction epidermolysis bullosa (junctional epidermolysis bullosa, JEB); malnutrition epidermolysis bullosa (dystrophic epidermolysis bullosa, DEB); kindler syndrome. Epidemiological surveys of different countries and regions internationally show that the prevalence of EB is 2-5 ten thousandths, and thus the estimated prevalence of China is about 3-7 ten thousand, and the global prevalence is probably 20-40 ten thousand, wherein more than 40% of the epidermolysis bullosa (recessive dystrophic epidermolysis bullosa, RDEB) is recessive dystrophy. Patient with RDEB has a large difference in clinical manifestations, including only involving the skin of hands and feet to damage the visceral mucosa and cornea, and even endangering patient lives. No effective treatment has been available so far, and symptomatic and supportive treatment is generally adopted. Cell therapy, protein replacement therapy and gene therapy are all being tried. Among them, gene therapy based on epidermal stem cells (epidermal stem cells, epSCs) is expected to be an effective therapeutic measure for EB. International gene therapy clinical trials of EB focused mainly on JEB and RDEB.

RDEB (recessive dystrophy) is caused by a mutation in the type VII collagen COL7A1 gene. COL7A1 collagen is the main component of anchor fibrils (anchoring fibrils, AFs), which play a vital role in maintaining the connection between epidermis and dermis, and therefore, mutation of the COL7A1 gene results in a loss of structural homeostasis between epidermis and dermis. The human COL7A1 locus is 32kb in length, is located on chromosome three 3p21, is composed of 118 exons, and mRNA transcripts are approximately 8.9kb, encoding a 2944 amino acid protein. At present, more than 700 mutations of COL7A1 genes are reported, most of the mutations are inherited in a recessive manner, and the mutations are expressed in a dominant manner.

Epidermal stem cells (epidermal stem cells, epSCs) are located in the basal layer of the epidermis and are responsible for maintaining the periodic keratinization process of the epidermis. The in vitro culture expansion and cloning analysis method of the epidermal stem cells is established by Howard Green of Harvard medical college and the last century of the team thereof, and is used for evaluating the in vitro dryness (stem) and the growth potential of EpSCs. EpSCs are capable of reconstructing intact epidermis in vivo and appear as a fully cloned (holoclone) growth pattern in vitro. Infant epidermal cells are mainly EpSCs, and are completely cloned in vitro, the proportion of the EpSCs of the elderly is very low, and the infant epidermal cells are partially cloned (meroclone) and finally cloned (paraclone) in vitro. Single epidermal stem cells can satisfy the epidermal reconstruction of patients with large-area burn by in-vitro amplified daughter cells, and have shown strong effects in gene therapy of single-gene genetic diseases such as EB which involve EpSCs.

RDEB can be treated in an in-vivo (ex vivo) mode, namely, epSCs of a patient are obtained, the in-vitro amplification is carried out, the COL7A1 gene is compensated and expressed normally through a gene transfer technology, skin flaps of epidermis are cultured in vitro, the skin flaps are transplanted to diseased parts, the skin structure of the patient is continuously restored, and long-term healing is realized. This strategy has several advantages: 1) Autologous cells, non-immunogenic; 2) EpSCs can be amplified in vitro in large quantity and can maintain dryness, and theoretically, one daughter cell of the EpSCs can meet the whole body treatment; 3) Exogenous gene integration sites can be clearly positioned, and genome toxicity caused by gene transfer is reduced to the minimum; 4) Local epidermolysis treatment, low risk; 5) The patent medicine is a slow virus vector plasmid carrying normal genes, can be produced in large scale and is used for all RDEB patients.

The clinical trials of RDEB cell gene therapy that have been developed internationally employ gene replacement (gene replacement) strategies in two main ways: "RV-mediated autologous dermal stem cell ex vivo gene corrected epidermal sheet transplantation therapy" and "LV-mediated autologous dermal fibroblast ex vivo gene corrected subcutaneous cell injection therapy". As a gene replacement introduction vector, LV (lentiviral vector plasmid) has the advantages of higher safety, larger capacity, wider applicability and the like compared with RV (retrovirus vector); compared with injection treatment of skin fibroblast, the skin graft transplantation method of the epidermal stem cells has high safety, large treatment area, lasting effect and less wound and pain of patients. The deficiency of RV and fibroblast injection schemes limits the application prospects of the existing clinical test therapies. However, there are currently no gene therapy products and techniques for LV binding to epidermal stem cells. Establishing RDEB clinical therapeutic products and techniques that combine the advantages of both LV and epidermal stem cells is one of the key technical problems that this project is intended to address.

Disclosure of Invention

First, the technical problem to be solved

In view of the above-mentioned shortcomings and disadvantages of the prior art, the present invention provides a codon-optimized COL7A1 gene, which can significantly increase the expression level of COL7A1 protein compared to the wild-type COL7A1 gene; in addition, the invention also provides a slow virus vector plasmid based on the COL7A1 gene optimized by codons and a slow virus used as an RDEB gene therapy drug, and an original purification method is adopted in the slow virus purification process to obtain the slow virus pseudovirus with high purity and high titer, so that the blank of the gene therapy technology of LV combined with epidermal stem cells is filled.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

in a first aspect, the invention provides a codon optimised COL7A1 gene sequence which is a nucleotide sequence having at least 80% homology to the sequence shown in SEQ ID No. 1.

Preferably, the codon optimized COL7A1 gene has at least 85% homology to the sequence shown in SEQ ID NO. 1, more preferably at least 90% homology to the sequence shown in SEQ ID NO. 1; it is further preferred that the sequence has at least 95% or at least 98% homology with the sequence shown in SEQ ID No. 1.

In a second aspect, the invention provides a lentiviral vector plasmid carrying a codon optimized COL7A1 gene, comprising: a lentiviral vector genome comprising an EFS promoter and a codon optimized COL7A1 gene sequence for encoding; wherein, the genome sequence of the lentiviral vector containing the EFS promoter is shown in SEQ ID No. 3; the codon optimized COL7A1 gene sequence is a nucleotide sequence having at least 80% homology with the sequence shown in SEQ ID NO. 1.

Preferably, the codon optimized COL7A1 gene has at least 85% homology to the sequence shown in SEQ ID NO. 1, more preferably at least 90% homology to the sequence shown in SEQ ID NO. 1; it is further preferred that the sequence has at least 95% homology with the sequence shown in SEQ ID No. 1.

In a third aspect, the invention provides a method for constructing a codon-optimized COL7A1 gene lentiviral vector plasmid, which comprises subcloning a codon-optimized COL7A1 gene fragment into a pCCL-EFS plasmid to obtain a pCCL-EFS-COL7A1-OPT plasmid; the method comprises the following steps:

(1) Double-enzyme cutting of pCCL-EFS plasmid with restriction enzymes EcoRI and SalI at 37+/-0.5 for 1 h+/-0.2, agarose electrophoresis and gel cutting to recover pCCL-EFS carrier fragment;

carrying out PCR amplification on the COL7A1 gene fragment with optimized coding codons, respectively adding a protective base and an EcoRI/SalI enzyme cutting site at the 5 'end and the 3' end, carrying out double enzyme cutting on the amplified PCR fragment at 37 ℃ plus or minus 0.5 by using EcoRI and SalI for 1h plus or minus 0.2, and cutting gel after agarose electrophoresis to recover the COL7A1 gene fragment with optimized coding codons;

(2) Connecting the pCCL-EFS carrier fragment recovered by the gel recovery kit and the codon-optimized COL7A1 gene fragment by adopting T4DNA ligase, and reacting for 10-20min at room temperature;

(3) Transformation of ligation products into E.coli: taking a ligation product transformation competent DH5a, gently mixing, and carrying out ice bath for 25-35min; heat shock at 42+/-0.5 for 70-100s, immediately ice-bathing for 2-5min, adding antibiotic-free LB culture solution at 37+/-0.5, oscillating for 40-80min, uniformly coating the bacterial liquid on an LB agar plate containing ampicillin by using a sterile glass coater, and inversely culturing at 37+/-0.5 for 12-16h;

(4) Selecting a monoclonal colony, inoculating the monoclonal colony into an ampicillin-containing LB liquid culture solution, and oscillating for 14-18h at 37+/-0.5 ℃; the pCCL-EFS-COL7A1-OPT plasmid is extracted by a plasmid extraction kit, DNA sequencing identification is carried out after preliminary identification by EcoRI and SalI double enzyme digestion, and the construction of the lentivirus expression vector plasmid is successful.

In a fourth aspect, the invention provides a Lentivirus carrying a codon optimized COL7A1 gene having a Lentivirus capsid, a Lentivirus genome packaged in the capsid, an EFS promoter and a COL7A1 gene sequence encoding a codon optimized; the codon optimized COL7A1 gene sequence is a nucleotide sequence with at least 80% homology with the sequence shown in SEQ ID NO. 1.

Preferably, at least 85% homology to the sequence shown in SEQ ID No. 1, more preferably at least 90% homology to the sequence shown in SEQ ID No. 1; it is further preferred that the sequence has at least 95% homology with the sequence shown in SEQ ID No. 1.

In a fifth aspect, the present invention provides a method for preparing a lentivirus carrying a codon optimized COL7A1 gene, the method comprising a packaging method and a purification method, wherein the packaging process is as follows:

co-transfecting 293T cells with a vector plasmid pCCL-EFS-COL7A1-OPT and a packaging plasmid psPAX2 and pMD2.G, and collecting culture supernatant for purification after transfection;

wherein, the purification process is as follows:

purifying by using AKTA avant chromatography system of GE, adopting DEAE chromatography, tangential flow filtration and core700 chromatography purification process, purifying to obtain GFP pseudo virus, specifically purifying as follows:

(1) Clarifying, namely filtering the virus harvest liquid to remove insoluble particles, and improving the clarity of the solution so as to facilitate the subsequent chromatographic purification treatment;

(2) Nuclease digestion: treating with 25U/ml Benzonase at 37+ -1deg.C for 50-70min to remove nucleic acid contaminants; or concentrating the clarified liquid in the step (1) and then carrying out the step to reduce the use cost of digestive enzymes and improve the removal rate of nucleic acid;

(3) Anion exchange chromatography: anion exchange chromatography is performed by adopting a monolithic column CIM DEAE to remove protein and DNA pollutants;

(4) Concentrating and changing liquid: the eluent of anion exchange chromatography adopts a membrane with the molecular weight cut-off of 500kDa as a tangential flow filtration, and the eluent is concentrated and exchanged to remove small molecular impurities;

(5) Size exclusion chromatography: adopting Core700 chromatographic packing, discharging particles containing virus with the molecular weight of more than 700kDa from the external water to obtain a purified solution of the molecular exclusion chromatography, and allowing impurities with smaller molecular weight to enter the pores of the packing and be adsorbed therein;

(6) And (3) preserving: filtering the purified solution of the molecular exclusion chromatography by a membrane with the aperture smaller than 0.5um to obtain the slow virus of the COL7A1 gene with optimized codons, and preserving at-80 ℃.

Preferably, the transfection conditions and process conditions during packaging are: 293T cells were seeded 24 hours prior to transfection in a 10-layered cell factory; preparing a transfection system containing opti-MEM culture medium, pCCL-EFS-COL7A1-OPT plasmid, psPAX2 plasmid and pSpike plasmid; the transfection system was added to a 10-layer cell factory and gently mixed. Fresh DMEM medium was changed 6 hours after transfection, cells were continued to be cultured, and medium supernatant was collected after 72 hours of transfection, and purified.

In a fifth aspect, a lentivirus carrying a codon-optimized COL7A1 gene is used in the preparation of an RDEB therapeutic agent.

(III) beneficial effects

The beneficial effects of the invention are as follows: the codon optimized COL7A1 gene provided by the invention has obviously better expressivity than the wild COL7A1 gene, and is verified in 239T cells. After the human epidermal stem cells are infected by the slow virus carrying the codon optimized COL7A1 gene, the slow virus can be successfully cultured in vitro to form an epidermal sheet. Therefore, lentiviruses carrying the codon optimized COL7A1 gene can be used as RDEB gene therapy drugs for establishing RDEB clinical therapy products that bind to the advantages of both LV and epidermal stem cells. In addition, an original purification method is adopted in the lentivirus purification process, so that the lentivirus pseudovirus with high purity and high titer is obtained.

Drawings

FIG. 1 is a photograph of normal human and RDEB subject epidermal stem cell clone formation observed under a microscope.

FIG. 2 shows the result of Western Blot detection of COL7A1-WT gene expression in human epidermal stem cells after infection of human epidermal stem cells with lentivirus carrying exogenous COL7A1-WT, demonstrating that COL7A1 gene can be expressed in epidermal cells by lentivirus carrying exogenous COL7A1 gene.

FIG. 3 is a photograph showing successful in vitro culture of human epidermal stem cells infected with a lentiviral vector carrying COL7A1-OPT to form an epidermal sheet.

FIG. 4 is a comparison of the results of Western Blot detection of COL7A1 gene expression in cells after infection of cells with lentivirus carrying the wild type and codon optimized COL7A1 gene.

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

The invention optimizes the codon of the wild COL7A1 gene to obtain the codon-optimized COL7A1 gene, and then clones the COL7A1 gene to a pCCL-LV vector to obtain the pCCL-EFS-COL7A1 vector for encoding the COL7A1 gene. The 293T transient transfection system is utilized to co-transfect 293T cells with vector plasmid pCCL-EFS-COL7A1-OPT and packaging plasmid psPAX2 and pMD2.G to produce slow virus carrying COL7A1 gene with optimized codons, and the purification process of LentiQ slow virus independently developed by the company is combined to obtain high-titer high-purity virus particles, after the virus particles are infected with human epidermal stem cells, the COL7A1 gene with optimized codons is expressed in the human epidermal stem cells, and the virus particles are successfully cultured in vitro to form epidermal sheets. The lentivirus carrying the COL7A1 gene with optimized codons, which is obtained by the invention, can be used as an RDEB gene therapeutic drug.

The codon optimized COL7A1 gene (COL 7A 1-OPT) has a nucleotide sequence with at least 80% homology to the sequence shown in SEQ ID No. 1. Preferably at least 85% homology to the sequence shown in SEQ ID No. 1, more preferably at least 90% homology to the sequence shown in SEQ ID No. 1; it is further preferred that the sequence shown in SEQ ID No. 1 has at least 95% homology, even 98% homology. The nucleotide sequence shown in SEQ ID No. 1 is obtained by partial codon optimization based on the wild type COL7A1 gene sequence (COL 7A 1-WT). The comparison of the wild-type COL7A1 gene sequence (nucleotide sequence shown as SEQ ID NO: 2) with the nucleotide sequence shown as SEQ ID NO:1 is shown in the comparison table at the end of the specification. The comparison of the wild COL7A1 gene sequence and the nucleotide sequence shown in SEQ ID NO. 1 shows that the homology is only 75.5%, which shows that the base change of the optimized codon is large, but the expressed amino acid sequence is identical and the nucleotide sequence shown in SEQ ID NO. 1 has significantly better expression in cells. It can thus also be reasonably deduced that sequences having at least 80%, 85%, 90%, 95% and even 98% homology to SEQ ID No. 1 should also have an expression similar to the sequences shown in SEQ ID No. 1.

To verify that the codon-optimized COL7A1 gene (COL 7A 1-OPT) had higher expression levels in human epidermal cells, a lentiviral expression plasmid was constructed from the nucleotide sequence shown in SEQ ID NO. 1 and the wild-type COL7A1 gene sequence (as shown in SEQ ID NO. 2), respectively, and 293T cells were transfected with the lentiviral expression plasmid for a predetermined period of time, and after transfection, cells were harvested and lysed and Western Blot detection results were compared. The specific operation is as follows:

constructing a wild type and code codon optimized COL7A1 gene lentiviral expression plasmid, wherein the wild type COL71 lentiviral expression plasmid comprises a lentiviral vector genome containing an EFS promoter and a wild type COL7A1 gene sequence for coding; the codon optimized COL7A1 lentiviral expression plasmid includes a lentiviral vector genome comprising an EFS promoter and a codon optimized COL7A1 gene sequence for encoding. Wherein the sequence of the slow virus vector containing the EFS promoter is SEQ ID No. 3, the sequence of the wild type COL7A1 (WT) gene is SEQ ID No. 2, and the sequence of the codon optimized CO7A1 (OPT) gene is SEQ ID No. 1.

The method for constructing the lentivirus expression plasmid comprises the following steps: subcloning the wild type COL7A1 gene and the COL7A1 gene fragment with optimized coding codons into pCCL-EFS plasmids to obtain pCCL-EFS-COL7A1-WT and pCCL-EFS-COL7A1-OPT plasmids; the cloning process comprises the following steps:

(1) The pCCL-EFS plasmid was digested simultaneously with restriction enzymes EcoRI and SalI at 37℃for 1 hour, and the pCCL-EFS vector fragment was recovered by agarose electrophoresis. PCR amplification is carried out on COL7A1 gene fragments encoding wild type COL7A1 genes and codon optimization, protective base and EcoRI/SalI enzyme cutting sites are added to the 5 'end and the 3' end respectively, double enzyme cutting is carried out on the amplified PCR fragments at 37 ℃ for 1h by using EcoRI and SalI, agarose electrophoresis is carried out, and gel cutting is carried out to recover COL7A1 gene fragments encoding wild type COL7A1 genes and codon optimization.

(2) The pCCL-EFS vector fragment and the COL7A1 gene fragment which codes for the wild-type COL7A1 gene and is codon-optimized are respectively recovered by the gel recovery kit and are connected by adopting T4DNA ligase, and the reaction is carried out for 15min at room temperature.

(3) Transformation of ligation products into E.coli: taking a ligation product transformation competent DH5a, gently mixing, and carrying out ice bath for 30min; heat shock at 42 deg.C for 80s, immediately ice-bathing for 3min, adding antibiotic-free LB culture solution, shaking at 37 deg.C for 60min, uniformly coating the bacterial solution onto LB agar plate containing ampicillin with sterile glass coater, and culturing at 37 deg.C for 14 hr.

(4) Picking a monoclonal colony, inoculating the monoclonal colony into an ampicillin-containing LB liquid culture solution, and oscillating for 16 hours at 37 ℃; the plasmid extraction kit is used for extracting pCCL-EFS-COL7A1-WT and pCCL-EFS-COL7A1-OPT vector plasmids, and DNA sequencing identification is carried out after preliminary identification by EcoRI and SalI double enzyme digestion, so that the construction of the lentivirus expression vector plasmids is successful.

(II) wild-type COL7A1 and codon-optimized COL7A1 lentiviral plasmid expression levels were compared. The comparison method comprises the following steps: the wild type COL7A1 gene and the lentiviral plasmid encoding codon optimized COL7A1 were transfected into 293T cells, and after a predetermined period of transfection, the cells were harvested and lysed and Western Blot comparison was performed as follows:

(1) 293T was 1X 10 as per day before transfection ⁶ Hole, laying a 6 plates;

(2) On the day of transfection, 293T cells were transfected with 3. Mu.g of the wild-type COL7A1 gene and a lentiviral plasmid encoding codon-optimized COL7A1, and after 48h the cells were collected and lysed to obtain 293T COL7A1-WT lysates and 293T COL7A1-OPT lysates.

(3) A6% SDS-PAGE gel was prepared, the protein concentration of the 293T COL7A1-WT lysate and the 293T COL7A1-OPT lysate were measured by the BCA method, the same amount of protein of the cell lysate was added to the 6% SDS-PAGE gel, the gel was run at 80V to the junction between the compression gel and the separation gel, and the gel was run at 120V to the bottom.

(4) SDS-PAGE gels containing the 293T COL7A1-WT protein and the 293T COL7A1-OPT protein were transferred onto NC membrane with 400mA constant current.

(5) NC membranes transformed with the 293T COL7A1-WT protein and the 293T COL7A1-OPT protein were incubated with 5% skimmed milk for 1h at room temperature, and the membranes were washed 3 times with TBST for 5min each.

(6) Incubation with primary antibody to COL7A1 was carried out overnight at 4 ℃.

(7) The following day the membranes were washed 3 times with TBST for 10min each, incubated with HRP-containing secondary antibody for 1h at room temperature, and washed 3 times with TBST for 5min each.

(8) Development was performed by ECL to obtain a comparison of 293T expression levels of COL7A1-WT and 293T COL7A 1-OPT. The Western Blot detection results are shown in FIG. 4: the expression level of the codon optimized COL7A1 gene is obviously higher than that of the wild type COL7A1 gene.

In order to verify whether the codon-optimized COL7A1 gene (COL 7A 1-OPT) has practical application prospect, the invention further packages and purifies the lentivirus of the written COL7A1-OPT gene, and carries out culture experiments in vitro after the lentivirus is infected with human epidermal stem cells. The method comprises the following steps: plasmid construction, lentivirus packaging, lentivirus purification, infection of human epidermal stem cells and in vitro culture. The specific procedure for "plasmid construction" referred to above for construction of wild-type and codon-optimized COL7A1 gene lentiviral expression plasmids "is not described in detail herein. The lentivirus packaging and purification process is as follows.

(1) Lentivirus package

Purifying COL7A1 virus expression vector plasmid (pCCL-EFS-COL 7A1-WT and pCCL-EFS-COL7A1-OPT vector plasmid), packaging plasmid pSPAX2 and plasmid pMD2.G by chromatography, co-transfecting 293T cells, and collecting virus supernatant after transfection for a predetermined time to obtain RDEB gene therapeutic drug based on Lentivirus vector; the method adopts the LentiQ lentivirus purification technology independently developed by applicant company to purify the virus, and comprises the following steps:

the vector plasmids pCCL-EFS-COL7A1-WT or pCCL-EFS-COL7A1-OPT, and the packaging plasmids psPAX2 and pMD2.G (purchased from Addgene under the trade designations Addgene #12259 and Addgene # 12260) were inoculated into 293T cells of a 10-layer cell factory one day before co-transfection, fresh DMEM medium was changed 6 hours after transfection, and supernatants were harvested for chromatographic purification 72 hours later. Wherein, the transfection conditions are as follows: 1.00E+09293T cells were seeded 24h before transfection in a 10-layer cell factory; a transfection system reagent containing 80ml of the pti-MEM medium, 750ug of pCCL-EFS-COL7A1-WT or pCCL-EFS-COL7A1-OPT plasmid, 500ug of the psPAX2 plasmid, 250ug of the pSpike plasmid was prepared. The transfection system was added to a 10-layer cell factory and gently mixed. Fresh DMEM 1000ml was replaced 6 hours after transfection. Cells were further cultured, and after 72 hours of transfection, the supernatant was collected, and lentiviruses were harvested from the supernatant.

(2) Virus purification

(1) MF (clarification) the virus harvest is filtered (filter membrane pore size 0.45 μm) to remove insoluble particles such as 293T cells and debris, and to improve the clarity of the solution for subsequent chromatographic purification.

(2) Benzonase treatment (nuclease digestion): the plasmid DNA remaining during transfection and the genome released by dead cells were removed by more than 85% by treatment with 25U/ml Benzonase for 1 hour at 37 ℃. The first step of clarifying solution may be concentrated before this step, so that the cost of digestive enzyme may be lowered and the nucleic acid eliminating rate may be further raised.

(3) IEX (anion exchange chromatography): the process adopts monolithic column CIM DEAE to perform anion exchange chromatography, and can remove 99% of protein and DNA pollutants, the virus yield is as high as more than 80%, and the higher loading capacity can be maintained at high flow rate.

(4) UF/DF (concentrate change): because the slow virus particles are large (the diameter is about 80-120 nm), a membrane with the molecular weight cut-off of 500kDa is needed to be used for TFF (tangential flow filtration), so that the purpose of concentrating and changing liquid is achieved. In the concentrating and liquid changing process, some small molecular impurities can be effectively removed, thereby achieving the aim of purification. The eluent of IEX chromatography is concentrated and changed, and then subjected to final size exclusion chromatography purification.

(5) SEC (size exclusion chromatography): by adopting Core700 chromatographic packing, virus and other particles with the molecular weight larger than 700kDa are discharged through the external water volume, and impurities with smaller molecular weight enter the pores of the packing and are adsorbed in the pores. Core700 chromatography has the advantages of large sample loading amount and good impurity removal capability. The yield of the step can reach 40 percent.

(6) Storage (save): filtering SEC purified solution with 0.2-0.25um membrane to obtain slow virus carrying COL7A1-WT gene and COL7A1-OPT gene, respectively, packaging, and storing at-80deg.C. Since the infection capacity of pseudoviruses is greatly reduced in repeated freeze thawing, they are preserved in DMEM medium or 1mg/ml BSA, providing protection for the freeze thawing process.

(3) Infecting human epidermic stem cells and culturing in vitro

As shown in FIG. 1, normal human and RDEB subject epidermal stem cells were cultured in vitro and the clonal formation of the epidermal stem cells was observed under a microscope. As a result, as shown in FIG. 1, the RDEB subjects had significantly less clonal formation of epidermal stem cells than normal.

The human epidermal stem cells were infected with the lentivirus carrying the COL7A1-WT gene obtained as described above, and Western Blot was used to detect the expression of the COL7A1 gene in human epidermal stem cells. As a result, as shown in FIG. 2, when the lentivirus carrying COL7A1-WT or COL7A1-FLAG was infected with the epidermal stem cell, the expression level of the COL7A1 gene was significantly increased as compared with the epidermal stem cell (Control) not infected with the lentivirus. The following is explained: the exogenous COL7A1 gene carried by lentiviruses can be expressed in human epidermal stem cells.

Carrying COL obtained as described aboveThe lentivirus of 7A1-OPT gene infects human epidermis stem cells for in vitro culture. The culture process is as follows: 1) Co-culturing the epidermal stem cells and the 3T3-J2feeder cells in an epidermal stem culture medium; 2) When the cell density is between 30% and 40%, the utilization titer is 2×10 ⁹ TU/ml lentivirus infects cells; 3) Epidermal stem cells were harvested by digestion after 48 hours, according to at least 1 x 10 ⁶ Seeding the treated 3T3-J2 cells with the epidermal stem cells at the density of 10cm dish; 4) After 10-14 days of culture, the epidermal stem clones were connected into pieces and feeder cells were extruded into lines. As shown in FIG. 3, the epidermis sheet (the forceps in the figure stretched the spread epidermis sheet) was successfully obtained after in vitro culture. The lentivirus carrying the COL7A1 gene with optimized codons, which is obtained by the invention, can be used as an RDEB gene therapeutic drug.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

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Sequence listing

<110> Jizhi medicine (Nanjing) Biotechnology Co., ltd

<120> a codon-optimized COL7A1 gene, lentivirus and application

<130> EJS201351I

<141> 2020-12-23

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

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tttctgttag acggcagtag ctctattggg agatctaact tccgggaggt cagaagcttc 180

cttgagggcc tggtgctgcc ttttagcggg gccgcttctg cccagggcgt gaggttcgcc 240

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ggagatgtga ttagggcaat cagagagttg agctacaaag gcggcaatac tagaaccggc 360

gccgccatcc tgcacgtggc tgaccacgtg tttctgcccc aactggccag gcccggcgtc 420

cccaaagtgt gtatcctgat tactgacggc aaatcccagg acctggtaga caccgccgca 480

cagcgcctaa agggacaggg agtgaagttg ttcgcagtgg ggatcaagaa tgctgacccc 540

gaggaactga agcgggtggc cagtcagccc acctccgact tctttttctt cgtgaatgat 600

ttctcaatct tgaggaccct gctgcctctg gtgagccgta gagtgtgcac aacagcaggc 660

ggcgtacctg tgactagacc ccctgacgat tccacctcag ctcctagaga cctggtactc 720

tccgagccgt cctcccagag tctcagagtg cagtggactg ccgccagcgg gcccgtgaca 780

gggtacaagg tgcagtatac acctctgaca ggattgggac agcccctacc aagtgagcgg 840

caagaggtga acgtgccagc aggagagaca tcagtgagac tcagaggtct gaggcctctc 900

actgagtatc aggtcactgt gatcgcactc tatgccaatt ctataggaga ggccgttagt 960

ggaaccgccc gcaccacagc ccttgagggg cccgaactga ccattcagaa caccaccgcc 1020

cactcactgc tcgtggcctg gcggtctgtg ccaggtgcta ctgggtacag agtgacttgg 1080

agagtgctta gcggaggccc tacccagcag caggagttag gtcctggcca ggggagcgtg 1140

ctactcagag atctggagcc cggcaccgat tacgaagtga cagtaagtac cctctttggc 1200

agatccgttg ggcccgctac atcactcatg gctcgcaccg atgcctcagt ggagcaaaca 1260

ctgcggcccg tgatcttggg acctacctct attttgttaa gttggaacct ggtgcctgaa 1320

gccagggggt accggctgga atggcgcaga gagaccggac tggagcctcc acaaaaagtt 1380

gtgctgcctt ccgacgttac cagatatcag ttagacggat tacagcccgg aaccgaatac 1440

aggctgacgc tctacaccct cttggagggg cacgaggtcg ccacccccgc aactgttgtc 1500

ccaaccggac ctgaactgcc agtttctccc gtgaccgatc tgcaggcaac agagctgccc 1560

ggccagcggg tgcgcgtcag ctggtcccca gtgcctgggg ccacacagta cagaatcatc 1620

gtgcgatcta ctcagggcgt ggagcggact cttgtgctcc caggctccca gacagccttt 1680

gatctcgacg acgttcaggc cgggctctcc tatacagtga gagtcagtgc cagagtgggc 1740

cctagggagg ggtccgccag tgtgcttact gtgagaagag agccagagac ccctctggct 1800

gttcccggct tgcgcgtggt cgtgtctgac gccacaagag tgagggtggc ctggggaccc 1860

gtcccaggag cttctgggtt tcgcatatct tggagtaccg gatcaggccc cgaaagttct 1920

cagaccttgc cacctgactc aaccgctacc gacattactg gcctgcagcc cggtaccacc 1980

tatcaggtgg cagtctcagt gctgcgtgga agagaagaag gacctgcagc cgttattgtg 2040

gctcggaccg acccactcgg acctgtcagg accgtgcatg tcacgcaggc aagcagctct 2100

agcgtaacaa ttacatggac tcgagttccc ggagccacag gctaccgagt gtcctggcac 2160

agcgcccatg ggcctgagaa gtctcagctg gtcagcgggg aggctactgt agcagaactg 2220

gatggactcg agccggatac tgagtatacg gttcacgttc gtgctcacgt ggccggtgta 2280

gacggccctc ccgccagcgt tgtggtcaga actgcccctg agcccgtcgg cagggtgtca 2340

aggttacaaa ttttaaacgc aagctccgac gtcttgcgta tcacctgggt tggcgtgact 2400

ggggccaccg cctaccggtt agcttggggt cgatctgagg gaggacctat gaggcatcag 2460

atcctgcctg gtaatactga ttctgccgag atacgcggcc tggaaggtgg tgtgagctat 2520

tctgtgcgcg tcactgccct ggtgggtgac agagagggta cccctgtcag catcgtggtc 2580

accaccccac cagaagcacc acctgcactg ggaacactgc atgtcgtcca gagaggcgag 2640

cacagtctga gactgaggtg ggagcctgtc cccagggccc agggctttct gctccactgg 2700

cagcccgaag gaggccagga gcagagccgg gtcttgggtc ctgaactgtc ctcttaccac 2760

ctggacggcc tggagcctgc aacccagtac cgggtgaggc tttctgttct aggacccgcc 2820

ggggaaggac catcagccga agtcaccgcg aggacagaaa gtcctagggt accaagcatt 2880

gagctgcggg tggttgatac ttccatcgac agcgtgactc tcgcttggac ccctgtctct 2940

cgggcctcca gctatatcct ctcatggcgt cctctgcgag gacctggaca ggaagtccca 3000

ggctcccccc aaactctgcc aggtattagc tcttctcagc gggtaacagg cctggaaccc 3060

ggagttagct acatattcag cttgacccct gttctggatg gagtgagggg acctgaagct 3120

tcagtgacac agacacccgt gtgccctcgc ggtctcgcgg acgtcgtgtt tctgccccac 3180

gccactcagg acaacgcaca ccgagccgag gcaactagac gcgtgctcga gagactggtc 3240

ctggcccttg ggcctttggg cccccaggct gttcaggtag gactgttgag ttattcacac 3300

cggccatccc ccctatttcc cctgaacgga agccatgatc tcggaatcat cctacagcga 3360

atccgtgata tgccttacat ggacccttct ggaaacaatc tggggactgc agtggtcaca 3420

gcccacaggt acatgcttgc acctgatgca ccaggccgca ggcagcacgt gcccggagtt 3480

atggtgctcc tcgtcgatga acccctgcgg ggcgatattt tctctcccat ccgagaggca 3540

caagcaagtg gactcaacgt cgtgatgctg ggcatggccg gggctgaccc agagcagctt 3600

agacgccttg ctccaggaat ggattctgtt cagaccttct ttgctgtgga tgatggcccc 3660

tcactagacc aggcagtgag cggcctggcc actgctctct gccaggccag tttcacaacg 3720

caaccacgcc ccgagccatg tcctgtgtat tgccctaaag gccagaaggg cgaaccaggg 3780

gagatgggcc tgcgcggaca agtggggcca ccaggagacc ccggactccc tgggcgaacc 3840

ggggctccgg gcccacaagg cccacctgga agcgctaccg caaagggcga aagggggttt 3900

ccaggagccg atggcaggcc aggttctccc ggaagagctg gtaatcctgg tactcctggc 3960

gcgcccggac taaaaggtag tcctggactg ccaggtccta ggggagatcc aggagaaaga 4020

ggcccaagag gtcctaaggg agagcctggg gcaccagggc aagtgattgg aggagaaggg 4080

ccaggcctgc caggaaggaa aggcgatcct ggaccttctg gccctccagg gccacgcggg 4140

cctcttggag atcctggccc tcgcggccct cctggcctgc caggtacagc catgaaggga 4200

gataagggag accggggaga gaggggcccc cccggaccag gcgagggagg catcgcccca 4260

ggggaaccag gtctcccagg cctgcctggt tccccaggtc cccagggtcc agtgggacct 4320

ccaggcaaga agggggagaa gggagattcg gaagatggcg ctcctggcct acctggccag 4380

cccggtagtc ctggggagca gggtccaaga ggaccgccag gcgccattgg ccccaaagga 4440

gaccgcggct tccctggccc attaggcgaa gccggcgaga aaggtgagcg gggacctccc 4500

ggccccgccg gctccagagg cctgcccggt gtggccgggc gtcccggtgc gaaaggacct 4560

gaagggcccc ctggccctac cggcaggcag ggagagaagg gggagccagg acggcctgga 4620

gaccccgctg tggtgggacc tgccgtcgcc ggtccaaagg gcgagaaagg cgatgttgga 4680

cctgctggac cgaggggcgc tacaggcgtg cagggagagc gagggccacc cggattggtt 4740

ctgccagggg atccaggtcc aaagggagac cctggcgata ggggcccaat cggtttaaca 4800

ggcagagccg gaccccctgg ggattccgga ccaccaggcg aaaaagggga ccctggtagg 4860

cctggccctc ctggtccagt gggcccacgc ggccgagatg gcgaagtagg cgaaaaggga 4920

gatgaaggcc ctccaggcga tccagggttg cccggaaagg ctggggaacg cggcctacga 4980

ggagcacctg gggtgcgcgg ccccgtcggg gagaagggcg accaaggcga cccaggggaa 5040

gatgggcgga atgggtcccc tggctccagt ggtcccaaag gggacagagg ggaaccaggc 5100

ccacccggac cacccggcag gctggttgat acaggacctg gggccaggga gaaaggcgaa 5160

ccgggagaca gagggcagga gggaccacgc ggacctaaag gagacccagg acttcctgga 5220

gcacctgggg aacgcggtat cgaggggttc agaggacccc ctggacctca aggagaccct 5280

ggcgtccgcg gccctgcagg tgagaaggga gatagaggcc cccccggatt ggatgggaga 5340

tccggactgg atggcaaacc cggtgctgcc gggccaagtg gaccaaacgg agccgccgga 5400

aaagcaggag acccaggtcg cgatggtctg cctggcctta gaggcgaaca gggcctccca 5460

ggcccaagcg gacctcccgg actgcccggt aaaccaggtg aagatggcaa gcctggtctg 5520

aacgggaaga atggcgaacc aggcgaccct ggagaggatg gacggaaggg cgagaaaggc 5580

gactccggcg cctccggacg cgagggacgg gacggtccca agggcgagag aggcgctccc 5640

ggcattctgg gtccccaagg accacccgga ctccctggcc ctgtcggccc acctggacaa 5700

ggcttcccag gcgtgcccgg tgggactgga cctaagggcg acaggggtga gacaggttcc 5760

aaaggcgagc agggcctacc tggagagcgg ggactgcgtg gcgaacctgg cagcgtgccc 5820

aatgttgaca ggctgcttga aaccgctgga attaaggcct ctgcccttcg cgaaatcgtt 5880

gagacatggg atgaaagtag cggcagcttt cttcctgtcc ctgagcggag gcgggggcct 5940

aagggagact ccggcgaaca aggacctcct ggcaaggagg ggcccatcgg ttttccaggc 6000

gaacgggggc tgaaaggaga ccgaggggac cctggccccc agggcccccc cggtctggcc 6060

ctcggcgaac gtggcccacc agggcctagc ggcctggctg gagagcctgg caagccgggg 6120

atcccagggt tacctggccg agccggcggc gtcggcgagg ctggccgtcc aggcgagcga 6180

ggggaacggg gagagaaagg agagaggggg gagcagggca gggacggccc ccccggcctg 6240

ccaggaacac ccggcccacc tggtcctccc ggacctaaag taagcgtgga tgagcccgga 6300

cctggactga gcggagagca agggcctcct ggactgaagg gagctaaggg cgagccgggt 6360

agcaatggcg atcaggggcc caaaggcgac cgaggcgtcc ccggtatcaa gggcgaccgc 6420

ggggagccag ggcctcgggg ccaggatggt aatcctggcc tgcccggcga gagaggaatg 6480

gccgggcccg aaggaaagcc aggccttcag ggacctaggg gcccaccggg gcccgtgggt 6540

ggacacgggg atccaggccc acctggcgcc cctggcttgg ctggccccgc tggcccccag 6600

ggcccgtcag gcctgaaagg cgagccaggg gagaccggac ccccaggaag aggactaaca 6660

ggccccacag gcgctgtggg cctgcccgga ccgccaggac catcaggcct ggtgggccct 6720

cagggaagtc ctggcttgcc tggacaggta ggggagactg gaaagccagg ggctcctgga 6780

agagatgggg cttcaggtaa agacggcgac aggggaagtc caggggtgcc tgggtcccct 6840

ggccttccag gccctgtggg cccaaagggc gaacccggcc caactggcgc accaggacaa 6900

gccgtggtgg ggctgccagg cgccaagggc gaaaaggggg cccctggagg cctggcgggc 6960

gacctggtgg gagaaccagg cgctaaaggg gacagaggtc tccctgggcc tcgcggagag 7020

aagggggagg ctgggagagc tggggagccc ggcgacccag gcgaggatgg ccagaagggg 7080

gcaccaggcc ctaaggggtt taagggcgac ccaggggttg gggtgccagg ctcaccaggg 7140

cccccaggtc cccctggcgt gaaaggcgat ttggggcttc ccggactgcc tggggcccct 7200

ggcgtggtgg gttttcccgg acagacagga cccagaggag agatggggca accaggcccg 7260

tcaggcgaga gaggattagc aggccctcca ggaagggaag gcatccccgg cccactgggg 7320

cccccaggac ctccaggtag cgtgggtccc cctggcgctt ccggcttgaa gggagataag 7380

ggcgatcctg gcgtcggact gcctgggcca agaggcgagc ggggagaacc cggaatcagg 7440

ggggaagatg ggcgcccagg acaggaaggc cccaggggac tgacaggtcc tcctggcagc 7500

agaggggagc gtggtgaaaa gggcgatgtg ggctcagctg gccttaaggg cgataaaggg 7560

gattccgcag taattctggg cccaccaggt cccagagggg caaaaggaga catgggggaa 7620

aggggccctc ggggactgga tggcgacaaa ggccctcgcg gcgacaacgg ggaccccggc 7680

gacaagggga gcaagggaga acccggagat aagggatctg ccgggcttcc aggattgaga 7740

gggctcctgg gcccccaagg acagcccggc gccgctggga ttcctggtga ccccggaagc 7800

cccggtaaag atggcgtgcc cggtatccgc ggcgagaagg gcgacgtggg ctttatgggc 7860

ccacgcggcc tgaagggtga gcgcggcgtg aagggcgcat gtgggctgga cggcgagaag 7920

ggcgataagg gtgaggcagg tcccccaggc cggcctggcc tggccgggca caagggcgaa 7980

atgggcgagc caggcgtgcc aggtcaatcc ggagccccag gcaaggaggg gctgattggg 8040

cccaagggcg accggggctt tgacggtcag ccggggccaa agggcgacca gggggagaaa 8100

ggggagcgtg gtacacctgg aatcggcggc ttccccggac ccagtgggaa tgacgggtct 8160

gccggccccc ctggccctcc cggctccgtg ggacctaggg gacccgaagg actgcaggga 8220

cagaaggggg aacgcgggcc tccaggcgag cgcgtggtcg gcgctcccgg cgtgcctggg 8280

gccccaggtg agagagggga gcagggccgg cctggccctg ccggcccaag aggagagaag 8340

ggagaggcag cactcactga ggatgatatc cgcggattcg ttaggcagga aatgagtcaa 8400

cattgtgcct gccagggcca gtttatcgcc tcaggatccc gccctctccc ctcctatgcc 8460

gccgacacag cagggagcca gctgcacgca gtccccgtgc tgagagtgtc tcatgcagag 8520

gaggaggaaa gggtgccacc tgaagatgat gagtacagcg agtatagtga gtacagtgtc 8580

gaggagtacc aggaccctga ggctccctgg gacagcgacg acccatgcag cctgcctctg 8640

gacgagggct catgcactgc ctacacactc cgctggtacc atagggccgt gactggcagc 8700

actgaggcct gccacccctt tgtgtacggc gggtgcggag gcaatgctaa tagattcggg 8760

acaagagaag cctgcgaaag gcgctgtcct ccgagggtgg tgcagtccca gggcacaggc 8820

acagcccagg attga 8835

<210> 2

<211> 8835

<212> DNA

<213> Artificial Sequence

<400> 2

atgacgctgc ggcttctggt ggccgcgctc tgcgccggga tcctggcaga ggcgccccga 60

gtgcgagccc agcacaggga gagagtgacc tgcacgcgcc tttacgccgc tgacattgtg 120

ttcttactgg atggctcctc atccattggc cgcagcaatt tccgcgaggt ccgcagcttt 180

ctcgaagggc tggtgctgcc tttctctgga gcagccagtg cacagggtgt gcgctttgcc 240

acagtgcagt acagcgatga cccacggaca gagttcggcc tggatgcact tggctctggg 300

ggtgatgtga tccgcgccat ccgtgagctt agctacaagg ggggcaacac tcgcacaggg 360

gctgcaattc tccatgtggc tgaccatgtc ttcctgcccc agctggcccg acctggtgtc 420

cccaaggtct gcatcctgat cacagacggg aagtcccagg acctggtgga cacagctgcc 480

caaaggctga aggggcaggg ggtcaagcta tttgctgtgg ggatcaagaa tgctgaccct 540

gaggagctga agcgagttgc ctcacagccc accagtgact tcttcttctt cgtcaatgac 600

ttcagcatct tgaggacact actgcccctc gtttcccgga gagtgtgcac gactgctggt 660

ggcgtgcctg tgacccgacc tccggatgac tcgacctctg ctccacgaga cctggtgctg 720

tctgagccaa gcagccaatc cttgagagta cagtggacag cggccagtgg ccctgtgact 780

ggctacaagg tccagtacac tcctctgacg gggctgggac agccactgcc gagtgagcgg 840

caggaggtga acgtcccagc tggtgagacc agtgtgcggc tgcggggtct ccggccactg 900

accgagtacc aagtgactgt gattgccctc tacgccaaca gcatcgggga ggctgtgagc 960

gggacagctc ggaccactgc cctagaaggg ccggaactga ccatccagaa taccacagcc 1020

cacagcctcc tggtggcctg gcggagtgtg ccaggtgcca ctggctaccg tgtgacatgg 1080

cgggtcctca gtggtgggcc cacacagcag caggagctgg gccctgggca gggttcagtg 1140

ttgctgcgtg acttggagcc tggcacggac tatgaggtga ccgtgagcac cctatttggc 1200

cgcagtgtgg ggcccgccac ttccctgatg gctcgcactg acgcttctgt tgagcagacc 1260

ctgcgcccgg tcatcctggg ccccacatcc atcctccttt cctggaactt ggtgcctgag 1320

gcccgtggct accggttgga atggcggcgt gagactggct tggagccacc gcagaaggtg 1380

gtactgccct ctgatgtgac ccgctaccag ttggatgggc tgcagccggg cactgagtac 1440

cgcctcacac tctacactct gctggagggc cacgaggtgg ccacccctgc aaccgtggtt 1500

cccactggac cagagctgcc tgtgagccct gtaacagacc tgcaagccac cgagctgccc 1560

gggcagcggg tgcgagtgtc ctggagccca gtccctggtg ccacccagta ccgcatcatt 1620

gtgcgcagca cccagggggt tgagcggacc ctggtgcttc ctgggagtca gacagcattc 1680

gacttggatg acgttcaggc tgggcttagc tacactgtgc gggtgtctgc tcgagtgggt 1740

ccccgtgagg gcagtgccag tgtcctcact gtccgccggg agccggaaac tccacttgct 1800

gttccagggc tgcgggttgt ggtgtcagat gcaacgcgag tgagggtggc ctggggaccc 1860

gtccctggag ccagtggatt tcggattagc tggagcacag gcagtggtcc ggagtccagc 1920

cagacactgc ccccagactc tactgccaca gacatcacag ggctgcagcc tggaaccacc 1980

taccaggtgg ctgtgtcggt actgcgaggc agagaggagg gccctgctgc agtcatcgtg 2040

gctcgaacgg acccactggg cccagtgagg acggtccatg tgactcaggc cagcagctca 2100

tctgtcacca ttacctggac cagggttcct ggcgccacag gatacagggt ttcctggcac 2160

tcagcccacg gcccagagaa atcccagttg gtttctgggg aggccacggt ggctgagctg 2220

gatggactgg agccagatac tgagtatacg gtgcatgtga gggcccatgt ggctggcgtg 2280

gatgggcccc ctgcctctgt ggttgtgagg actgcccctg agcctgtggg tcgtgtgtcg 2340

aggctgcaga tcctcaatgc ttccagcgac gttctacgga tcacctgggt aggggtcact 2400

ggagccacag cttacagact ggcctggggc cggagtgaag gcggccccat gaggcaccag 2460

atactcccag gaaacacaga ctctgcagag atccggggtc tcgaaggtgg agtcagctac 2520

tcagtgcgag tgactgcact tgtcggggac cgcgagggca cacctgtctc cattgttgtc 2580

actacgccgc ctgaggctcc gccagccctg gggacgcttc acgtggtgca gcgcggggag 2640

cactcgctga ggctgcgctg ggagccggtg cccagagcgc agggcttcct tctgcactgg 2700

caacctgagg gtggccagga acagtcccgg gtcctggggc ccgagctcag cagctatcac 2760

ctggacgggc tggagccagc gacacagtac cgcgtgaggc tgagtgtcct agggccagct 2820

ggagaagggc cctctgcaga ggtgactgcg cgcactgagt cacctcgtgt tccaagcatt 2880

gaactacgtg tggtggacac ctcgatcgac tcggtgactt tggcctggac tccagtgtcc 2940

agggcatcca gctacatcct atcctggcgg ccactcagag gccctggcca ggaagtgcct 3000

gggtccccgc agacacttcc agggatctca agctcccagc gggtgacagg gctagagcct 3060

ggcgtctctt acatcttctc cctgacgcct gtcctggatg gtgtgcgggg tcctgaggca 3120

tctgtcacac agacgccagt gtgcccccgt ggcctggcgg atgtggtgtt cctaccacat 3180

gccactcaag acaatgctca ccgtgcggag gctacgagga gggtcctgga gcgtctggtg 3240

ttggcacttg ggcctcttgg gccacaggca gttcaggttg gcctgctgtc ttacagtcat 3300

cggccctccc cactgttccc actgaatggc tcccatgacc ttggcattat cttgcaaagg 3360

atccgtgaca tgccctacat ggacccaagt gggaacaacc tgggcacagc cgtggtcaca 3420

gctcacagat acatgttggc accagatgct cctgggcgcc gccagcacgt accaggggtg 3480

atggttctgc tagtggatga acccttgaga ggtgacatat tcagccccat ccgtgaggcc 3540

caggcttctg ggcttaatgt ggtgatgttg ggaatggctg gagcggaccc agagcagctg 3600

cgtcgcttgg cgccgggtat ggactctgtc cagaccttct tcgccgtgga tgatgggcca 3660

agcctggacc aggcagtcag tggtctggcc acagccctgt gtcaggcatc cttcactact 3720

cagccccggc cagagccctg cccagtgtat tgtccaaagg gccagaaggg ggaacctgga 3780

gagatgggcc tgagaggaca agttgggcct cctggcgacc ctggcctccc gggcaggacc 3840

ggtgctcccg gcccccaggg gccccctgga agtgccactg ccaagggcga gaggggcttc 3900

cctggagcag atgggcgtcc aggcagccct ggccgcgccg ggaatcctgg gacccctgga 3960

gcccctggcc taaagggctc tccagggttg cctggccctc gtggggaccc gggagagcga 4020

ggacctcgag gcccaaaggg ggagccgggg gctcccggac aagtcatcgg aggtgaagga 4080

cctgggcttc ctgggcggaa aggggaccct ggaccatcgg gcccccctgg acctcgtgga 4140

ccactggggg acccaggacc ccgtggcccc ccagggcttc ctggaacagc catgaagggt 4200

gacaaaggcg atcgtgggga gcggggtccc cctggaccag gtgaaggtgg cattgctcct 4260

ggggagcctg ggctgccggg tcttcccgga agccctggac cccaaggccc cgttggcccc 4320

cctggaaaga aaggagaaaa aggtgactct gaggatggag ctccaggcct cccaggacaa 4380

cctgggtctc cgggtgagca gggcccacgg ggacctcctg gagctattgg ccccaaaggt 4440

gaccggggct ttccagggcc cctgggtgag gctggagaga agggcgaacg tggaccccca 4500

ggcccagcgg gatcccgggg gctgccaggg gttgctggac gtcctggagc caagggtcct 4560

gaagggccac caggacccac tggccgccaa ggagagaagg gggagcctgg tcgccctggg 4620

gaccctgcag tggtgggacc tgctgttgct ggacccaaag gagaaaaggg agatgtgggg 4680

cccgctgggc ccagaggagc taccggagtc caaggggaac ggggcccacc cggcttggtt 4740

cttcctggag accctggccc caagggagac cctggagacc ggggtcccat tggccttact 4800

ggcagagcag gacccccagg tgactcaggg cctcctggag agaagggaga ccctgggcgg 4860

cctggccccc caggacctgt tggcccccga ggacgagatg gtgaagttgg agagaaaggt 4920

gacgagggtc ctccgggtga cccgggtttg cctggaaaag caggcgagcg tggccttcgg 4980

ggggcacctg gagttcgggg gcctgtgggt gaaaagggag accagggaga tcctggagag 5040

gatggacgaa atggcagccc tggatcatct ggacccaagg gtgaccgtgg ggagccgggt 5100

cccccaggac ccccgggacg gctggtagac acaggacctg gagccagaga gaagggagag 5160

cctggggacc gcggacaaga gggtcctcga gggcccaagg gtgatcctgg cctccctgga 5220

gcccctgggg aaaggggcat tgaagggttt cggggacccc caggcccaca gggggaccca 5280

ggtgtccgag gcccagcagg agaaaagggt gaccggggtc cccctgggct ggatggccgg 5340

agcggactgg atgggaaacc aggagccgct gggccctctg ggccgaatgg tgctgcaggc 5400

aaagctgggg acccagggag agacgggctt ccaggcctcc gtggagaaca gggcctccct 5460

ggcccctctg gtccccctgg attaccggga aagccaggcg aggatggcaa acctggcctg 5520

aatggaaaaa acggagaacc tggggaccct ggagaagacg ggaggaaggg agagaaagga 5580

gattcaggcg cctctgggag agaaggtcgt gatggcccca agggtgagcg tggagctcct 5640

ggtatccttg gaccccaggg gcctccaggc ctcccagggc cagtgggccc tcctggccag 5700

ggttttcctg gtgtcccagg aggcacgggc cccaagggtg accgtgggga gactggatcc 5760

aaaggggagc agggcctccc tggagagcgt ggcctgcgag gagagcctgg aagtgtgccg 5820

aatgtggatc ggttgctgga aactgctggc atcaaggcat ctgccctgcg ggagatcgtg 5880

gagacctggg atgagagctc tggtagcttc ctgcctgtgc ccgaacggcg tcgaggcccc 5940

aagggggact caggcgaaca gggcccccca ggcaaggagg gccccatcgg ctttcctgga 6000

gaacgcgggc tgaagggcga ccgtggagac cctggccctc aggggccacc tggtctggcc 6060

cttggggaga ggggcccccc cgggccttcc ggccttgccg gggagcctgg aaagcctggt 6120

attcccgggc tcccaggcag ggctgggggt gtgggagagg caggaaggcc aggagagagg 6180

ggagaacggg gagagaaagg agaacgtgga gaacagggca gagatggccc tcctggactc 6240

cctggaaccc ctgggccccc cggaccccct ggccccaagg tgtctgtgga tgagccaggt 6300

cctggactct ctggagaaca gggaccccct ggactcaagg gtgctaaggg ggagccgggc 6360

agcaatggtg accaaggtcc caaaggagac aggggtgtgc caggcatcaa aggagaccgg 6420

ggagagcctg gaccgagggg tcaggacggc aacccgggtc taccaggaga gcgtggtatg 6480

gctgggcctg aagggaagcc gggtctgcag ggtccaagag gcccccctgg cccagtgggt 6540

ggtcatggag accctggacc acctggtgcc ccgggtcttg ctggccctgc aggaccccaa 6600

ggaccttctg gcctgaaggg ggagcctgga gagacaggac ctccaggacg gggcctgact 6660

ggacctactg gagctgtggg acttcctgga ccccccggcc cttcaggcct tgtgggtcca 6720

caggggtctc caggtttgcc tggacaagtg ggggagacag ggaagccggg agccccaggt 6780

cgagatggtg ccagtggaaa agatggagac agagggagcc ctggtgtgcc agggtcacca 6840

ggtctgcctg gccctgtcgg acctaaagga gaacctggcc ccacgggggc ccctggacag 6900

gctgtggtcg ggctccctgg agcaaaggga gagaagggag cccctggagg ccttgctgga 6960

gacctggtgg gtgagccggg agccaaaggt gaccgaggac tgccagggcc gcgaggcgag 7020

aagggtgaag ctggccgtgc aggggagccc ggagaccctg gggaagatgg tcagaaaggg 7080

gctccaggac ccaaaggttt caagggtgac ccaggagtcg gggtcccggg ctcccctggg 7140

cctcctggcc ctccaggtgt gaagggagat ctgggcctcc ctggcctgcc cggtgctcct 7200

ggtgttgttg ggttcccggg tcagacaggc cctcgaggag agatgggtca gccaggccct 7260

agtggagagc ggggtctggc aggcccccca gggagagaag gaatcccagg acccctgggg 7320

ccacctggac caccggggtc agtgggacca cctggggcct ctggactcaa aggagacaag 7380

ggagaccctg gagtagggct gcctgggccc cgaggcgagc gtggggagcc aggcatccgg 7440

ggtgaagatg gccgccccgg ccaggaggga ccccgaggac tcacggggcc ccctggcagc 7500

aggggagagc gtggggagaa gggtgatgtt gggagtgcag gactaaaggg tgacaaggga 7560

gactcagctg tgatcctggg gcctccaggc ccacggggtg ccaaggggga catgggtgaa 7620

cgagggcctc ggggcttgga tggtgacaaa ggacctcggg gagacaatgg ggaccctggt 7680

gacaagggca gcaagggaga gcctggtgac aagggctcag ccgggttgcc aggactgcgt 7740

ggactcctgg gaccccaggg tcaacctggt gcagcaggga tccctggtga cccgggatcc 7800

ccaggaaagg atggagtgcc tggtatccga ggagaaaaag gagatgttgg cttcatgggt 7860

ccccggggcc tcaagggtga acggggagtg aagggagcct gtggccttga tggagagaag 7920

ggagacaagg gagaagctgg tcccccaggc cgccccgggc tggcaggaca caaaggagag 7980

atgggggagc ctggtgtgcc gggccagtcg ggggcccctg gcaaggaggg cctgatcggt 8040

cccaagggtg accgaggctt tgacgggcag ccaggcccca agggtgacca gggcgagaaa 8100

ggggagcggg gaaccccagg aattgggggc ttcccaggcc ccagtggaaa tgatggctct 8160

gctggtcccc cagggccacc tggcagtgtt ggtcccagag gccccgaagg acttcagggc 8220

cagaagggtg agcgaggtcc ccccggagag agagtggtgg gggctcctgg ggtccctgga 8280

gctcctggcg agagagggga gcaggggcgg ccagggcctg ccggtcctcg aggcgagaag 8340

ggagaagctg cactgacgga ggatgacatc cggggctttg tgcgccaaga gatgagtcag 8400

cactgtgcct gccagggcca gttcatcgca tctggatcac gacccctccc tagttatgct 8460

gcagacactg ccggctccca gctccatgct gtgcctgtgc tccgcgtctc tcatgcagag 8520

gaggaagagc gggtaccccc tgaggatgat gagtactctg aatactccga gtattctgtg 8580

gaggagtacc aggaccctga agctccttgg gatagtgatg acccctgttc cctgccactg 8640

gatgagggct cctgcactgc ctacaccctg cgctggtacc atcgggctgt gacaggcagc 8700

acagaggcct gtcacccttt tgtctatggt ggctgtggag ggaatgccaa ccgttttggg 8760

acccgtgagg cctgcgagcg ccgctgccca ccccgggtgg tccagagcca ggggacaggt 8820

actgcccagg actga 8835

<210> 3

<211> 5782

<212> DNA

<213> Artificial Sequence

<400> 3

ggccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa 60

cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt 120

cattagttca tagcccatat atgggttaca taacttacgg taaatggccc gcctggctga 180

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 240

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 300

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 360

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 420

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 480

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 540

ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 600

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg 660

aaccgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 720

cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 780

gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 840

tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga 900

cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt 960

acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt 1020

attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag 1080

aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt 1140

aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca 1200

tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat 1260

tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa 1320

gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg 1380

aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga 1440

accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc 1500

agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc 1560

agcctcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca 1620

gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg 1680

catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct 1740

cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc 1800

tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga 1860

cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca 1920

gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg 1980

gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt 2040

ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata 2100

ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg 2160

aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc 2220

tcgacggtat cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 2280

aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 2340

aaaaattcaa aattttatcg atcacgagac tagcctcgag ggctccggtg cccgtcagtg 2400

ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac 2460

cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 2520

cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 2580

ttttcgcaac gggtttgccg ccagaacaca ggtgaattcc cgggatcctt aattaacgcg 2640

tgtcgacggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 2700

taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt 2760

ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 2820

tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa taaaggaaat 2880

ttattttcat tgcaatagtg tgttggtttt ttgtgtgctc tcacctatag tgagtcgtat 2940

tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 3000

caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 3060

cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc 3120

tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 3180

gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 3240

ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 3300

cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 3360

tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 3420

ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 3480

ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 3540

tttaacaaaa tattaacgtt tacaatttcc caggtggcac ttttcgggga aatgtgcgcg 3600

gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 3660

aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 3720

gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 3780

cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 3840

tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 3900

tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 3960

agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 4020

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 4080

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 4140

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 4200

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 4260

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 4320

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 4380

ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 4440

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 4500

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 4560

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 4620

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 4680

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 4740

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 4800

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 4860

cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 4920

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 4980

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5040

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5100

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5160

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5220

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5280

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 5340

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 5400

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 5460

gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 5520

cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 5580

ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 5640

aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 5700

ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 5760

gggaacaaaa gctggagctg ca 5782

Claims

1. A lentiviral vector plasmid carrying a codon optimized COL7A1 gene for infection of human epidermal stem cells, comprising: a lentiviral vector genome comprising an EFS promoter and a codon optimized COL7A1 gene sequence for encoding; wherein, the genome sequence of the lentiviral vector containing the EFS promoter is shown in SEQ ID No. 3; the codon optimized COL7A1 gene sequence is the nucleotide sequence shown in SEQ ID NO. 1.

2. A method for constructing a codon optimized COL7A1 gene lentiviral vector plasmid according to claim 1, comprising: subcloning the COL7A1 gene fragment with optimized coding codons into a pCCL-EFS plasmid to obtain a pCCL-EFS-COL7A1-OPT plasmid; the method comprises the following steps:

3. A Lentivirus carrying a codon optimized COL7A1 gene for infecting human epidermal stem cells, characterized in that the Lentivirus comprises a Lentivirus capsid, a Lentivirus genome packaged in the capsid, an EFS promoter and a sequence of COL7A1 genes encoding a codon optimization; the codon optimized COL7A1 gene sequence is a nucleotide sequence shown as SEQ ID NO. 1.

4. A method of preparing a lentivirus carrying a codon optimized COL7A1 gene according to claim 3, the method comprising a packaging method and a purification method, wherein the packaging process is as follows:

wherein, the purification process is as follows:

purifying by using GE AKTAavant chromatography system, adopting DEAE chromatography, tangential flow filtration and core700 chromatography purification process, and purifying to obtain GFP pseudo virus, wherein the specific purification process is as follows:

5. The method of claim 4, wherein the transfection conditions and process conditions during the packaging process are: inoculating 293T cells in a 10-layer cell factory 24h before transfection; preparing a transfection system containing opti-MEM culture medium, pCCL-EFS-COL7A1-OPT plasmid, psPAX2 plasmid and pSpike plasmid; adding the transfection system into a 10-layer cell factory, and gently and uniformly mixing; fresh DMEM medium was changed 6 hours after transfection, cells were continued to be cultured, and medium supernatant was collected after 72 hours of transfection, and purified.

6. Use of a lentivirus carrying a codon optimized COL7A1 gene of claim 3 for infecting human epidermal stem cells in the preparation of an RDEB therapeutic drug.