CN112852882A - System and method for producing AAV gene medicine by infecting insect cell with baculovirus - Google Patents
System and method for producing AAV gene medicine by infecting insect cell with baculovirus Download PDFInfo
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- CN112852882A CN112852882A CN202110159142.2A CN202110159142A CN112852882A CN 112852882 A CN112852882 A CN 112852882A CN 202110159142 A CN202110159142 A CN 202110159142A CN 112852882 A CN112852882 A CN 112852882A
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- baculovirus
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Abstract
The present invention relates to a system for producing AAV by infecting insect cells with baculovirus, comprising: a first baculovirus, a second baculovirus, a third baculovirus and Sf9 cell; the genome of the first baculovirus integrates an expression cassette for coding Rep78 protein and an expression cassette for coding Cap protein VP2, wherein the gene for coding Rep78 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP2 is driven to express by p10 promoter; the genome of the second baculovirus integrates an expression cassette for coding Rep52 protein and an expression cassette for coding Cap protein VP1, wherein the gene for coding Rep52 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP1 is driven to express by p10 promoter; the third baculovirus has integrated AAV vector sequence carrying the target gene expression cassette in its genome; co-infecting Sf9 cells with the first baculovirus, the second baculovirus and the third baculovirus to produce AAV carrying the target gene.
Description
Technical Field
The invention relates to the technical field of gene medicines, in particular to a system and a method for producing AAV gene medicines by infecting insect cells with baculovirus.
Background
Adeno-associated virus (AAV) is a member of the Parvoviridae family (paraviridae) and is a class of tiny, non-enveloped and icosahedral-structured viruses. The virus particles have a diameter of 20 to 26nm and contain a linear single-stranded DNA genome having a size of about 4.70 kb. The recombinant adeno-associated virus designed based on AAV is an important gene therapy vector and has safety and controllability.
AAV acts as a replication-defective DNA virus, replication of which is accomplished by coinfection with a helper virus. The genome structure is shown in FIG. 1. In the process of constructing rAAV, rep and cap genes of WtAAV are replaced by the expression system of the target gene for research, and only two ITRs at both ends of AAV genome are reserved. The ITRs have important biological functions in the process of replication and packaging of the rAAV and are necessary DNA sequences of the rAAV vector. The construction of recombinant viral vectors containing target genes and the easy and efficient production are the foundation for the development of rAAV-mediated gene therapy drugs. One of the methods for producing rAAV that is widely adopted at present is a three-plasmid cotransfection method, namely, a target gene and related functional fragments are used for replacing rep and cap genes of WtAAV virus to form a virus vector plasmid; the adenovirus helper genes necessary for production form another helper plasmid; and (3) forming a third plasmid by using an adeno-associated virus packaging structural gene (cap) and a non-structural replication gene (rep), and transfecting the third plasmid into a packaging cell line HEK293 which stably expresses Ela and EIb helper virus genes to produce the rAAV with the infection activity. In recent years, researchers have also reported that two helper plasmids are integrated into one plasmid for two-plasmid transfection, but there is a need to overcome the inhibitory effect of Rep proteins on helper viruses. Since rAAV is used as a gene vector, researchers have tried various ways of providing functional elements to conveniently, flexibly and massively produce rAAV that meets the needs of research and clinical application, and the means of providing elements so far mainly include plasmids, viruses and cell lines, and the existing methods are simply combined in one way or several ways.
Because plasmid can not be replicated in eukaryotic cells, progeny can not be generated to infect more host cells, so that the plasmid transfection method can not realize the capacity of transforming uninfected cells into infected cells by infected cells, and the scale of the system can be enlarged only by enlarging the culture area or volume and simultaneously enlarging the plasmid dosage in both an adherent system and a suspension culture system. The maximum reported scale is that 100 layers of Cell Cube are used as a container, and the Cell capacity is 1010The yield is 1014-1015And (c) viral particles. By optimizing the transfection method and the cell culture conditions, the yield of the current plasmid transfection method can reach 10 vg/cell. However, this system has a low input-output ratio and is difficult to remove rAAV plasmid.
Compared with the prior transfection method, the virus infection method does not need transient transfection or establishment of a stably transfected cell line, all elements are provided by virus, rAAV produced by adopting human cells needs amplification and transfection of adherent cells, and the restriction step of transfecting the adherent cells by plasmid cannot be completely separated in the production process. A recombinant baculovirus obtained from Autographa californica nuclear polyhedrosis virus is applied to an rAAV production system for insect cell production, and the above difficulties are thoroughly solved. The method has great improvement on the production process of rAAV, including completely getting rid of transient plasmid infection adherent cells, needing no auxiliary virus or genes thereof, having flexibility (producing different types of rAAV), low production cost (being capable of producing in serum-free culture medium), being capable of enduring a series of molar osmotic pressures, being capable of continuous large-scale production in suspension culture medium, the production yield and total amount completely meeting the requirements of clinic and marketization, and the exogenous gene being capable of high-efficiency expression.
3 different baculoviruses are needed in a baculovirus/insect cell production system, namely Bac-Rep, Rep52 containing rAAV and Rep78 which are respectively regulated and controlled by Polyhedrin promoter and IE (intermediate-early gene) promoter; (iv) Bav-VP or Bac-Cap: VP1, VP2 and VP3 containing 3 Cap genes, which are regulated by polyhedrin promoter; Bac-Transgene contains Transgene wrapped by rAAV terminal repetitive sequence, and is regulated and controlled by insect p10 and mammalian Cytomegalovirus (CMV) promoter. In a specific procedure, 3 kinds of baculovirus are first infected with Spodoptera frugiperda (Sf 9) cells, respectively, and then amplified, and the purified 3 kinds of baculovirus co-infect the rAAV insect-producing cells cultured in suspension. The parameters of infected cell quantity, survival rate, rAAV output and the like are regularly detected in the production process so as to optimize the production process. Cells are harvested and rAAV virus is extracted and purified at optimal time periods, after which the yield and quality of rAAV is tested. The titer after purification of baculovirus-based rAAV production systems to date can reach 2.00X 1015A vg/50L reactor, which can substantially satisfy preclinical studies and clinical studies of local therapy. However, Sf9 cells are non-human cells, and the virus prepared by the method has reduced infection capacity, and may influence the clinical application of the virus.
This approach has gained increasing attention since the first report of a baculovirus-based expression system for AAV production in insect cells. The use of insect cells to produce AAV vectors has been reported by several groups. However, since AAV introns and promoters do not function normally in insect cells, most of these studies use two separate Rep coding sequences in two expression cassettes, one Rep78 and the other Rep52, which are constructed in baculovirus with the two coding sequences arranged in a head-to-tail palindrome. In addition, these studies used the Cap coding sequence for the VP1 start codon mutation (AUG to ACG). Since Rep78 and Rep52 have large homologous repeats, it has been reported that recombinant baculoviruses carrying the homologous repeats of the Rep are unstable (homologous recombination brings instability), and the Rep protein expression is lost after several generations. AAV vectors produced in insect cells with mutations in the VP1 initiation codon, AUG-to-ACG, have also been reported to have reduced infection activity due to relatively poor expression of VP1 protein. In addition, scientists have designed and constructed a new artificial intron, which is inserted into the coding sequence of Rep and Cap to express Rep and Cap proteins. The single Rep coding sequence containing the artificial intron expresses both the Rep78 and Rep52 proteins, which avoids the use of a large number of homologous Rep repeats, and thus provides higher stability to recombinant baculoviruses carrying Rep coding sequences. At the same time, the authentic VP1 initiation codon AUG (to avoid a decrease in infection activity) was retained, since a single intron-containing Cap coding sequence would allow expression of all three Cap proteins (VP1, VP2, and VP 3). Finally, methods for producing AAV vectors in insect cells using these intron-inserted Rep and Cap coding sequences were established, and the obtained AAV vectors were produced in yields comparable to those of the previous methods and were highly infectious. However, the insertion of foreign introns into Rep and Cap proteins increases the complexity of gene regulation and is inefficient.
Disclosure of Invention
Technical problem to be solved
In view of the above disadvantages and shortcomings of the prior art, the present invention provides a system and method for producing AAV gene drugs by infecting insect cells with baculovirus, which avoids the problem of baculovirus instability caused by homologous recombination using two independent Rep coding sequences (Rep78 and Rep52), eliminates the need to insert exogenous introns into Rep and Cap proteins, reduces the complexity of gene regulation, and improves production efficiency while the obtained AAV exogenous genes can be efficiently expressed.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
in a first aspect, the present invention provides a method for producing AAV by infecting insect cells with a baculovirus, comprising:
s1, connecting one of two expression cassettes for coding a Rep78 protein and a Rep52 protein with an expression cassette for coding a Cap protein VP2(VP2 comprises a VP3 sequence), cloning the two expression cassettes on a first baculovirus vector, and respectively driving the two expression cassettes by a polyhedrin promoter and a p10 promoter to obtain a first baculovirus;
connecting the other of the two expression cassettes for coding the Rep78 protein and the Rep52 protein with an expression cassette for coding the Cap protein VP1, cloning the other expression cassette on a second baculovirus vector, and respectively driving the two expression cassettes by a polyhedrin promoter and a p10 promoter to obtain a second baculovirus;
s2, cloning the AAV vector sequence carrying the target gene expression cassette to a third baculovirus vector to obtain a third baculovirus;
s3, culturing Sf9 cells in an adherent manner, and co-infecting the Sf9 cells by using the first baculovirus, the second baculovirus and the third baculovirus when AAV needs to be produced to produce AAV carrying the target gene.
The above steps S1-S2 are not limited in sequence, and may be completed simultaneously or in steps.
According to a preferred embodiment of the present invention, in step S2, the genome of the third baculovirus has integrated thereon a plasmid sequence pFast-Bac-AAV-Target Gene, wherein Target Gene represents a Target Gene and AAV is an expression cassette sequence of adeno-associated virus.
According to a preferred embodiment of the invention, in step S2, the Target Gene is SMN1 Gene, the sequence of which is shown in SEQ ID No. 7, and is used for preparing AAV Gene medicine for treating spinal muscular atrophy;
or the Target Gene is a Gene sequence SEQ ID No. 8 or SEQ ID No. 9 for coding a soluble extracellular region ACE2 protein, the sequence shown in the SEQ ID No. 8 codes a soluble extracellular region ACE21-620 fragment, and the SEQ ID No. 9 codes a soluble extracellular region ACE21-740 fragment, so that the prepared AAV infects Target cells, and then the Target cells generate soluble ACE2 protein to the extracellular (the ACE2 protein can be specifically combined with coronavirus S protein), and the AAV Gene medicine for antagonizing SARS-CoV infection is prepared.
According to a preferred embodiment of the present invention, in step S2, the third baculovirus is prepared by: constructing a pFast-Bac-AAV-Target Gene plasmid vector, transfecting the plasmid vector into competent DH10Bac, and obtaining a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene to obtain the third baculovirus.
According to a preferred embodiment of the present invention, in step S1, the expression cassette encoding the Rep78 protein and the Cap protein VP2 are cloned into a first baculovirus vector to obtain a first baculovirus; connecting the Rep52 encoding protein and an expression cassette of the Cap protein VP1 and cloning the Rep52 encoding protein and the expression cassette onto a second baculovirus vector to obtain a second baculovirus;
the first baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep78/VP2 plasmid vector, wherein the plasmid vector contains a p10-VP2-polyhedrin-Rep78 sequence (shown as SEQ ID No: 6); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP 2; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP2 to obtain a first baculovirus;
the second baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep52/VP1 plasmid vector, wherein the plasmid vector contains a p10-VP1-polyhedrin-Rep52 sequence (shown as SEQ ID No: 5); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP 1; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP1 to obtain the second baculovirus.
According to the preferred embodiment of the present invention, in S1, the method for constructing pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmid vectors is as follows:
step 1: after the pFast-Bac-dual plasmid is subjected to double digestion treatment by using restriction enzyme, agarose electrophoresis is carried out, gel cutting is carried out, a pFast-Bac-dual carrier fragment is recovered, and the pFast-Bac-dual carrier fragment is respectively connected with a gene fragment for coding VP2 and a gene fragment for coding VP1 by adopting ligase;
transforming the ligation product into competent DH5a, mixing uniformly, performing ice bath and heat shock, immediately bathing, culturing by using a liquid culture medium without antibiotics, transferring to an agar plate containing ampicillin for continuous culture, selecting a monoclonal colony, inoculating into a liquid culture medium containing ampicillin for shake culture, extracting plasmids, performing double enzyme digestion identification, performing sequencing identification, and correspondingly obtaining pFast-Bac-dual-VP2 plasmid and pFast-Bac-dual-VP1 plasmid;
step 2: carrying out double digestion on pFast-Bac-dual-VP2 plasmid and pFast-Bac-dual-VP1 plasmid by using restriction enzymes respectively, carrying out agarose electrophoresis respectively, and cutting gel to recover pFast-Bac-dual-VP2 carrier fragment and pFast-Bac-dual-VP1 carrier fragment;
connecting a pFast-Bac-dual-VP2 vector fragment with a gene fragment for coding Rep78, and connecting the pFast-Bac-dual-VP1 vector fragment with the gene fragment for coding Rep52 by adopting ligase;
respectively transforming the connecting products into competent DH5a, mixing uniformly, performing ice bath and heat shock, then immediately bathing, firstly culturing by using a liquid culture medium without antibiotics, then transferring to an agar plate containing ampicillin for continuous culture, then selecting a monoclonal colony to inoculate in a liquid culture solution containing ampicillin for oscillation culture, extracting plasmids, performing double enzyme digestion identification, and then performing sequencing identification to successfully construct pFast-Bac-dual-Rep78/VP2 plasmid vectors and pFast-Bac-dual-Rep52/VP1 plasmid vectors;
after double digestion and ligase ligation by the above restriction enzymes, the pFast-Bac-dual-Rep78/VP2 plasmid vector contains a p10-VP2-polyhedrin-Rep78 sequence (shown as SEQ ID No:6), and the pFast-Bac-dual-Rep52/VP1 plasmid vector contains a p10-VP1-polyhedrin-Rep52 sequence (shown as SEQ ID No: 5).
According to a preferred embodiment of the present invention, the method for preparing the recombinant baculovirus plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 in S1 is:
plasmid vectors of pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 are diluted, transfected into competent DH10Bac respectively, mixed uniformly, subjected to ice bath and heat shock, immediately bathed, cultured by a liquid culture medium without antibiotics, transferred to an agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, and cultured continuously for screening blue and white spots;
selecting white large single clone to a fresh antibiotic-free plate culture medium, carrying out secondary streaking to an agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, carrying out culture and then screening again;
then, a monoclonal colony is selected and inoculated in a liquid culture solution containing kanamycin, gentamicin and tetracycline for culture, recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 are respectively extracted by phenol chloroform and are identified by M13F and M13R, and thus, recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-52/VP 1 are respectively obtained.
According to a preferred embodiment of the present invention, in S1, the method for obtaining the first baculovirus and the second baculovirus is:
diluting recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1, adding a diluted transfection reagent, simultaneously adding a mixture of the recombinant plasmids and the transfection reagent into adherent Sf9 cells, culturing for a period of time, and replacing a fresh culture medium; observing cells, and centrifuging to collect supernatant when most of the cells are enlarged and more than half of the cells are suspended to obtain a first baculovirus and a second baculovirus respectively; the pFast-Bac-dual-Rep78/VP2 plasmid sequence is integrated on the first baculovirus genome, and the pFast-Bac-dual-Rep52/VP1 plasmid sequence is integrated on the second baculovirus genome.
According to the preferred embodiment of the present invention, in step S3, the first baculovirus, the second baculovirus and the third baculovirus are used to co-infect Sf9 insect cells, the cells are harvested 72 hours after infection, the cells are repeatedly frozen and thawed to lyse the cells, the cell lysate is obtained, and the AAV carrying the target gene is produced through chromatography purification.
As a parallel scheme of the above scheme, the following is provided:
in step S1, connecting the Rep78 protein and the expression cassette of the Cap protein VP1 to clone onto a first baculovirus vector to obtain a first baculovirus; connecting the Rep52 encoding protein and an expression cassette of the Cap protein VP2 and cloning the Rep52 encoding protein and the expression cassette onto a second baculovirus vector to obtain a second baculovirus;
the first baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep78/VP1 plasmid vector, wherein the plasmid vector contains a p10-VP1-polyhedrin-Rep78 sequence; transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP 1; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP1 to obtain a first baculovirus;
the second baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep52/VP2 plasmid vector, wherein the plasmid vector contains a p10-VP2-polyhedrin-Rep52 sequence; transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP 2; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP2 to obtain a second baculovirus; the pFast-Bac-dual-Rep78/VP1 plasmid sequence is integrated on the first baculovirus genome, and the pFast-Bac-dual-Rep52/VP2 plasmid sequence is integrated on the second baculovirus genome.
In a second aspect, the present invention provides a system for producing AAV by infecting insect cells with a baculovirus, comprising: a first baculovirus, a second baculovirus, a third baculovirus and Sf9 cell;
the genome of the first baculovirus integrates an expression cassette for encoding Rep78 protein and an expression cassette for encoding Cap protein VP2 (containing VP3 sequence), wherein the gene for encoding Rep78 protein is driven by polyhedrin promoter to express, and the gene for encoding Cap protein VP2 is driven by p10 promoter to express;
the genome of the second baculovirus integrates an expression cassette for coding Rep52 protein and an expression cassette for coding Cap protein VP1, wherein the gene for coding Rep52 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP1 is driven to express by p10 promoter;
an AAV vector sequence (pFast-Bac-AAV-Target Gene) carrying a Target Gene expression cassette is integrated on the genome of the third baculovirus;
co-infecting Sf9 cells with the first baculovirus, the second baculovirus and the third baculovirus to produce AAV carrying the target gene.
In a third aspect, the present invention provides a system for producing AAV by infecting insect cells with a baculovirus, comprising: a first baculovirus, a second baculovirus, a third baculovirus and Sf9 cell;
the genome of the first baculovirus integrates an expression cassette for encoding Rep52 protein and an expression cassette for encoding Cap protein VP2 (containing VP3 sequence), wherein the gene for encoding Rep52 protein is driven by polyhedrin promoter to express, and the gene for encoding Cap protein VP2 is driven by p10 promoter to express;
the genome of the second baculovirus integrates an expression cassette for coding Rep78 protein and an expression cassette for coding Cap protein VP1, wherein the gene for coding Rep78 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP1 is driven to express by p10 promoter;
an AAV vector sequence (pFast-Bac-AAV-Target Gene) carrying a Target Gene expression cassette is integrated on the genome of the third baculovirus;
co-infecting Sf9 cells with the first baculovirus, the second baculovirus and the third baculovirus to produce AAV carrying the target gene.
(III) advantageous effects
The invention has the technical effects that:
through three cloning baculoviruses, Rep78 and Rep52 were cloned separately into two different cloning baculoviruses, and then AAV vector sequences carrying expression cassettes of the genes of interest were cloned onto a third baculovirus, and Sf9 cells were infected simultaneously with the three baculoviruses to produce AAV. The method of the invention avoids the problem of baculovirus instability caused by homologous recombination of two independent Rep coding sequences (Rep78 and Rep52) cloned into the same baculovirus. The invention also needs no insertion of exogenous intron in Rep and Cap proteins, reduces the complexity of gene regulation, and can efficiently express the obtained AAV exogenous gene while improving the production efficiency.
Drawings
FIG. 1 shows the genome structure of adeno-associated virus.
FIG. 2 is a schematic diagram of the p10-VP2-polyhedrin-Rep78 sequence (SEQ ID No:6) contained in the pFast-Bac-dual-Rep78/VP2 plasmid vector.
FIG. 3 is a schematic diagram of the p10-VP1-polyhedrin-Rep52 sequence (SEQ ID No:2) contained in the pFast-Bac-dual-Rep52/VP1 plasmid vector.
FIG. 4 is a graph showing the expression levels of Rep, Cap in cell lysates after Sf9 cells were separately administered using a first baculovirus and a second baculovirus in examples of the present invention.
FIG. 5 shows the demonstration of the expression of GFP in AAV by infecting U87MG cells with Sf9 cell lysate after co-infecting Sf9 cells with the first, second and third baculoviruses in the present example.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
The method for producing AAV by infecting insect cells with baculovirus of the embodiment of the invention comprises the following steps:
(1) the expression cassette coding the Rep78 protein and the expression cassette coding the Cap protein VP2(VP2 comprises VP3 sequence) are linked and cloned on a first baculovirus vector, and are driven by polyhedrin and p10 promoters respectively to obtain a first baculovirus.
The first baculovirus preparation process comprises:
constructing a pFast-Bac-dual-Rep78/VP2 plasmid vector, wherein the plasmid vector contains a p10-VP2-polyhedrin-Rep78 sequence (shown as SEQ ID No: 6); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP 2; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP2 to obtain a first baculovirus.
(2) And (3) connecting the expression cassette for coding the Rep52 protein and the expression cassette for coding the Cap protein VP1, cloning the expression cassettes on a second baculovirus vector, and driving the vectors by polyhedrin and p10 promoters respectively to obtain a second baculovirus.
The second baculovirus preparation process comprises:
constructing a pFast-Bac-dual-Rep52/VP1 plasmid vector, wherein the plasmid vector contains a p10-VP1-polyhedrin-Rep52 sequence (shown as SEQ ID No: 5); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP 1; and (3) transfecting adherent Sf9 cells by using the recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP1 to obtain a second baculovirus.
(3) Cloning the AAV vector sequence carrying the target gene expression cassette to a third baculovirus vector to obtain a third baculovirus.
The third baculovirus preparation process is as follows:
constructing a pFast-Bac-AAV-Target Gene plasmid vector, transfecting the plasmid vector into competent DH10Bac, and obtaining a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene to obtain the third baculovirus.
(4) Culturing Sf9 cells by adherence, and co-infecting Sf9 cells by using a first baculovirus, a second baculovirus and a third baculovirus when AAV needs to be produced to produce AAV, wherein the method specifically comprises the following steps:
the method for determining the ratio of the first baculovirus, the second baculovirus and the third baculovirus by using MOI1, 1: 1: 1, infecting Sf9 cells together, collecting the cells after infecting for 72h, repeatedly freezing and thawing the lysed cells to obtain cell lysate, and carrying out chromatography purification to produce AAV carrying target genes.
The above steps (1) - (3) can be performed in parallel.
Wherein, a plasmid sequence pFast-Bac-AAV-Target Gene is integrated on the genome of the third baculovirus, wherein the Target Gene represents a Target Gene, and AAV is an expression cassette sequence of adeno-associated virus. The Target Gene can be various Target genes, for example, when the Target Gene is SMN1 Gene (shown as SEQ ID No: 7), AAV Gene medicine for treating spinal muscular atrophy can be produced, or the Target Gene is a Gene sequence (SEQ ID No:8 or SEQ ID No:9, the former encodes soluble extracellular region ACE21-620 fragment, the latter encodes soluble extracellular region ACE21-740 fragment) encoding soluble extracellular region ACE2 protein, both of which can make cells express soluble ACE2 protein to the outside of the cells (ACE2 protein can be specifically combined with coronavirus S protein), so as to prepare AAV Gene medicine for antagonizing SARS-CoV infection.
Now, the scheme, realizability and technical effects of the present invention will be described by taking GFP as an example of a target gene. The implementation method comprises the following steps:
firstly, constructing pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmid vectors
The vector plasmid is composed of a commercial vector pFast-Bac-dual (invitrogen 10712024), a Rep78 gene sequence of AAV2, a Rep52 gene sequence, a VP1 gene sequence and a VP2 gene sequence; p10-VP1-polyhedrin-Rep52, p10-VP2-polyhedrin-Rep78 constitute the core expression cassette of two plasmids.
The sequence of Rep78 is SEQ ID No. 1, the sequence of Rep52 is SEQ ID No. 2, the sequence of VP1 is SEQ ID No. 3, the sequence of VP2 is SEQ ID No. 4, the sequence of p10-VP1-polyhedrin-Rep52 is SEQ ID No. 5, and the sequence of p10-VP2-polyhedrin-Rep78 is SEQ ID No. 6.
The pFast-Bac-dual has two promoters of p10 and polyhedrin, and double digestion and ligase connection are carried out by two times of restriction enzymes, so that VP2 (or VP1) is connected to one side of p10, Rep78 (or Rep52) is connected to one side of the polyhedrin, the p10 promoter drives VP2 (or VP1), and the polyhedrin promoter drives Rep78 (or Rep 52).
The sequence structure of the p10-VP1-polyhedrin-Rep52 sequence (SEQ ID No:5) is shown in FIG. 3, and the sequence structure of the p10-VP2-polyhedrin-Rep78 sequence (SEQ ID No:6) is shown in FIG. 2.
The construction method of pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 is as follows:
(1) the pFast-Bac-dual plasmid is subjected to double digestion for 1h at 37 ℃ by using restriction endonucleases NheI and KpnI, and a pFast-Bac-dual vector fragment is recovered by cutting gel after agarose electrophoresis.
Carrying out PCR amplification on fragments of the coding VP1 gene and the coding VP2 gene, respectively adding a protective base and a NheI/KpnI enzyme digestion site at the 5 'end and the 3' end, carrying out double enzyme digestion on the amplified PCR fragments at 37 ℃ for 1h by using NheI and KpnI, carrying out agarose electrophoresis, cutting gel, and recovering the fragments of the coding VP1 gene and the coding VP2 gene.
(2) And (3) respectively recovering the pFast-Bac-dual vector fragment, the gene coding VP1 and the gene coding VP2 from the gel recovery kit, connecting by adopting T4 DNA ligase, and reacting for 15min at room temperature.
(3) The ligation product was transformed into E.coli: taking the ligation product transformation competent DH5a, gently mixing uniformly, and carrying out ice bath for 30 min; performing heat shock at 42 deg.C for 80s, immediately ice-bathing for 5min, adding LB culture solution without antibiotic, shaking at 37 deg.C for 60min, uniformly coating the bacterial solution on LB agar plate containing ampicillin with sterile glass coater, and performing inverted culture at 37 deg.C for 14 h.
(4) Selecting a monoclonal colony, inoculating the colony in an LB liquid culture solution containing ampicillin, and oscillating for 16h at 37 ℃; plasmids pFast-Bac-dual-VP1 and pFast-Bac-dual-VP2 are extracted by using a plasmid extraction kit, and sequencing identification is carried out after double enzyme digestion identification of NheI and KpnI, so that plasmids pFast-Bac-dual-VP1 and pFast-Bac-dual-VP2 are obtained.
(5) The pFast-Bac-dual-VP1 and pFast-Bac-dual-VP2 plasmids are subjected to double digestion for 1h at 37 ℃ by using restriction enzymes NotI and XbaI, and pFast-Bac-dual-VP1 and pFast-Bac-dual-VP2 vector fragments are recovered by cutting gel after agarose electrophoresis.
Carrying out PCR amplification on the Rep78 coding gene and the Rep52 coding gene fragments, respectively adding a protective base and NotI/XbaI enzyme cutting sites at the 5 'end and the 3' end, carrying out double enzyme cutting on the PCR fragments obtained by amplification for 1h at 37 ℃ by using NotI and XbaI, cutting gel after agarose electrophoresis, and recovering the Rep78 coding gene and the Rep52 coding gene fragments.
(6) And (3) respectively recovering the pFast-Bac-dual-VP1 vector fragment and the Rep52 gene which are recovered by the gel recovery kit, connecting by adopting T4 DNA ligase, and reacting for 15min at room temperature.
And (3) respectively recovering the pFast-Bac-dual-VP2 vector fragment and the Rep78 gene which are recovered by the gel recovery kit, connecting by adopting T4 DNA ligase, and reacting for 15min at room temperature.
(7) The ligation product was transformed into E.coli: taking the ligation product to convert into competent DH5a, mixing gently, and freezing for 30 min; performing heat shock at 42 deg.C for 80s, immediately ice-bathing for 5min, adding LB culture solution without antibiotic, shaking at 37 deg.C for 60min, uniformly coating the bacterial solution on LB agar plate containing ampicillin with sterile glass coater, and performing inverted culture at 37 deg.C for 14 h.
(8) Selecting a monoclonal colony, inoculating the colony in an LB liquid culture solution containing ampicillin, and oscillating for 16h at 37 ℃; plasmids pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 are extracted by using a plasmid extraction kit, and sequencing identification is carried out after NotI and XhoI double enzyme digestion identification until the pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmids are successfully constructed.
Secondly, acquiring the baculovirus pFast-Bac-dual-Rep78/VP2, pFast-Bac-dual-Rep52/VP1
The pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmid vectors are firstly transfected with competent DH10Bac to obtain recombinant baculovirus plasmids, and then the recombinant baculovirus plasmids are transfected with SF9 cells to obtain corresponding baculovirus, and the specific steps are as follows:
(1) the pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmids were transformed into DH10Bac Escherichia coli:
diluting pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmids according to different concentrations, transfecting competent DH10Bac, gently mixing uniformly, and carrying out ice bath for 30 min; performing heat shock at 42 ℃ for 45s, immediately performing ice bath for 4min, adding LB culture solution without antibiotics to 1ml, oscillating at 37 ℃ for 60min, uniformly coating the bacterial solution on an LB agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG by using a sterile glass coater, performing inverted culture at 37 ℃ for 55h, and performing blue-white spot screening.
(2) White large single clones were picked into fresh non-resistant LB, streaked twice onto LB agar plates containing kanamycin, gentamicin, tetracycline, X-gal, IPTG, and subjected to inverted culture at 37 ℃ for 55h, followed by re-screening.
(3) Selecting a monoclonal colony to be inoculated in LB liquid culture solution containing kanamycin, gentamicin and tetracycline, and oscillating for 16h at 37 ℃; the recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 are extracted by phenol chloroform and identified by M13F and M13R, so that the recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 are obtained.
(4) Insect cell Sf9 at 2X 106One 35mm plate was placed in each well, and cultured at 27 ℃ for 1 hour to allow cells to adhere to the wall.
(5) And (2) diluting 20 mu l of Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 recombinant plasmid to 100 mu l by using Grace culture medium, gently flicking and uniformly mixing, standing for 5min at room temperature, diluting 8 mu l of Cellffectin II transfection reagent to 100 mu l by using Grace culture medium, gently flicking and uniformly mixing, standing for 5min at room temperature, adding the diluted transfection reagent into the diluted plasmid, gently flicking and uniformly mixing, and standing for 15min at room temperature.
(6) The transfection reagent plasmid mixture was added to adherent Sf9 cells and replaced with fresh medium after 6h incubation at 27 ℃.
(7) Cells were observed daily after transfection, and when most of the cells became large in volume and more than half of the cells were suspended off the surface of the dish, the supernatant was harvested by centrifugation at 4000rpm for 5min to obtain pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 baculoviruses of P1 generation.
And thirdly, verifying the protein expression of baculovirus of pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1
Sf9 cells were infected with baculovirus, and the expression of Rep78, Rep52, VP1 and VP2 was examined by Western Blot after harvesting the cells. The specific experimental steps are as follows:
(1) insect cell Sf9 at 2X 106One 35mm plate was placed in each well, and cultured at 27 ℃ for 1 hour to allow cells to adhere to the wall.
(2) And (3) respectively taking pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 baculovirus to infect Sf9 cells with MOI 1.
(3) After infection for 72h, collected cells are respectively collected and lysed to obtain Sf9pFast-Bac-dual-Rep78/VP2 lysate and Sf9pFast-Bac-dual-Rep 52/VP1 lysate.
(4) Preparing 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) glue, carrying out protein concentration determination on Sf9pFast-Bac-dual-Rep78/VP2 lysate and Sf9pFast-Bac-dual-Rep 52/VP1 lysate by using a BCA (brain-opening cell protein) method, adding cell lysate with the same protein amount into 6% SDS-PAGE glue, running to the junction of the compression glue and the separation glue by using 80V voltage, and running to the bottom of the glue by using 120V voltage.
(5) SDS-PAGE gels containing Sf9pFast-Bac-dual-Rep78/VP2 protein and Sf9pFast-Bac-dual-Rep 52/VP1 protein were transferred to NC membranes using a 400mA constant current.
(6) NC membranes transferred with Sf9pFast-Bac-dual-Rep78/VP2 protein and Sf9pFast-Bac-dual-Rep 52/VP1 protein were incubated with 5% skim milk at room temperature for 1h, and the membranes were washed 3 times with TBST for 5min each.
(7) The primary antibody to Cap was incubated with Rep overnight at 4 ℃.
(8) The following day the membranes were washed 3 times with TBST for 10min each, incubated with secondary HRP containing antibody for 1h at room temperature, and washed 3 times with TBST for 5min each.
(9) The result of Sf9 expression level of baculovirus pFast-Bac-dual-Rep78/VP2 and baculovirus pFast-Bac-dual-Rep52/VP1 is obtained by developing by ECL method.
As shown in FIG. 4, both baculoviruses infected sf9 cells, and the packaging components Rep, Cap of the adeno-associated virus were well expressed.
And fourthly, infecting Sf9 with pFast-Bac-dual-Rep78/VP2, pFast-Bac-dual-Rep52/VP1 and pFast-Bac-AAV-GFP (addge #40868) baculovirus to generate AAV feasibility verification
Three baculoviruses are used for co-infecting Sf9 cells, the cells are harvested after infection for 72h, cell lysates are obtained by repeated freeze-thaw lysis of the cells, and the feasibility of GFP expression in AAV is verified by infecting target cells U87MG with the cell lysates. The specific experimental steps are as follows:
(1) insect cell Sf9 at 2X 106Laying a 6-hole plate in each hole, and culturing at 27 ℃ for 1h to make the cells adhere to the wall;
(2) pFast-Bac-dual-Rep78/VP2, pFast-Bac-dual-Rep52/VP1 and pFast-Bac-AAV-GFP baculovirus were expressed using MOI1, 1: 1: 1 to co-infect Sf9 cells, and simultaneously taking the same amount of pFast-Bac-AAV-GFP baculovirus infected Sf9 cells as negative control;
(3) after infection for 72h, Sf9 cells were harvested and frozen at-80 deg.C, thawed rapidly at 37 deg.C, frozen again at-80 deg.C with shaking on a vortex shaker for 5s, repeated 3 times, centrifuged at 12000rpm for 10min, and cell supernatants were filtered through a 0.22 μm filter.
(4) The U87MG cells are paved into a 6-hole plate one day in advance, the confluency of the cells reaches 60% the next day, filtered cell supernatants are infected with target cells U87MG, GFP fluorescence is observed for 72 hours, and the feasibility of the system is verified by comparing the number of GFP fluorescence of infected U87MG by Sf9 cell lysate co-infected with three baculoviruses and Sf9 cell lysate infected with pFast-Bac-AAV-GFP baculovirus only.
As shown in FIG. 5, when sf9 was infected with the three baculoviruses, and the purified cell lysate infected with U87MG, green fluorescence was generated in the target cells, indicating that the AAV carried GFP protein sequence was expressed in the target cells. Thus, it was demonstrated that the virus production system in which three baculoviruses of the present invention infect insect cells can efficiently produce AAV.
The above examples are described by taking the objective gene as GFP, and the baculovirus of pFast-Bac-AAV-GFP (addge #40868) used in the examples is an existing baculovirus. When preparing AAV gene medicine in practical production and application, different target genes are carried according to the treatment requirement to achieve the treatment purpose of gene repair or specific protein production. Thus, based on the above examples, the Target Gene plasmid can be represented by the general formula of pFast-Bac-AAV-Target Gene, wherein Target Gene can be various Target genes, for example, when Target Gene is SMN1 Gene (as shown in SEQ ID No: 7), AAV Gene drug for treating spinal muscular atrophy can be produced, or Target Gene is a Gene sequence (SEQ ID No:8 or SEQ ID No:9) encoding soluble extracellular region ACE2 protein, which can make cells express soluble ACE2 protein to the extracellular (ACE2 protein can be specifically combined with coronavirus S protein), and AAV Gene drug for antagonizing SARS-CoV infection can be prepared.
Baculoviruses of different target gene plasmids can be prepared as follows: constructing a pFast-Bac-AAV-Target Gene plasmid vector, transfecting the plasmid vector into competent DH10Bac, and obtaining a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene to obtain the third baculovirus.
In a parallel embodiment of the above embodiment, an expression cassette encoding Rep52 protein and an expression cassette encoding Cap protein VP2(VP2 comprises VP3 sequence) may be ligated and cloned into a first baculovirus vector, and the first baculovirus vector may be obtained by driving with polyhedrin and p10 promoter, respectively. Meanwhile, an expression cassette for coding Rep78 protein and an expression cassette for coding Cap protein VP1 are connected and cloned to a second baculovirus vector, and are driven by polyhedrin and a p10 promoter respectively to obtain a second baculovirus. The specific preparation process of baculovirus is similar to the above scheme, and is not described again.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Zhongji Zhi medicine (Nanjing) Biotech Co., Ltd
<120> a system and method for producing AAV gene drug by infecting insect cell with baculovirus
<141> 2021-02-04
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3208
<212> DNA/RNA
<213> Artificial Sequence
<400> 1
actcgacgat ggagctggtc gggtggctcg tggacaaggg gattacctcg gagaagcagt 60
ggatccagga ggaccaggcc tcatacatct ccttcaatgc ggcctccaac tcgcggtccc 120
aaatcaaggc tgccttggac aatgcgggaa agattatgag cctgactaaa accgcccccg 180
actacctggt gggccagcag cccgtggagg acatttccag caatcggatt tataaaattt 240
tggaactaaa cgggtacgat ccccaatatg cggcttccgt ctttctggga tgggccacga 300
aaaagttcgg caagaggaac accatctggc tgtttgggcc tgcaactacc gggaagacca 360
acatcgcgga ggccatagcc cacactgtgc ccttctacgg gtgcgtaaac tggaccaatg 420
agaactttcc cttcaacgac tgtgtcgaca agatggtgat ctggtgggag gaggggaaga 480
tgaccgccaa ggtcgtggag tcggccaaag ccattctcgg aggaagcaag gtgcgcgtgg 540
accagaaatg caagtcctcg gcccagatag acccgactcc cgtgatcgtc acctccaaca 600
ccaacatgtg cgccgtgatt gacgggaact caacgacctt cgaacaccag cagccgttgc 660
aagaccggat gttcaaattt gaactcaccc gccgtctgga tcatgacttt gggaaggtca 720
ccaagcagga agtcaaagac tttttccggt gggcaaagga tcacgtggtt gaggtggagc 780
atgaattcta cgtcaaaaag ggtggagcca agaaaagacc cgcccccagt gacgcagata 840
taagtgagcc caaacgggtg cgcgagtcag ttgcgcagcc atcgacgtca gacgcggaag 900
cttcgatcaa ctacgcagac aggtaccaaa acaaatgttc tcgtcacgtg ggcatgaatc 960
tgatgctgtt tccctgcaga caatgcgaga gaatgaatca gaattcaaat atctgcttca 1020
ctcacggaca gaaagactgt ttagagtgct ttcccgtgtc agaatctcaa cccgtttctg 1080
tcgtcaaaaa ggcgtatcag aaactgtgct acattcatca tatcatggga aaggtgccag 1140
acgcttgcac tgcctgcgat ctggtcaatg tggatttgga tgactgcatc tttgaacaat 1200
aaatgattta aatcaggtat ggctgccgat ggttatcttc cagattggct cgaggacact 1260
ctctctgaac tcgacgatgc cggggtttta cgagattgtg attaaggtcc ccagcgacct 1320
tgacgagcat ctgcccggca tttctgacag ctttgtgaac tgggtggccg agaaggaatg 1380
ggagttgccg ccagattctg acatggatct gaatctgatt gagcaggcac ccctgaccgt 1440
ggccgagaag ctgcagcgcg actttctgac ggaatggcgc cgtgtgagta aggccccgga 1500
ggcccttttc tttgtgcaat ttgagaaggg agagagctac ttccacatgc acgtgctcgt 1560
ggaaaccacc ggggtgaaat ccatggtttt gggacgtttc ctgagtcaga ttcgcgaaaa 1620
actgattcag agaatttacc gcgggatcga gccgactttg ccaaactggt tcgcggtcac 1680
aaagaccaga aatggcgccg gaggcgggaa caaggtggtg gatgagtgct acatccccaa 1740
ttacttgctc cccaaaaccc agcctgagct ccagtgggcg tggactaata tggaacagta 1800
tttaagcgcc tgtttgaatc tcacggagcg taaacggttg gtggcgcagc atctgacgca 1860
cgtgtcgcag acgcaggagc agaacaaaga gaatcagaat cccaattctg atgcgccggt 1920
gatcagatca aaaacttcag ccaggtacat ggagctggtc gggtggctcg tggacaaggg 1980
gattacctcg gagaagcagt ggatccagga ggaccaggcc tcatacatct ccttcaatgc 2040
ggcctccaac tcgcggtccc aaatcaaggc tgccttggac aatgcgggaa agattatgag 2100
cctgactaaa accgcccccg actacctggt gggccagcag cccgtggagg acatttccag 2160
caatcggatt tataaaattt tggaactaaa cgggtacgat ccccaatatg cggcttccgt 2220
ctttctggga tgggccacga aaaagttcgg caagaggaac accatctggc tgtttgggcc 2280
tgcaactacc gggaagacca acatcgcgga ggccatagcc cacactgtgc ccttctacgg 2340
gtgcgtaaac tggaccaatg agaactttcc cttcaacgac tgtgtcgaca agatggtgat 2400
ctggtgggag gaggggaaga tgaccgccaa ggtcgtggag tcggccaaag ccattctcgg 2460
aggaagcaag gtgcgcgtgg accagaaatg caagtcctcg gcccagatag acccgactcc 2520
cgtgatcgtc acctccaaca ccaacatgtg cgccgtgatt gacgggaact caacgacctt 2580
cgaacaccag cagccgttgc aagaccggat gttcaaattt gaactcaccc gccgtctgga 2640
tcatgacttt gggaaggtca ccaagcagga agtcaaagac tttttccggt gggcaaagga 2700
tcacgtggtt gaggtggagc atgaattcta cgtcaaaaag ggtggagcca agaaaagacc 2760
cgcccccagt gacgcagata taagtgagcc caaacgggtg cgcgagtcag ttgcgcagcc 2820
atcgacgtca gacgcggaag cttcgatcaa ctacgcagac aggtaccaaa acaaatgttc 2880
tcgtcacgtg ggcatgaatc tgatgctgtt tccctgcaga caatgcgaga gaatgaatca 2940
gaattcaaat atctgcttca ctcacggaca gaaagactgt ttagagtgct ttcccgtgtc 3000
agaatctcaa cccgtttctg tcgtcaaaaa ggcgtatcag aaactgtgct acattcatca 3060
tatcatggga aaggtgccag acgcttgcac tgcctgcgat ctggtcaatg tggatttgga 3120
tgactgcatc tttgaacaat aaatgattta aatcaggtat ggctgccgat ggttatcttc 3180
cagattggct cgaggacact ctctctga 3208
<210> 2
<211> 1268
<212> DNA/RNA
<213> Artificial Sequence
<400> 2
actcgacgat ggagctggtc gggtggctcg tggacaaggg gattacctcg gagaagcagt 60
ggatccagga ggaccaggcc tcatacatct ccttcaatgc ggcctccaac tcgcggtccc 120
aaatcaaggc tgccttggac aatgcgggaa agattatgag cctgactaaa accgcccccg 180
actacctggt gggccagcag cccgtggagg acatttccag caatcggatt tataaaattt 240
tggaactaaa cgggtacgat ccccaatatg cggcttccgt ctttctggga tgggccacga 300
aaaagttcgg caagaggaac accatctggc tgtttgggcc tgcaactacc gggaagacca 360
acatcgcgga ggccatagcc cacactgtgc ccttctacgg gtgcgtaaac tggaccaatg 420
agaactttcc cttcaacgac tgtgtcgaca agatggtgat ctggtgggag gaggggaaga 480
tgaccgccaa ggtcgtggag tcggccaaag ccattctcgg aggaagcaag gtgcgcgtgg 540
accagaaatg caagtcctcg gcccagatag acccgactcc cgtgatcgtc acctccaaca 600
ccaacatgtg cgccgtgatt gacgggaact caacgacctt cgaacaccag cagccgttgc 660
aagaccggat gttcaaattt gaactcaccc gccgtctgga tcatgacttt gggaaggtca 720
ccaagcagga agtcaaagac tttttccggt gggcaaagga tcacgtggtt gaggtggagc 780
atgaattcta cgtcaaaaag ggtggagcca agaaaagacc cgcccccagt gacgcagata 840
taagtgagcc caaacgggtg cgcgagtcag ttgcgcagcc atcgacgtca gacgcggaag 900
cttcgatcaa ctacgcagac aggtaccaaa acaaatgttc tcgtcacgtg ggcatgaatc 960
tgatgctgtt tccctgcaga caatgcgaga gaatgaatca gaattcaaat atctgcttca 1020
ctcacggaca gaaagactgt ttagagtgct ttcccgtgtc agaatctcaa cccgtttctg 1080
tcgtcaaaaa ggcgtatcag aaactgtgct acattcatca tatcatggga aaggtgccag 1140
acgcttgcac tgcctgcgat ctggtcaatg tggatttgga tgactgcatc tttgaacaat 1200
aaatgattta aatcaggtat ggctgccgat ggttatcttc cagattggct cgaggacact 1260
ctctctga 1268
<210> 3
<211> 2211
<212> DNA/RNA
<213> Artificial Sequence
<400> 3
atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60
gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120
aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180
aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240
cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300
caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360
gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420
ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480
aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540
tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600
cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660
gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780
tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840
tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900
ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960
caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020
acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080
gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140
acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200
ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260
cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320
gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380
ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440
ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500
tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560
ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620
ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680
accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740
gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800
atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860
aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920
aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980
gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040
gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100
tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160
tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211
<210> 4
<211> 1800
<212> DNA/RNA
<213> Artificial Sequence
<400> 4
acggctcctg gaaagaagag gcctgtagag cagtctcctc aggaaccgga ctcctccgcg 60
ggtattggca aatcgggtgc acagcccgct aaaaagagac tcaatttcgg tcagactggc 120
gacacagagt cagtcccaga ccctcaacca atcggagaac ctcccgcagc cccctcaggt 180
gtgggatctc ttacaatggc ttcaggtggt ggcgcaccag tggcagacaa taacgaaggt 240
gccgatggag tgggtagttc ctcgggaaat tggcattgcg attcccaatg gctgggggac 300
agagtcatca ccaccagcac ccgaacctgg gccctgccca cctacaacaa tcacctctac 360
aagcaaatct ccaacagcac atctggagga tcttcaaatg acaacgccta cttcggctac 420
agcaccccct gggggtattt tgacttcaac agattccact gccacttctc accacgtgac 480
tggcagcgac tcatcaacaa caactgggga ttccggccta agcgactcaa cttcaagctc 540
ttcaacattc aggtcaaaga ggttacggac aacaatggag tcaagaccat cgccaataac 600
cttaccagca cggtccaggt cttcacggac tcagactatc agctcccgta cgtgctcggg 660
tcggctcacg agggctgcct cccgccgttc ccagcggacg ttttcatgat tcctcagtac 720
gggtatctga cgcttaatga tggaagccag gccgtgggtc gttcgtcctt ttactgcctg 780
gaatatttcc cgtcgcaaat gctaagaacg ggtaacaact tccagttcag ctacgagttt 840
gagaacgtac ctttccatag cagctacgct cacagccaaa gcctggaccg actaatgaat 900
ccactcatcg accaatactt gtactatctc tcaaagacta ttaacggttc tggacagaat 960
caacaaacgc taaaattcag tgtggccgga cccagcaaca tggctgtcca gggaagaaac 1020
tacatacctg gacccagcta ccgacaacaa cgtgtctcaa ccactgtgac tcaaaacaac 1080
aacagcgaat ttgcttggcc tggagcttct tcttgggctc tcaatggacg taatagcttg 1140
atgaatcctg gacctgctat ggccagccac aaagaaggag aggaccgttt ctttcctttg 1200
tctggatctt taatttttgg caaacaagga actggaagag acaacgtgga tgcggacaaa 1260
gtcatgataa ccaacgaaga agaaattaaa actactaacc cggtagcaac ggagtcctat 1320
ggacaagtgg ccacaaacca ccagagtgcc caagcacagg cgcagaccgg ctgggttcaa 1380
aaccaaggaa tacttccggg tatggtttgg caggacagag atgtgtacct gcaaggaccc 1440
atttgggcca aaattcctca cacggacggc aactttcacc cttctccgct gatgggaggg 1500
tttggaatga agcacccgcc tcctcagatc ctcatcaaaa acacacctgt acctgcggat 1560
cctccaacgg ccttcaacaa ggacaagctg aactctttca tcacccagta ttctactggc 1620
caagtcagcg tggagatcga gtgggagctg cagaaggaaa acagcaagcg ctggaacccg 1680
gagatccagt acacttccaa ctattacaag tctaataatg ttgaatttgc tgttaatact 1740
gaaggtgtat atagtgaacc ccgccccatt ggcaccagat acctgactcg taatctgtaa 1800
<210> 5
<211> 4256
<212> DNA/RNA
<213> Artificial Sequence
<400> 5
gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60
gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtacct 120
tacagattac gagtcaggta tctggtgcca atggggcggg gttcactata tacaccttca 180
gtattaacag caaattcaac attattagac ttgtaatagt tggaagtgta ctggatctcc 240
gggttccagc gcttgctgtt ttccttctgc agctcccact cgatctccac gctgacttgg 300
ccagtagaat actgggtgat gaaagagttc agcttgtcct tgttgaaggc cgttggagga 360
tccgcaggta caggtgtgtt tttgatgagg atctgaggag gcgggtgctt cattccaaac 420
cctcccatca gcggagaagg gtgaaagttg ccgtccgtgt gaggaatttt ggcccaaatg 480
ggtccttgca ggtacacatc tctgtcctgc caaaccatac ccggaagtat tccttggttt 540
tgaacccagc cggtctgcgc ctgtgcttgg gcactctggt ggtttgtggc cacttgtcca 600
taggactccg ttgctaccgg gttagtagtt ttaatttctt cttcgttggt tatcatgact 660
ttgtccgcat ccacgttgtc tcttccagtt ccttgtttgc caaaaattaa agatccagac 720
aaaggaaaga aacggtcctc tccttctttg tggctggcca tagcaggtcc aggattcatc 780
aagctattac gtccattgag agcccaagaa gaagctccag gccaagcaaa ttcgctgttg 840
ttgttttgag tcacagtggt tgagacacgt tgttgtcggt agctgggtcc aggtatgtag 900
tttcttccct ggacagccat gttgctgggt ccggccacac tgaattttag cgtttgttga 960
ttctgtccag aaccgttaat agtctttgag agatagtaca agtattggtc gatgagtgga 1020
ttcattagtc ggtccaggct ttggctgtga gcgtagctgc tatggaaagg tacgttctca 1080
aactcgtagc tgaactggaa gttgttaccc gttcttagca tttgcgacgg gaaatattcc 1140
aggcagtaaa aggacgaacg acccacggcc tggcttccat cattaagcgt cagatacccg 1200
tactgaggaa tcatgaaaac gtccgctggg aacggcggga ggcagccctc gtgagccgac 1260
ccgagcacgt acgggagctg atagtctgag tccgtgaaga cctggaccgt gctggtaagg 1320
ttattggcga tggtcttgac tccattgttg tccgtaacct ctttgacctg aatgttgaag 1380
agcttgaagt tgagtcgctt aggccggaat ccccagttgt tgttgatgag tcgctgccag 1440
tcacgtggtg agaagtggca gtggaatctg ttgaagtcaa aataccccca gggggtgctg 1500
tagccgaagt aggcgttgtc atttgaagat cctccagatg tgctgttgga gatttgcttg 1560
tagaggtgat tgttgtaggt gggcagggcc caggttcggg tgctggtggt gatgactctg 1620
tcccccagcc attgggaatc gcaatgccaa tttcccgagg aactacccac tccatcggca 1680
ccttcgttat tgtctgccac tggtgcgcca ccacctgaag ccattgtaag agatcccaca 1740
cctgaggggg ctgcgggagg ttctccgatt ggttgagggt ctgggactga ctctgtgtcg 1800
ccagtctgac cgaaattgag tctcttttta gcgggctgtg cacccgattt gccaataccc 1860
gcggaggagt ccggttcctg aggagactgc tctacaggcc tcttctttcc aggagccgtc 1920
ttagccgctt cctcaaccag accaagaggt tcaagaagcc tctttttggc ctggaagact 1980
gctcgcccga ggttgccccc aaaagacgta tcttctttga gccgctcctg gaactcggcg 2040
tcggcgtggt tgtacttgag gtacgggttg tctccggcct tgagctgctg gtcgtaggcc 2100
ttgtcgtgct cgagggccgc cgcgtctgct gcgttgaccg gctccccctt gtcgagtccg 2160
ttgccgggtc caaggtattt gtaacccgga agcacaagac ctcgagcgtt gtcttgatgt 2220
tgttgatttg ccttgggttg aggggctcca ggtttcaaag cccaccactc gcgaattcct 2280
tcactaaggt tgtcctcgag ccaatctgga agataaccat cggcagccat gctagcacca 2340
tggctcgaga tcccgggtga tcaagtcttc gtcgagtgat tgtaaataaa atgtaattta 2400
cagtatagta ttttaattaa tatacaaatg atttgataat aattcttatt taactataat 2460
atattgtgtt gggttgaatt aaaggtccgt atactccgga atattaatag atcatggaga 2520
taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc gtaacagttt 2580
tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca tcgggcgcgg 2640
atcccggtcc gaagcgcgcg gaattcaaag gcctacgtcg acgagctcac tagtcgcggc 2700
cgcactcgac gatggagctg gtcgggtggc tcgtggacaa ggggattacc tcggagaagc 2760
agtggatcca ggaggaccag gcctcataca tctccttcaa tgcggcctcc aactcgcggt 2820
cccaaatcaa ggctgccttg gacaatgcgg gaaagattat gagcctgact aaaaccgccc 2880
ccgactacct ggtgggccag cagcccgtgg aggacatttc cagcaatcgg atttataaaa 2940
ttttggaact aaacgggtac gatccccaat atgcggcttc cgtctttctg ggatgggcca 3000
cgaaaaagtt cggcaagagg aacaccatct ggctgtttgg gcctgcaact accgggaaga 3060
ccaacatcgc ggaggccata gcccacactg tgcccttcta cgggtgcgta aactggacca 3120
atgagaactt tcccttcaac gactgtgtcg acaagatggt gatctggtgg gaggagggga 3180
agatgaccgc caaggtcgtg gagtcggcca aagccattct cggaggaagc aaggtgcgcg 3240
tggaccagaa atgcaagtcc tcggcccaga tagacccgac tcccgtgatc gtcacctcca 3300
acaccaacat gtgcgccgtg attgacggga actcaacgac cttcgaacac cagcagccgt 3360
tgcaagaccg gatgttcaaa tttgaactca cccgccgtct ggatcatgac tttgggaagg 3420
tcaccaagca ggaagtcaaa gactttttcc ggtgggcaaa ggatcacgtg gttgaggtgg 3480
agcatgaatt ctacgtcaaa aagggtggag ccaagaaaag acccgccccc agtgacgcag 3540
atataagtga gcccaaacgg gtgcgcgagt cagttgcgca gccatcgacg tcagacgcgg 3600
aagcttcgat caactacgca gacaggtacc aaaacaaatg ttctcgtcac gtgggcatga 3660
atctgatgct gtttccctgc agacaatgcg agagaatgaa tcagaattca aatatctgct 3720
tcactcacgg acagaaagac tgtttagagt gctttcccgt gtcagaatct caacccgttt 3780
ctgtcgtcaa aaaggcgtat cagaaactgt gctacattca tcatatcatg ggaaaggtgc 3840
cagacgcttg cactgcctgc gatctggtca atgtggattt ggatgactgc atctttgaac 3900
aataaatgat ttaaatcagg tatggctgcc gatggttatc ttccagattg gctcgaggac 3960
actctctctg atctagagcc tgcagtctcg acaagcttgt cgagaagtac tagaggatca 4020
taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 4080
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 4140
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 4200
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatc 4256
<210> 6
<211> 4517
<212> DNA/RNA
<213> Artificial Sequence
<400> 6
gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt ttattgccgt catagcgcgg 60
gttccttccg gtattgtctc cttccgtgtt tcagttagcc tcccccatct cccggtacct 120
tacagattac gagtcaggta tctggtgcca atggggcggg gttcactata tacaccttca 180
gtattaacag caaattcaac attattagac ttgtaatagt tggaagtgta ctggatctcc 240
gggttccagc gcttgctgtt ttccttctgc agctcccact cgatctccac gctgacttgg 300
ccagtagaat actgggtgat gaaagagttc agcttgtcct tgttgaaggc cgttggagga 360
tccgcaggta caggtgtgtt tttgatgagg atctgaggag gcgggtgctt cattccaaac 420
cctcccatca gcggagaagg gtgaaagttg ccgtccgtgt gaggaatttt ggcccaaatg 480
ggtccttgca ggtacacatc tctgtcctgc caaaccatac ccggaagtat tccttggttt 540
tgaacccagc cggtctgcgc ctgtgcttgg gcactctggt ggtttgtggc cacttgtcca 600
taggactccg ttgctaccgg gttagtagtt ttaatttctt cttcgttggt tatcatgact 660
ttgtccgcat ccacgttgtc tcttccagtt ccttgtttgc caaaaattaa agatccagac 720
aaaggaaaga aacggtcctc tccttctttg tggctggcca tagcaggtcc aggattcatc 780
aagctattac gtccattgag agcccaagaa gaagctccag gccaagcaaa ttcgctgttg 840
ttgttttgag tcacagtggt tgagacacgt tgttgtcggt agctgggtcc aggtatgtag 900
tttcttccct ggacagccat gttgctgggt ccggccacac tgaattttag cgtttgttga 960
ttctgtccag aaccgttaat agtctttgag agatagtaca agtattggtc gatgagtgga 1020
ttcattagtc ggtccaggct ttggctgtga gcgtagctgc tatggaaagg tacgttctca 1080
aactcgtagc tgaactggaa gttgttaccc gttcttagca tttgcgacgg gaaatattcc 1140
aggcagtaaa aggacgaacg acccacggcc tggcttccat cattaagcgt cagatacccg 1200
tactgaggaa tcatgaaaac gtccgctggg aacggcggga ggcagccctc gtgagccgac 1260
ccgagcacgt acgggagctg atagtctgag tccgtgaaga cctggaccgt gctggtaagg 1320
ttattggcga tggtcttgac tccattgttg tccgtaacct ctttgacctg aatgttgaag 1380
agcttgaagt tgagtcgctt aggccggaat ccccagttgt tgttgatgag tcgctgccag 1440
tcacgtggtg agaagtggca gtggaatctg ttgaagtcaa aataccccca gggggtgctg 1500
tagccgaagt aggcgttgtc atttgaagat cctccagatg tgctgttgga gatttgcttg 1560
tagaggtgat tgttgtaggt gggcagggcc caggttcggg tgctggtggt gatgactctg 1620
tcccccagcc attgggaatc gcaatgccaa tttcccgagg aactacccac tccatcggca 1680
ccttcgttat tgtctgccac tggtgcgcca ccacctgaag ccattgtaag agatcccaca 1740
cctgaggggg ctgcgggagg ttctccgatt ggttgagggt ctgggactga ctctgtgtcg 1800
ccagtctgac cgaaattgag tctcttttta gcgggctgtg cacccgattt gccaataccc 1860
gcggaggagt ccggttcctg aggagactgc tctacaggcc tcttctttcc aggagccgtg 1920
ctagcaccat ggctcgagat cccgggtgat caagtcttcg tcgagtgatt gtaaataaaa 1980
tgtaatttac agtatagtat tttaattaat atacaaatga tttgataata attcttattt 2040
aactataata tattgtgttg ggttgaatta aaggtccgta tactccggaa tattaataga 2100
tcatggagat aattaaaatg ataaccatct cgcaaataaa taagtatttt actgttttcg 2160
taacagtttt gtaataaaaa aacctataaa tattccggat tattcatacc gtcccaccat 2220
cgggcgcgga tcccggtccg aagcgcgcgg aattcaaagg cctacgtcga cgagctcact 2280
agtcgcggcc gcactcgacg atgccggggt tttacgagat tgtgattaag gtccccagcg 2340
accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg 2400
aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga 2460
ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc 2520
cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc 2580
tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg 2640
aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg 2700
tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc 2760
ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac 2820
agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga 2880
cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc 2940
cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca 3000
aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca 3060
atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta 3120
tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt 3180
ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt 3240
ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg 3300
ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct 3360
acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg 3420
tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc 3480
tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga 3540
ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga 3600
ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc 3660
tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa 3720
aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa 3780
gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc 3840
agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat 3900
gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga 3960
atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg 4020
tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc 4080
atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt 4140
tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat 4200
cttccagatt ggctcgagga cactctctct gatctagagc ctgcagtctc gacaagcttg 4260
tcgagaagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 4320
taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 4380
ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 4440
caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 4500
cttatcatgt ctggatc 4517
<210> 7
<211> 885
<212> DNA/RNA
<213> Artificial Sequence
<400> 7
atggccatga gctccggagg aagcggcgga ggagtgcccg aacaagagga ctccgtgctg 60
tttcgtagag gcaccggcca gagcgatgac tccgacatct gggacgacac cgctttaatc 120
aaggcctacg acaaggccgt ggctagcttc aagcacgctt taaagaatgg cgacatctgc 180
gagaccagcg gcaagcccaa gaccaccccc aagagaaaac ccgccaagaa gaataagagc 240
cagaagaaga acaccgccgc ctctttacag cagtggaaag tcggcgacaa gtgcagcgcc 300
atctggagcg aggacggatg catctacccc gccaccatcg ccagcatcga cttcaagagg 360
gagacttgtg tggtggtgta cactggttac ggcaatcgtg aggagcagaa tctgagcgat 420
ttactgtccc ccatctgtga ggtcgccaac aatatcgagc agaacgccca agaaaacgag 480
aacgagagcc aagttagcac cgacgagagc gagaactctc gttcccccgg caacaagtcc 540
gacaacatca aacccaagtc cgccccttgg aactcctttc tgcctccccc ccctcctatg 600
cccggcccta gactgggacc cggtaagccc ggtttaaaat tcaacggtcc ccctcctcct 660
cctcctcccc cccctcccca tttactgagc tgttggctgc cccctttccc cagcggacct 720
cccatcatcc ccccccctcc ccccatctgc cccgatagcc tcgacgatgc cgacgcttta 780
ggcagcatgc tgatcagctg gtacatgagc ggctaccaca ccggctacta catgggcttt 840
cgtcagaatc agaaggaggg tcgttgctcc cactctttaa actga 885
<210> 8
<211> 1863
<212> DNA/RNA
<213> Artificial Sequence
<400> 8
atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
taa 1863
<210> 9
<211> 2223
<212> DNA/RNA
<213> Artificial Sequence
<400> 9
atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
taa 2223
Claims (10)
1. A system for producing AAV by infecting insect cells with a baculovirus, comprising: a first baculovirus, a second baculovirus, a third baculovirus and Sf9 cell;
the genome of the first baculovirus integrates an expression cassette for coding Rep78 protein and an expression cassette for coding Cap protein VP2, wherein the gene for coding Rep78 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP2 is driven to express by p10 promoter;
the genome of the second baculovirus integrates an expression cassette for coding Rep52 protein and an expression cassette for coding Cap protein VP1, wherein the gene for coding Rep52 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP1 is driven to express by p10 promoter;
the third baculovirus has integrated AAV vector sequence carrying the target gene expression cassette in its genome;
co-infecting Sf9 cells with the first baculovirus, the second baculovirus and the third baculovirus to produce AAV carrying the target gene.
2. A method for producing AAV by infecting insect cells with a baculovirus, comprising:
s1, connecting one of two expression cassettes for coding the Rep78 protein and the Rep52 protein with an expression cassette for coding the Cap protein VP2, cloning the two expression cassettes on a first baculovirus vector, and respectively driving the two expression cassettes by polyhedrin and a p10 promoter to obtain a first baculovirus;
connecting the other of the two expression cassettes for coding the Rep78 protein and the Rep52 protein with an expression cassette for coding the Cap protein VP1, cloning the other expression cassette on a second baculovirus vector, and respectively driving the two expression cassettes by a polyhedrin promoter and a p10 promoter to obtain a second baculovirus;
s2, cloning the AAV vector sequence carrying the target gene expression cassette to a third baculovirus vector to obtain a third baculovirus;
s3, culturing Sf9 cells in an adherent manner, and co-infecting the Sf9 cells by using the first baculovirus, the second baculovirus and the third baculovirus when AAV needs to be produced to produce AAV carrying the target gene.
3. The method of claim 2, wherein in step S2, the third baculovirus has integrated into its genome a plasmid sequence pFast-Bac-AAV-Target Gene, wherein Target Gene represents a Gene of interest and AAV is an adeno-associated virus expression cassette sequence; the third baculovirus was prepared as follows: constructing a pFast-Bac-AAV-Target Gene plasmid vector, transfecting the plasmid vector into competent DH10Bac, and obtaining a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-AAV-Target Gene to obtain the third baculovirus.
4. The method according to claim 3, wherein the Target Gene is SMN1 Gene, and the sequence thereof is shown as SEQ ID No. 7; or the Target Gene is a Gene sequence SEQ ID No. 8 or SEQ ID No. 9 of an ACE2 protein which is a soluble extracellular region.
5. The method according to claim 2, wherein in step S1, the expression cassette encoding the Rep78 protein and the Cap protein VP2 are cloned in conjunction into a first baculovirus vector to obtain a first baculovirus; connecting the Rep52 encoding protein and an expression cassette of the Cap protein VP1 and cloning the Rep52 encoding protein and the expression cassette onto a second baculovirus vector to obtain a second baculovirus;
the first baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep78/VP2 plasmid vector, wherein the plasmid vector contains a p10-VP2-polyhedrin-Rep78 sequence (shown as SEQ ID No: 6); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP 2; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP2 to obtain a first baculovirus;
the second baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep52/VP1 plasmid vector, wherein the plasmid vector contains a p10-VP1-polyhedrin-Rep52 sequence (shown as SEQ ID No: 5); transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP 1; and (3) transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP1 to obtain the second baculovirus.
6. The method of claim 5,
in S1, the method for constructing pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 plasmid vectors comprises the following steps:
step 1: after the pFast-Bac-dual plasmid is subjected to double digestion treatment by using restriction enzyme, agarose electrophoresis is carried out, gel cutting is carried out, a pFast-Bac-dual carrier fragment is recovered, and the pFast-Bac-dual carrier fragment is respectively connected with a gene fragment for coding VP2 and a gene fragment for coding VP1 by adopting ligase;
transforming the ligation product into competent DH5a, mixing uniformly, performing ice bath and heat shock, immediately bathing, culturing by using a liquid culture medium without antibiotics, transferring to an agar plate containing ampicillin for continuous culture, selecting a monoclonal colony, inoculating into a liquid culture medium containing ampicillin for shake culture, extracting plasmids, performing double enzyme digestion identification, performing sequencing identification, and correspondingly obtaining pFast-Bac-dual-VP2 plasmid and pFast-Bac-dual-VP1 plasmid;
step 2: carrying out double digestion on pFast-Bac-dual-VP2 plasmid and pFast-Bac-dual-VP1 plasmid by using restriction enzymes respectively, carrying out agarose electrophoresis respectively, and cutting gel to recover pFast-Bac-dual-VP2 carrier fragment and pFast-Bac-dual-VP1 carrier fragment;
connecting a pFast-Bac-dual-VP2 vector fragment with a gene fragment for coding Rep78, and connecting the pFast-Bac-dual-VP1 vector fragment with the gene fragment for coding Rep52 by adopting ligase;
respectively transforming the connecting products into competent DH5a, mixing uniformly, performing ice bath and heat shock, then immediately bathing, firstly culturing by using a liquid culture medium without antibiotics, then transferring to an agar plate containing ampicillin for continuous culture, then selecting a monoclonal colony to inoculate in a liquid culture solution containing ampicillin for oscillation culture, extracting plasmids, performing double enzyme digestion identification, and then performing sequencing identification to successfully construct pFast-Bac-dual-Rep78/VP2 plasmid vectors and pFast-Bac-dual-Rep52/VP1 plasmid vectors;
after double digestion and ligase ligation by the restriction endonuclease, the pFast-Bac-dual-Rep78/VP2 plasmid vector contains a p10-VP2-polyhedrin-Rep78 sequence, and the pFast-Bac-dual-Rep52/VP1 plasmid vector contains a p10-VP1-polyhedrin-Rep52 sequence.
7. The method of claim 5,
in S1, the method for preparing the recombinant baculovirus plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 comprises the following steps:
plasmid vectors of pFast-Bac-dual-Rep78/VP2 and pFast-Bac-dual-Rep52/VP1 are diluted, transfected into competent DH10Bac respectively, mixed uniformly, subjected to ice bath and heat shock, immediately bathed, cultured by a liquid culture medium without antibiotics, transferred to an agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, and cultured continuously for screening blue and white spots;
selecting white large single clone to a fresh antibiotic-free plate culture medium, carrying out secondary streaking to an agar plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG, carrying out culture and then screening again;
then, a monoclonal colony is selected and inoculated in a liquid culture solution containing kanamycin, gentamicin and tetracycline for culture, recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1 are respectively extracted by phenol chloroform and are identified by M13F and M13R, and thus, recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-52/VP 1 are respectively obtained.
According to a preferred embodiment of the present invention, in S1, the method for obtaining the first baculovirus and the second baculovirus is:
diluting recombinant plasmids Bac-pFast-Bac-dual-Rep78/VP2 and Bac-pFast-Bac-dual-Rep52/VP1, adding a diluted transfection reagent, simultaneously adding a mixture of the recombinant plasmids and the transfection reagent into adherent Sf9 cells, culturing for a period of time, and replacing a fresh culture medium; observing cells, and centrifuging to collect supernatant when most of the cells are enlarged and more than half of the cells are suspended to obtain a first baculovirus and a second baculovirus respectively; the pFast-Bac-dual-Rep78/VP2 plasmid sequence is integrated on the first baculovirus genome, and the pFast-Bac-dual-Rep52/VP1 plasmid sequence is integrated on the second baculovirus genome.
8. The method according to claim 2, wherein in step S3, Sf9 insect cells are co-infected with the first baculovirus, the second baculovirus and the third baculovirus, the cells are harvested after infection for 72h, the cells are lysed by repeated freeze-thawing to obtain a cell lysate, and the cell lysate is subjected to chromatographic purification to produce AAV carrying the target gene.
9. The method according to claim 2, wherein in step S1, the expression cassette encoding the Rep78 protein and the Cap protein VP1 are cloned in conjunction into a first baculovirus vector to obtain a first baculovirus; connecting the Rep52 encoding protein and an expression cassette of the Cap protein VP2 and cloning the Rep52 encoding protein and the expression cassette onto a second baculovirus vector to obtain a second baculovirus;
the first baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep78/VP1 plasmid vector, wherein the plasmid vector contains a p10-VP1-polyhedrin-Rep78 sequence; transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP 1; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep78/VP1 to obtain a first baculovirus;
the second baculovirus was prepared as follows: constructing a pFast-Bac-dual-Rep52/VP2 plasmid vector, wherein the plasmid vector contains a p10-VP2-polyhedrin-Rep52 sequence; transfecting the plasmid vector with competent DH10Bac to obtain a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP 2; transfecting adherent Sf9 cells by using a recombinant baculovirus plasmid Bac-pFast-Bac-dual-Rep52/VP2 to obtain a second baculovirus; the pFast-Bac-dual-Rep78/VP1 plasmid sequence is integrated on the first baculovirus genome, and the pFast-Bac-dual-Rep52/VP2 plasmid sequence is integrated on the second baculovirus genome.
10. A system for producing AAV by infecting insect cells with a baculovirus, comprising: a first baculovirus, a second baculovirus, a third baculovirus and Sf9 cell;
the genome of the first baculovirus integrates an expression cassette for encoding Rep52 protein and an expression cassette for encoding Cap protein VP2 (containing VP3 sequence), wherein the gene for encoding Rep52 protein is driven by polyhedrin promoter to express, and the gene for encoding Cap protein VP2 is driven by p10 promoter to express;
the genome of the second baculovirus integrates an expression cassette for coding Rep78 protein and an expression cassette for coding Cap protein VP1, wherein the gene for coding Rep78 protein is driven to express by polyhedrin promoter, and the gene for coding Cap protein VP1 is driven to express by p10 promoter;
the third baculovirus has integrated AAV vector sequence carrying the target gene expression cassette in its genome;
co-infecting Sf9 cells with the first baculovirus, the second baculovirus and the third baculovirus to produce AAV carrying the target gene.
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