CN113549652A

CN113549652A - SFV-helper plasmid, pSFVCs-LacZ virus-like particle, and preparation method and application thereof

Info

Publication number: CN113549652A
Application number: CN202110822407.2A
Authority: CN
Inventors: 方倪冉; 杨斌; 郑航辉; 杨小云; 董楠
Original assignee: Zhaoqing Institute Of Biotechnology Co ltd
Current assignee: Zhaoqing Institute Of Biotechnology Co ltd
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2021-10-26

Abstract

The invention discloses an SFV-helper plasmid, which is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector. Correspondingly, the invention also provides pSFVCs-LacZ virus-like particles and a preparation method thereof, wherein the preparation method comprises the following steps: (1) constructing the SFV-helper plasmid; (2) pSFVCs-LacZ and SFV-helper plasmids are transcribed into mRNA in vitro, and then the mRNA is co-electroporated into BHK cells to obtain pSFVCs-LacZ virus-like particles. Correspondingly, the invention also provides the application of the SFV-helper plasmid and the pSFVCs-LacZ virus-like particle. The invention can efficiently express beta-galactosidase and has better biological safety.

Description

SFV-helper plasmid, pSFVCs-LacZ virus-like particle, and preparation method and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to SFV-helper plasmids, pSFVCs-LacZ virus-like particles, and a preparation method and application thereof.

Background

The saccharase, also called saccharan hydrolase, is a general name of enzymes capable of hydrolyzing saccharan bonds, can be used for synthesizing oligosaccharides, can also be used for synthesizing glycosyl and aromatic saccharan, plays an important role in catalyzing glycosylation of amino acid, polypeptide, alkaloid, antibiotics and other substances, and has wide application in the fields of medicines and foods. Beta-galactosidase is an important class of sugar aromatase, and beta-galactosidase encoded by the Lacz gene is widely found in microorganisms, animals, and plants.

An RNA replicon is an RNA that is derived from the genome of an RNA virus and is capable of autonomous replication. The viruses most commonly used for replicon development are alphaviruses (alphaviruses) in the togaviridae family, such as Sindbis virus (SIN), Semliki Forest Virus (SFV), and Venezuelan equine encephalitis Virus (VEE); in addition to alphaviruses, there are flaviviruses, picornaviruses, paramyxoviruses, caliciviruses, and the like. Expression vectors engineered for RNA replicon the alphavirus (Semliki forest virus) vectors are used herein as examples to illustrate the basic principles and features of RNA replicon vaccines. The alphavirus is a positive-strand RNA virus (75), the genome is about 11-12kb in length, the 5 'end of the genome has a cap structure, the 3' end of the genome has a polyadenylic acid structure and two open reading frames, and the alphavirus can encode 4 nonstructural proteins (NS1, NS2, NS3 and NS4) and 5 structural proteins (Core, E3, E2, 6K and E1). A stop codon is present in the alphavirus genome between NS3 and NS4, sometimes resulting in a larger polyprotein P1234 upon codon read. However, in some alphavirus genomes, such as SINV and O' n yong-nyong viruses (ONNV), this stop codon is replaced by an Arg or Cys codon and thus P123 polyprotein is not produced. This phenomenon is likely to be a potential pathogenesis. The polyprotein P1234, upon hydrolysis of the NS2 protein, self-cleaves to yield the P123 and NS4 proteins. Meanwhile, the P123 and NS4 proteins can be used as viral replicase to synthesize minus-strand RNA with genomic RNA as a template. Thereafter, the negative-strand RNA can also be used as a template to synthesize positive-strand genomic RNA and subgenomic RNA (subgenomic RNA). Subsequently, the P123 polyprotein is hydrolyzed to produce NS1 and P23 proteins, resulting in reduced negative strand RNA synthesis and increased positive strand genomic RNA synthesis. The P23 polyprotein has a very short half-life of only a few seconds and is then hydrolyzed to form NS2 and NS3 proteins. At this time, NS1, NS2, NS3, and NS4 proteins existing in a monomeric form bind to each other to form a replication complex, thereby further promoting replication of viral genome and subgenomic RNA.

The invention takes Semliki Forest Virus (SFV) as a vector, which comprises two independent RNA molecules, wherein one RNA molecule comprises an inserted RNA replicon vector. The other RNA molecule is a helper RNA. How to construct a packaging system by using Semliki Forest Virus (SFV) as a vector so that the SFV can efficiently express beta-galactosidase and has great significance for the application of the beta-galactosidase in the field of food. However, how to use semliki forest virus for high-efficiency expression of beta-galactosidase is not described in the literature.

Disclosure of Invention

The technical problem to be solved by the invention is that the SFV-helper plasmid, the pSFVCs-LacZ virus-like particles and the preparation method thereof are provided by taking Semliki forest virus as a vector, and can efficiently express beta-galactosidase.

The technical problem to be solved by the invention is also to provide the application of the SFV-helper plasmid and the pSFVCs-LacZ virus-like particle.

In order to solve the technical problem, the invention provides a preparation method of pSFVCs-LacZ virus-like particles, which comprises the following steps:

(1) constructing SFV-helper plasmid;

(2) in vitro transcribing pSFVCs-LacZ and SFV-helper plasmids into mRNA, and co-electroporating the mRNA into BHK cells to obtain pSFVCs-LacZ virus-like particles;

wherein the SFV-helper plasmid is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector.

As an improvement of the technical scheme, the step (1) comprises the following steps:

(1.1) constructing a recombinant vector, wherein the sequence of the constructed recombinant vector is SEQ ID NO. 1;

(1.2) synthesizing an insert gene, wherein the sequence of the insert is SEQ ID NO. 2;

(1.3) carrying out homologous recombination on the recombinant vector and the insert, transforming DH5 alpha competent bacteria by a recombined product, coating a flat plate, carrying out inverted culture, and extracting plasmids to obtain SFV-helper plasmids, wherein the sequence of the SFV-helper plasmids is SEQ ID NO. 3.

As an improvement of the technical scheme, the step (1.3) comprises the following steps:

homologous recombination is carried out on the recombinant vector and the insert, the recombinant product is transformed into DH5 alpha competent bacteria, an LB flat plate coated with ampicillin resistance is coated, inverted culture is carried out for 10-20 hours at 35-38 ℃, colony positive by colony PCR is subjected to shake, bacteria are preserved, and plasmids are extracted;

then the extracted plasmid is subjected to enzyme digestion identification and sequencing.

As an improvement of the technical scheme, the step (1.1) comprises the following steps:

designing and synthesizing primers, wherein the primers comprise a first section-F, a first section-R, a second section-F and a second section-R:

amplifying a fragment 1 by taking the pSFVCs-lacZ as a template and a primer of a first section-F and a first section-R as primers and amplifying a fragment 2 by taking a second section-F and a second section-R as primers to obtain a vector fragment 1 and a vector fragment 2;

carrying out homologous recombination on the vector fragment 1 and the vector fragment 2 to obtain a recombinant product;

adding the recombinant product into a competent cell DH5 alpha cell to carry out recombinant product transformation;

carrying out recombinant product identification on the transformed recombinant product;

extracting plasmids from the identified recombinant product;

and carrying out enzyme digestion identification and sequencing on the plasmid.

As an improvement of the technical scheme, the sequence of the primer is designed as follows:

the sequence of the first section-F is SEQ ID NO. 4;

the sequence of the first section-R is SEQ ID NO. 5;

the sequence of the second section-F is SEQ ID NO. 6;

the sequence of the second segment-R is SEQ ID NO. 7.

As an improvement of the technical scheme, the step (2) comprises the following steps:

linearizing the pSFVCs-LacZ and SFV-helper;

performing in vitro transcription on the linearized pSFVCs-LacZ and SFV-helper;

the pSFVCs-LacZ and the mRNA transcribed in vitro from the pSFVCs-helper plasmids were co-transfected into BHK cells to obtain pSFVCs-LacZ virus-like particles.

Correspondingly, the invention also provides an SFV-helper plasmid, which is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector.

Correspondingly, the invention also provides pSFVCs-LacZ virus-like particles prepared by the preparation method.

Correspondingly, the invention also discloses an SFV-helper plasmid, pSFVCs-LacZ virus-like particles and application of the preparation method of the pSFVCs-LacZ virus-like particles in expression of beta-galactosidase.

Correspondingly, the invention also discloses an SFV-helper plasmid, pSFVCs-LacZ virus-like particles and application of the preparation method of the pSFVCs-LacZ virus-like particles in foods, therapeutic drugs and animal models.

The implementation of the invention has the following beneficial effects:

the invention constructs the assistant plasmid SFV-helper, transcribes pSFVCs-LacZ and SFV-helper to mRNA in vitro, and co-electrifies the mRNA into BHK cell, which can successfully express beta-galactosidase. Moreover, the pSFVCs-LacZ virus-like particle is replication-defective virus due to the lack of the gene encoding the viral nucleocapsid, and has higher biological safety.

Drawings

FIG. 1 is a map of the template plasmid pSFVCs-LacZ of amplified

vector fragments

1, 2;

FIG. 2 is a schematic representation of the results of amplification of

vector fragments

1, 2;

FIG. 3 is a schematic representation of the results of recombinant product identification;

FIG. 4 is a SFV-helper recombinant plasmid map;

FIG. 5 is a schematic diagram showing the result of enzyme digestion and identification of the recombinant plasmid SFV-helper Ncol;

FIG. 6 is a schematic diagram of SpeI digestion of pSFVCS-LacZ and SFV-helper;

FIG. 7 is a schematic representation of linearized pSFVCS-LacZ in vitro transcription;

FIG. 8 is a schematic diagram of linearized SFV-helper in vitro transcription;

FIG. 9 is a schematic representation of the detected expression of β -galactosidase (. times.200).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

The invention provides a preparation method of pSFVCs-LacZ virus-like particles, which comprises the following steps:

(1) constructing SFV-helper plasmid;

the SFV-helper plasmid is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector.

Specifically, the step (1) comprises the following steps:

Preferably, step (1.1) comprises:

designing and synthesizing primers, wherein the primers comprise a first section-F, a first section-R, a second section-F and a second section-R: the sequence of the primers was designed as follows: the sequence of the first section-F is SEQ ID NO. 4; the sequence of the first section-R is SEQ ID NO. 5; the sequence of the second section-F is SEQ ID NO. 6; the sequence of the second segment-R is SEQ ID NO. 7.

extracting plasmids from the identified recombinant product;

and carrying out enzyme digestion identification and sequencing on the plasmid.

Preferably, step (1.3) comprises:

More preferably, step (1.3) comprises:

homologous recombination is carried out on the recombinant vector and the insert, the recombined product is transformed into DH5 alpha competent bacteria, an ampicillin resistant LB plate is coated, inverted culture is carried out for 10-20 hours at 37 ℃, colony positive by colony PCR is subjected to shake, bacteria are preserved, and plasmids are extracted; then the extracted plasmid is subjected to enzyme digestion identification and sequencing.

Step (1) is further illustrated below by means of a preferred embodiment

(1.1) construction of recombinant vector:

(A) primer design and Synthesis

Primers were designed according to SnapGene software using homologous recombination techniques, first-F, first-R, and second-F, second-R, synthesized in Kingwei technology Inc. (Suzhou), and the primers were designed as follows:

TABLE 1 primers for amplifying vector fragments

(B) Amplification of vector fragments

Amplifying fragment 1 by taking pSFVCs-lacZ (#92076) purchased from addrene as a template and taking a first segment-F and a first segment-R as primers respectively; and amplifying the fragment 2 by taking the second segment-F and the second segment-R as primers, wherein the PCR system and conditions are respectively as follows:

TABLE 2 fragment 1 amplification PCR System

TABLE 3 fragment 1 amplification PCR procedure

TABLE 4 fragment 2 amplification PCR System

TABLE 5 fragment 2 amplification PCR procedure

After PCR amplification is finished, 1% nucleic acid gel electrophoresis identification is carried out, obvious target bands appear around 3042bp and 1665bp respectively, the gel containing the target bands is cut under ultraviolet light, the size of the cut gel just contains all the target bands as much as possible, the gel is not too large, the ultraviolet light is cut off immediately after the gel is cut, and the target fragments are prevented from being degraded due to over irradiation of the ultraviolet light. The cut gel was transferred to a 2mL EP tube, gel recovery was performed, and the DNA concentration was measured.

(C)

Vector fragment

1, 2 homologous recombination

After the gel recovery, the following reaction systems were prepared on ice according to the appropriate volumes for

vector segments

1 and 2, as shown in table 6 below:

TABLE 6 homologous recombination System

Shaking gently, incubating in water bath at 37 deg.C for 30min, and cooling at 4 deg.C or on ice.

(D) Transformation of recombinant product

Chemically competent cells for cloning, DH 5. alpha. cells, were thawed on ice, 10. mu.l of the recombinant product was added to 100. mu.l of the competent cells, gently flicked against the vessel wall (Do not shake well), and allowed to stand on ice for 30 min. After heat shock in 42 deg.C water bath for 45sec, immediately cooling on ice for 2-3 min. Adding 900 μ l SOC, shaking the bacteria at 37 ℃ for 1h (rotation speed 200-. LB plate solid media, which are resistant to chloramphenicol, were preheated in a 37 ℃ incubator. Centrifuge at 5,000rpm for 5min and discard 900. mu.l of supernatant. The cells were resuspended in the remaining medium and spread gently on chloramphenicol resistant plates using a sterile spreading rod. Culturing in 37 deg.C incubator for 12-16 h.

(E) Identification of recombinant products

After overnight culture, colonies of the plate were examined and selected for bacterial liquid identification, and cultured in a shaker at 37 ℃ for about 5 hours in a 2mL EP tube containing a chloramphenicol-resistant LP medium, followed by bacterial liquid identification of 4691bp as an identification fragment, and PCR system was identified as shown in the following Table 7:

TABLE 7 bacteria liquid identification PCR system

And (4) taking 100uL of the bacteria liquid which is identified to be positive, adding the bacteria liquid into a 50mL centrifuge tube with the LP culture medium containing chloramphenicol resistance, shaking greatly, and culturing overnight.

(F) Extraction of plasmids

Column equilibration step: 500. mu.l of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, the waste solution in the collection tube was decanted, and the adsorption column was replaced in the collection tube.

15ml of overnight-cultured broth was added to a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated as much as possible.

To the tube containing the pellet was added 500. mu.l of solution P1, and the pellet was suspended thoroughly using a pipette or vortex shaker.

The cells were lysed by adding 500. mu.l of solution P2 to the tube and gently inverting the tube 6 to 8 times. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

Add 700. mu.l of solution P3 to the centrifuge tube, gently turn up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

Transferring the supernatant collected in the previous step to an adsorption column CP3 by using a pipette, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring the waste liquid in the collection tube, and placing the adsorption column CP3 in the collection tube.

Add 600. mu.l of rinsing solution PW (please check if absolute ethanol has been added) to the adsorption column CP3, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, dump the waste liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

Operation 7 is repeated.

The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was uncapped and left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

(G) Sequencing of recombinant vectors

The extracted plasmid is subjected to enzyme digestion, and after the plasmid is identified correctly, the plasmid is sent to Jinzhi Biotechnology GmbH for sequencing.

(1.2) Synthesis of an insert gene,

the sequence of the SFV-helper gene to be inserted is shown in SEQ ID NO.2, and the SFV-helper gene is subjected to gene synthesis.

(1.3) homologous recombination of the recombinant vector and the insert,

(A) homologous recombination, transformation and plasmid extraction

The recombinant vector is subjected to homologous recombination with the insert. The recombinant product was transformed into DH 5. alpha. competent bacteria, spread on ampicillin-resistant LB plates, and cultured in an inverted state at 37 ℃ for 16 hours. Shaking the colony with positive PCR, preserving the bacteria, and extracting the plasmid according to the instruction of the OMEGA plasmid miniprep kit

(B) Plasmid restriction enzyme identification and sequencing

Carrying out enzyme digestion identification on the extracted plasmid, and detecting the result by 1% agarose gel electrophoresis; the correct result is sent to Jinzhi Biotechnology GmbH for sequencing.

specifically, the step (2) comprises the following steps:

(2.1) linearizing the pSFVCS-LacZ and SFV-helper;

pSFVCs-LacZ and SFV-helper were digested separately with SpeI, in 300uL each, as shown in tables 8 and 9 below.

TABLE 8 SpeI enzyme digestion of pSFVCS-LacZ

TABLE 9 SpeI enzyme-cleaved replication plasmid SFV-helper

(2.2) in vitro transcription of the linearized pSFVCS-LacZ and SFV-helper;

according to mMESSAGE mMACHINE^TMIn vitro transcription was performed as described in the SP6 transcription kit (AM1340, purchased from semer plane) in the following system:

TABLE 10 pSFVCs-LacZ in vitro transcription System

TABLE 11 SFV-helper in vitro transcription System

After the system is prepared, the transcription is carried out for 40min at 37 ℃, then 1uL GTP is added for continuous transcription for 2h at 37 ℃, and then 1uL TURBO DNase37 ℃ is added for 15 min. After transcription is finished, 1uL of transcription product is respectively absorbed, 4uL of nucleic-free Water and 6 × loading buffer are added for dilution, aseptic operation is paid attention to and RNA degradation is prevented, and then electrophoresis experiment is carried out in 1% SDS-page to verify whether the band is correct or not.

(2.3) co-transfecting mRNA transcribed in vitro from pSFVCs-LacZ and pSFVCs-helper plasmids into BHK cells to obtain pSFVCs-LacZ virus-like particles.

Specifically, the method can be operated according to the following steps:

1. the cells are transferred to a T75 square bottle in advance, and when the cell confluence reaches 80%, the experiment is carried out;

2. 1.5ml EP tubes and PBS were pre-cooled on ice. Precooling a cuvette (electrode cup) of 4mm in a refrigerator at-20 ℃;

preheat 6ml of medium (DMEM with 1% FBS) at 3.37 ℃;

4. cells in T75 flasks were trypsinized and resuspended in 5ml complete medium;

5.500 g, centrifuging at 4 ℃ for 5 minutes;

6. the supernatant was discarded and the cell particles were washed with 10ml ice-cold PBS;

7.500 g, centrifuged at 4 ℃ for 5 minutes, and PBS washing was repeated again;

8. discarding the supernatant and completely removing residual PBS, and resuspending the cells with cold 520uL PBS, dividing into two equal parts, and placing each PBS cell suspension containing 260uL into a precooled 1.5mLEP tube;

9. 10uL of pSFVCs-sp6-LacZ and pSFVCs-helper transcripts were added to the first PBS cell suspension; the second PBS cell resuspension was also placed on ice without any added material;

10. respectively transferring the three mixtures into 3 precooled electrode cups (4mm), setting the conditions of an electric rotating instrument to be 100V and 25ms, electrically shocking once, and placing on ice for 1min after electric shock;

11. then, adding about 400uL of DMEM containing 1% FBS into the electrode cups, blowing, beating and uniformly mixing, then transferring to a T25 square bottle, adding 6mL of DMEM containing 1% FBS, and placing in a 5% CO2 incubator at 37 ℃ for culture;

12. observing by using an inverted fluorescence microscope after 8-36 h of electroporation, and collecting supernatant after 36 h;

1.3.3 detection expression of beta-galactosidase;

and (3) according to a beta-galactosidase in-situ staining kit (Biyun day), carrying out in-situ beta-galactosidase detection expression on the cells after the supernatant is collected, and taking the negative cells as a control after the supernatant is removed.

Correspondingly, the invention also provides an SFV-helper plasmid, which is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector. The sequence of the SFV-helper plasmid is SEQ ID NO. 3.

Correspondingly, the invention also provides pSFVCs-LacZ virus-like particles prepared by the preparation method. The pSFVCs-LacZ virus-like particle can successfully express beta-galactosidase. Moreover, the pSFVCs-LacZ virus-like particle is replication-defective virus due to the lack of the gene encoding the viral nucleocapsid, and has higher biological safety.

The experimental results are as follows: the SFV-helper plasmid and the pSFVCs-LacZ virus-like particles are subjected to experimental result verification, which is specifically as follows;

one, SFV-helper plasmid

(1) Amplification of

vector fragments

1, 2

Referring to FIGS. 1 and 2, FIG. 1 shows a map of the amplified

vector fragments

1 and 2, which are the pSFVCs-LacZ plasmids, and FIG. 2 shows the amplification results of the vector fragments 1 and 2, which are 3042bp and 1665bp, respectively, and correspond to the theoretical values, as shown in FIG. 2.

In FIG. 2, M is 8000, 5000, 3000, 1500, 1000, 500bp from top to bottom, 1 to 5 are amplified

vector fragments

2, 6 to 10 are amplified vector fragments 1.

(2) Identification of recombinant products

10 colonies were picked up and identified, as shown in FIG. 3, the sizes of 1-10 were 4691bp, which are consistent with the theoretical values.

In FIG. 3, M is 8000, 5000, 3000, 1500, 1000, 500bp from top to bottom, and 1 to 10 are single colony identification fragments.

(3) Sequencing of recombinant vectors

The correctly identified plasmids are sent to Jinzhi Biotechnology GmbH for sequencing, and the sequencing results are completely the same.

(4) Cloning of the Gene-synthesized insert into recombinant vectors

After the digestion of the recombinant plasmid Ncol, the recombinant plasmid was identified by 1% nucleic acid gel electrophoresis. With reference to FIGS. 4 and 5, FIG. 4 is a map of the SFV-helper recombinant plasmid, and FIG. 5 is a map of the restriction enzyme identification of the recombinant plasmid SFV-helper Ncol. As can be seen from FIG. 5, the target bands completely coincided with the theoretical values at about 1407bp and 6785bp, respectively, and the target bands were sent to Jinzhi Biotechnology GmbH for sequencing, and the sequencing results were completely identical to the original sequences.

Bis, pSFVCs-LacZ virus-like particles

(1) Linearized pSFVCs-LacZ and SFV-helper

The results of using SpeI to separately cleave pSFVCs-LacZ and SFV-helper are shown in FIG. 6, wherein M1 is 15000, 10000, 7500, 5000, 3000, 1500, 1000, 500bp from top to bottom; m2 is 8000, 5000, 3000, 1500, 1000, 500bp from top to bottom respectively, 1 is SpeI enzyme cutting pSFVCS-LacZ; 2 is SpeI enzyme SFV-helper. The size of the linearized target band corresponds to the theoretical value.

The linearized pSFVCs-LacZ and SFV-helper were transcribed in vitro

According to mMESSAGE mMACHINE^TMThe results of in vitro transcription of pSFVCs-LacZ and helper plasmid SFV-helper as described in the SP6 transcription kit (AM1340 from Saimerfei) are shown in FIGS. 7 and 8. In fig. 7, M is from top to bottom: 8000. 5000, 3000, 1500, 1000 and 500 bp; 1 is pSFVCS-LacZ in vitro transcription. In fig. 8, M is from top to bottom: 10000. 7000, 4000, 2000, 1000, 500, 200bp, 2 is SFV-helper in vitro transcription. From 7 and 8, the size of the band after in vitro transcription matched the theoretical value.

Detection expression of tri, beta-galactosidase

According to the beta-galactosidase in-situ staining kit (Biyun day), cells obtained after supernatant liquid is collected after the above electrotransformation culture for 36h are subjected to in-situ beta-galactosidase detection expression, and meanwhile, negative cells with supernatant liquid removed are used as a control, as shown in FIG. 9: the negative cells have no change of deep blue, and a large number of cells in the experimental group have the change of deep blue, so that the experimental group is proved to have the expression of a large number of beta-galactosidase. Meanwhile, the preparation of pSFVCs-LacZ virus-like particles is proved to be successful.

In conclusion, the invention constructs the helper plasmid SFV-helper, transcribes pSFVCs-LacZ and SFV-helper to mRNA in vitro, co-electrifies the mRNA into BHK cells, harvests the supernatant after 36h and detects and expresses beta-galactosidase in situ, and as a result, a large number of cells turn into dark blue, but negative cells do not have color change. Therefore, the virus packaging system is constructed by taking Semliki Forest Virus (SFV) as a vector, and the beta-galactosidase is successfully expressed.

The above description is only a preferred embodiment of the present invention, and certainly should not be taken as limiting the scope of the invention, which is defined by the claims and their equivalents.

Sequence listing

<110> Chongqing research institute of BioIndustrial technology, Inc

<120> SFV-helper plasmid, pSFVCs-LacZ virus-like particle, preparation method and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3538

<212> DNA

<213> Artificial Synthesis

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cactataccg acaggagcgg gcaaaccggg agacagtggc cggcccatct ttgacaacaa 60

gggtagggta gtcgctatcg tcctgggcgg ggccaacgag ggctcacgca cagcactgtc 120

ggtggtcacc tggaacaaag atatggtgac tagagtgacc cccgaggggt ccgaagagtg 180

gtccgccccg ctgattactg ccatgtgtgt ccttgccaat gctaccttcc cgtgcttcca 240

gcccccgtgt gtaccttgct gctatgaaaa caacgcagag gccacactac ggatgctcga 300

ggataacgtg gataggccag ggtactacga cctccttcag gcagccttga cgtgccgaaa 360

cggaacaaga caccggcgca gcgtgtcgca acacttcaac gtgtataagg ctacacgccc 420

ttacatcgcg tactgcgccg actgcggagc agggcactcg tgtcatagcc ccgtagcaat 480

tgaagcggtc aggtccgaag ctaccgacgg gatgctgaag attcagttct cggcacaaat 540

tggcatagat aagagtgaca atcatgacta cacgaagata aggtacgcag acgggcacgc 600

cattgagaat gccgtccggt catctttgaa ggtagccacc tccggagact gtttcgtcca 660

tggcacaatg ggacatttca tactggcaaa gtgcccaccg ggtgaattcc tgcaggtctc 720

gatccaggac accagaaacg cggtccgtgc ctgcagaata caatatcatc atgaccctca 780

accggtgggt agagaaaaat ttacaattag accacactat ggaaaagaga tcccttgcac 840

cacttatcaa cagaccacag cgaagaccgt ggaggaaatc gacatgcata tgccgccaga 900

tacgccggac aggacgttgc tatcacagca atctggcaat gtaaagatca cagtcggagg 960

aaagaaggtg aaatacaact gcacctgtgg aaccggaaac gttggcacta ctaattcgga 1020

catgacgatc aacacgtgtc taatagagca gtgccacgtc tcagtgacgg accataagaa 1080

atggcagttc aactcacctt tcgtcccgag agccgacgaa ccggctagaa aaggcaaagt 1140

ccatatccca ttcccgttgg acaacatcac atgcagagtt ccaatggcgc gcgaaccaac 1200

cgtcatccac ggcaaaagag aagtgacact gcaccttcac ccagatcatc ccacgctctt 1260

ttcctaccgc acactgggtg aggacccgca gtatcacgag gaatgggtga cagcggcggt 1320

ggaacggacc atacccgtac cagtggacgg gatggagtac cactggggaa acaacgaccc 1380

agtgaggctt tggtctcaac tcaccactga agggaaaccg cacggctggc cgcatcagat 1440

cgtacagtac tactatgggc tttacccggc cgctacagta tccgcggtcg tcgggatgag 1500

cttactggcg ttgatatcga tcttcgcgtc gtgctacatg ctggttgcgg cccgcagtaa 1560

gtgcttgacc ccttatgctt taacaccagg agctgcagtt ccgtggacgc tggggatact 1620

ctgctgcgcc ccgcgggcgc acgcagctag tgtggcagag actatggcct acttgtggga 1680

ccaaaaccaa gcgttgttct ggttggagtt tgcggcccct gttgcctgca tcctcatcat 1740

cacgtattgc ctcagaaacg tgctgtgttg ctgtaagagc ctttcttttt tagtgctact 1800

gagcctcggg gcaaccgcca gagcttacga acattcgaca gtaatgccga acgtggtggg 1860

gttcccgtat aaggctcaca ttgaaaggcc aggatatagc cccctcactt tgcagatgca 1920

ggttgttgaa accagcctcg aaccaaccct taatttggaa tacataacct gtgagtacaa 1980

gacggtcgtc ccgtcgccgt acgtgaagtg ctgcggcgcc tcagagtgct ccactaaaga 2040

gaagcctgac taccaatgca aggtttacac aggcgtgtac ccgttcatgt ggggaggggc 2100

atattgcttc tgcgactcag aaaacacgca actcagcgag gcgtacgtcg atcgatcgga 2160

cgtatgcagg catgatcacg catctgctta caaagcccat acagcatcgc tgaaggccaa 2220

agtgagggtt atgtacggca acgtaaacca gactgtggat gtttacgtga acggagacca 2280

tgccgtcacg atagggggta ctcagttcat attcgggccg ctgtcatcgg cctggacccc 2340

gttcgacaac aagatagtcg tgtacaaaga cgaagtgttc aatcaggact tcccgccgta 2400

cggatctggg caaccagggc gcttcggcga catccaaagc agaacagtgg agagtaacga 2460

cctgtacgcg aacacggcac tgaagctggc acgcccttca cccggcatgg tccatgtacc 2520

gtacacacag acaccttcag ggttcaaata ttggctaaag gaaaaaggga cagccctaaa 2580

tacgaaggct ccttttggct gccaaatcaa aacgaaccct gtcagggcca tgaactgcgc 2640

cgtgggaaac atccctgtct ccatgaattt gcctgacagc gcctttaccc gcattgtcga 2700

ggcgccgacc atcattgacc tgacttgcac agtggctacc tgtacgcact cctcggattt 2760

cggcggcgtc ttgacactga cgtacaagac cgacaagaac ggggactgct ctgtacactc 2820

gcactctaac gtagctactc tacaggaggc cacagcaaaa gtgaagacag caggtaaggt 2880

gaccttacac ttctccacgg caagcgcatc accttctttt gtggtgtcgc tatgcagtgc 2940

tagggccacc tgttcagcgt cgtgtgagcc cccgaaagac cacatagtcc catatgcggc 3000

tagccacagt aacgtagtgt ttccagacat gtcgggcacc gcactatcat gggtgcagaa 3060

aatctcgggt ggtctggggg ccttcgcaat cggcgctatc ctggtgctgg ttgtggtcac 3120

ttgcattggg ctccgcagat aagttagggt aggcaatggc attgatatag caagaaaatt 3180

gaaaacagaa aaagttaggg taagcaatgg catataacca taactgtata acttgtaaca 3240

aagcgcaaca agacctgcgc aattggcccc gtggtccgcc tcacggaaac tcggggcaac 3300

tcatattgac acattaattg gcaataattg gaagcttaca taagcttaat tcgacgaata 3360

attggatttt tattttattt tgcaattggt ttttaatatt tccaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactagtct 3480

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttcc 3538

<210> 2

<211> 3538

<212> DNA

<213> Artificial Synthesis

<400> 2

cactataccg acaggagcgg gcaaaccggg agacagtggc cggcccatct ttgacaacaa 60

gggtagggta gtcgctatcg tcctgggcgg ggccaacgag ggctcacgca cagcactgtc 120

ggtggtcacc tggaacaaag atatggtgac tagagtgacc cccgaggggt ccgaagagtg 180

gtccgccccg ctgattactg ccatgtgtgt ccttgccaat gctaccttcc cgtgcttcca 240

gcccccgtgt gtaccttgct gctatgaaaa caacgcagag gccacactac ggatgctcga 300

ggataacgtg gataggccag ggtactacga cctccttcag gcagccttga cgtgccgaaa 360

cggaacaaga caccggcgca gcgtgtcgca acacttcaac gtgtataagg ctacacgccc 420

ttacatcgcg tactgcgccg actgcggagc agggcactcg tgtcatagcc ccgtagcaat 480

tgaagcggtc aggtccgaag ctaccgacgg gatgctgaag attcagttct cggcacaaat 540

tggcatagat aagagtgaca atcatgacta cacgaagata aggtacgcag acgggcacgc 600

cattgagaat gccgtccggt catctttgaa ggtagccacc tccggagact gtttcgtcca 660

tggcacaatg ggacatttca tactggcaaa gtgcccaccg ggtgaattcc tgcaggtctc 720

gatccaggac accagaaacg cggtccgtgc ctgcagaata caatatcatc atgaccctca 780

accggtgggt agagaaaaat ttacaattag accacactat ggaaaagaga tcccttgcac 840

cacttatcaa cagaccacag cgaagaccgt ggaggaaatc gacatgcata tgccgccaga 900

tacgccggac aggacgttgc tatcacagca atctggcaat gtaaagatca cagtcggagg 960

aaagaaggtg aaatacaact gcacctgtgg aaccggaaac gttggcacta ctaattcgga 1020

catgacgatc aacacgtgtc taatagagca gtgccacgtc tcagtgacgg accataagaa 1080

atggcagttc aactcacctt tcgtcccgag agccgacgaa ccggctagaa aaggcaaagt 1140

ccatatccca ttcccgttgg acaacatcac atgcagagtt ccaatggcgc gcgaaccaac 1200

cgtcatccac ggcaaaagag aagtgacact gcaccttcac ccagatcatc ccacgctctt 1260

ttcctaccgc acactgggtg aggacccgca gtatcacgag gaatgggtga cagcggcggt 1320

ggaacggacc atacccgtac cagtggacgg gatggagtac cactggggaa acaacgaccc 1380

agtgaggctt tggtctcaac tcaccactga agggaaaccg cacggctggc cgcatcagat 1440

cgtacagtac tactatgggc tttacccggc cgctacagta tccgcggtcg tcgggatgag 1500

cttactggcg ttgatatcga tcttcgcgtc gtgctacatg ctggttgcgg cccgcagtaa 1560

gtgcttgacc ccttatgctt taacaccagg agctgcagtt ccgtggacgc tggggatact 1620

ctgctgcgcc ccgcgggcgc acgcagctag tgtggcagag actatggcct acttgtggga 1680

ccaaaaccaa gcgttgttct ggttggagtt tgcggcccct gttgcctgca tcctcatcat 1740

cacgtattgc ctcagaaacg tgctgtgttg ctgtaagagc ctttcttttt tagtgctact 1800

gagcctcggg gcaaccgcca gagcttacga acattcgaca gtaatgccga acgtggtggg 1860

gttcccgtat aaggctcaca ttgaaaggcc aggatatagc cccctcactt tgcagatgca 1920

ggttgttgaa accagcctcg aaccaaccct taatttggaa tacataacct gtgagtacaa 1980

gacggtcgtc ccgtcgccgt acgtgaagtg ctgcggcgcc tcagagtgct ccactaaaga 2040

gaagcctgac taccaatgca aggtttacac aggcgtgtac ccgttcatgt ggggaggggc 2100

atattgcttc tgcgactcag aaaacacgca actcagcgag gcgtacgtcg atcgatcgga 2160

cgtatgcagg catgatcacg catctgctta caaagcccat acagcatcgc tgaaggccaa 2220

agtgagggtt atgtacggca acgtaaacca gactgtggat gtttacgtga acggagacca 2280

tgccgtcacg atagggggta ctcagttcat attcgggccg ctgtcatcgg cctggacccc 2340

gttcgacaac aagatagtcg tgtacaaaga cgaagtgttc aatcaggact tcccgccgta 2400

cggatctggg caaccagggc gcttcggcga catccaaagc agaacagtgg agagtaacga 2460

cctgtacgcg aacacggcac tgaagctggc acgcccttca cccggcatgg tccatgtacc 2520

gtacacacag acaccttcag ggttcaaata ttggctaaag gaaaaaggga cagccctaaa 2580

tacgaaggct ccttttggct gccaaatcaa aacgaaccct gtcagggcca tgaactgcgc 2640

cgtgggaaac atccctgtct ccatgaattt gcctgacagc gcctttaccc gcattgtcga 2700

ggcgccgacc atcattgacc tgacttgcac agtggctacc tgtacgcact cctcggattt 2760

cggcggcgtc ttgacactga cgtacaagac cgacaagaac ggggactgct ctgtacactc 2820

gcactctaac gtagctactc tacaggaggc cacagcaaaa gtgaagacag caggtaaggt 2880

gaccttacac ttctccacgg caagcgcatc accttctttt gtggtgtcgc tatgcagtgc 2940

tagggccacc tgttcagcgt cgtgtgagcc cccgaaagac cacatagtcc catatgcggc 3000

tagccacagt aacgtagtgt ttccagacat gtcgggcacc gcactatcat gggtgcagaa 3060

aatctcgggt ggtctggggg ccttcgcaat cggcgctatc ctggtgctgg ttgtggtcac 3120

ttgcattggg ctccgcagat aagttagggt aggcaatggc attgatatag caagaaaatt 3180

gaaaacagaa aaagttaggg taagcaatgg catataacca taactgtata acttgtaaca 3240

aagcgcaaca agacctgcgc aattggcccc gtggtccgcc tcacggaaac tcggggcaac 3300

tcatattgac acattaattg gcaataattg gaagcttaca taagcttaat tcgacgaata 3360

attggatttt tattttattt tgcaattggt ttttaatatt tccaaaaaaa aaaaaaaaaa 3420

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactagtct 3480

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttcc 3538

<210> 3

<211> 8192

<212> DNA

<213> Artificial Synthesis

<400> 3

atttaggtga cactatagat ggcggatgtg tgacatacac gacgccaaaa gattttgttc 60

cagctcctgc cacctccgct acgcgagaga ttaaccaccc acgatggccg ccaaagtgca 120

tgttgatatt gaggctgaca gcccattcat caagtctttg cagaaggcat ttccgtcgtt 180

cgaggtggag tcattgcagg tcacaccaaa tgaccatgca aatgccagag cattttcgca 240

cctggctacc aaattgatcg agcaggagac tgacaaagac acactcatct tggatatcgg 300

cagtgcgcct tccaggagaa tgatgtcgac atgaaacgag atgtcaaagt cactccaggg 360

acgaaacaca cagaggaaag acccaaagtc caggtaattc aagcagcgga gccattggcg 420

accgcttacc tgtgcggcat ccacagggaa ttagtaagga gactaaatgc tgtgttacgc 480

cctaacgtgc acacattgtt tgatatgtcg gccgaagact ttgacgcgat catcgcctct 540

cacttccacc caggagaccc ggttctagag acggacattg catcattcga caaaagccag 600

gacgactcct tggctcttac aggtttaatg atcctcgaag atctaggggt ggatcagtac 660

ctgctggact tgatcgaggc agcctttggg gaaatatcca gctgtcacct accaactggc 720

acgcgcttca agttcggagc tatgatgaaa tcgggcatgt ttctgacttt gtttattaac 780

actgttttga acatcaccat agcaagcagg gtactggagc agagactcac tgactccgcc 840

tgtgcggcct tcatcggcga cgacaacatc gttcacggag tgatctccga caagctgatg 900

gcggagaggt gcgcgtcgtg ggtcaacatg gaggtgaaga tcattgacgc tgtcatgggc 960

gaaaaacccc catatttttg tgggggattc atagtttttg acagcgtcac acagaccgcc 1020

tgccgtgttt cagacccact taagcgcctg ttcaagttgg gtaagccgct aacagctgaa 1080

gacaagcagg acgaagacag gcgacgagca ctgagtgacg aggttagcaa gtggttccgg 1140

acaggcttgg gggccgaact ggaggtggca ctaacatcta ggtatgaggt agagggctgc 1200

aaaagtatcc tcatagccat ggccaccttg gcgagggaca ttaaggcgtt taagaaattg 1260

agaggacctg ttatacacct ctacggcggt cctagattgg tgcgttaata cacagaattc 1320

tgattatagc gcactattat agcaccatga attacatccc tacgcaaacg ttttacggcc 1380

gccggtggcg cccgcgcccg gcggcccgtc cctggccgtt gcaggccact ccggtggctc 1440

ccgtcgtccc cgacttccag gcccagcaga tgcagcaact catcagcgcc gtaaatgcgc 1500

tgacaatgag acagaacgca attgctcctg ctaggcctcc caaaccaaag aagaagaaga 1560

caaccaaacc aaagccgaaa acgcagccca agaagatcaa cggaaaaacg cagcagcaaa 1620

agaagaaaga caagcaagcc gacaagaaga agaagaaacc cggaaaaaga gaaagaatgt 1680

gcatgaagat tgaaaatgac tgtatcttcg aagtcaaaca cgaaggaaag gtcactgggt 1740

acgcctgcct ggtgggcgac aaagtcatga aacctgccca cgtgaaagga gtcatcgaca 1800

acgcggacct ggcaaagcta gctttcaaga aatcgagcaa gtatgacctt gagtgtgccc 1860

agataccagt tcacatgagg tcggatgcct caaagtacac gcatgagaag cccgagggac 1920

actataactg gcaccacggg gctgttcagt acagcggagg taggttcact ataccgacag 1980

gagcgggcaa accgggagac agtggccggc ccatctttga caacaagggt agggtagtcg 2040

ctatcgtcct gggcggggcc aacgagggct cacgcacagc actgtcggtg gtcacctgga 2100

acaaagatat ggtgactaga gtgacccccg aggggtccga agagtggtcc gccccgctga 2160

ttactgccat gtgtgtcctt gccaatgcta ccttcccgtg cttccagccc ccgtgtgtac 2220

cttgctgcta tgaaaacaac gcagaggcca cactacggat gctcgaggat aacgtggata 2280

ggccagggta ctacgacctc cttcaggcag ccttgacgtg ccgaaacgga acaagacacc 2340

ggcgcagcgt gtcgcaacac ttcaacgtgt ataaggctac acgcccttac atcgcgtact 2400

gcgccgactg cggagcaggg cactcgtgtc atagccccgt agcaattgaa gcggtcaggt 2460

ccgaagctac cgacgggatg ctgaagattc agttctcggc acaaattggc atagataaga 2520

gtgacaatca tgactacacg aagataaggt acgcagacgg gcacgccatt gagaatgccg 2580

tccggtcatc tttgaaggta gccacctccg gagactgttt cgtccatggc acaatgggac 2640

atttcatact ggcaaagtgc ccaccgggtg aattcctgca ggtctcgatc caggacacca 2700

gaaacgcggt ccgtgcctgc agaatacaat atcatcatga ccctcaaccg gtgggtagag 2760

aaaaatttac aattagacca cactatggaa aagagatccc ttgcaccact tatcaacaga 2820

ccacagcgaa gaccgtggag gaaatcgaca tgcatatgcc gccagatacg ccggacagga 2880

cgttgctatc acagcaatct ggcaatgtaa agatcacagt cggaggaaag aaggtgaaat 2940

acaactgcac ctgtggaacc ggaaacgttg gcactactaa ttcggacatg acgatcaaca 3000

cgtgtctaat agagcagtgc cacgtctcag tgacggacca taagaaatgg cagttcaact 3060

cacctttcgt cccgagagcc gacgaaccgg ctagaaaagg caaagtccat atcccattcc 3120

cgttggacaa catcacatgc agagttccaa tggcgcgcga accaaccgtc atccacggca 3180

aaagagaagt gacactgcac cttcacccag atcatcccac gctcttttcc taccgcacac 3240

tgggtgagga cccgcagtat cacgaggaat gggtgacagc ggcggtggaa cggaccatac 3300

ccgtaccagt ggacgggatg gagtaccact ggggaaacaa cgacccagtg aggctttggt 3360

ctcaactcac cactgaaggg aaaccgcacg gctggccgca tcagatcgta cagtactact 3420

atgggcttta cccggccgct acagtatccg cggtcgtcgg gatgagctta ctggcgttga 3480

tatcgatctt cgcgtcgtgc tacatgctgg ttgcggcccg cagtaagtgc ttgacccctt 3540

atgctttaac accaggagct gcagttccgt ggacgctggg gatactctgc tgcgccccgc 3600

gggcgcacgc agctagtgtg gcagagacta tggcctactt gtgggaccaa aaccaagcgt 3660

tgttctggtt ggagtttgcg gcccctgttg cctgcatcct catcatcacg tattgcctca 3720

gaaacgtgct gtgttgctgt aagagccttt cttttttagt gctactgagc ctcggggcaa 3780

ccgccagagc ttacgaacat tcgacagtaa tgccgaacgt ggtggggttc ccgtataagg 3840

ctcacattga aaggccagga tatagccccc tcactttgca gatgcaggtt gttgaaacca 3900

gcctcgaacc aacccttaat ttggaataca taacctgtga gtacaagacg gtcgtcccgt 3960

cgccgtacgt gaagtgctgc ggcgcctcag agtgctccac taaagagaag cctgactacc 4020

aatgcaaggt ttacacaggc gtgtacccgt tcatgtgggg aggggcatat tgcttctgcg 4080

actcagaaaa cacgcaactc agcgaggcgt acgtcgatcg atcggacgta tgcaggcatg 4140

atcacgcatc tgcttacaaa gcccatacag catcgctgaa ggccaaagtg agggttatgt 4200

acggcaacgt aaaccagact gtggatgttt acgtgaacgg agaccatgcc gtcacgatag 4260

ggggtactca gttcatattc gggccgctgt catcggcctg gaccccgttc gacaacaaga 4320

tagtcgtgta caaagacgaa gtgttcaatc aggacttccc gccgtacgga tctgggcaac 4380

cagggcgctt cggcgacatc caaagcagaa cagtggagag taacgacctg tacgcgaaca 4440

cggcactgaa gctggcacgc ccttcacccg gcatggtcca tgtaccgtac acacagacac 4500

cttcagggtt caaatattgg ctaaaggaaa aagggacagc cctaaatacg aaggctcctt 4560

ttggctgcca aatcaaaacg aaccctgtca gggccatgaa ctgcgccgtg ggaaacatcc 4620

ctgtctccat gaatttgcct gacagcgcct ttacccgcat tgtcgaggcg ccgaccatca 4680

ttgacctgac ttgcacagtg gctacctgta cgcactcctc ggatttcggc ggcgtcttga 4740

cactgacgta caagaccgac aagaacgggg actgctctgt acactcgcac tctaacgtag 4800

ctactctaca ggaggccaca gcaaaagtga agacagcagg taaggtgacc ttacacttct 4860

ccacggcaag cgcatcacct tcttttgtgg tgtcgctatg cagtgctagg gccacctgtt 4920

cagcgtcgtg tgagcccccg aaagaccaca tagtcccata tgcggctagc cacagtaacg 4980

tagtgtttcc agacatgtcg ggcaccgcac tatcatgggt gcagaaaatc tcgggtggtc 5040

tgggggcctt cgcaatcggc gctatcctgg tgctggttgt ggtcacttgc attgggctcc 5100

gcagataagt tagggtaggc aatggcattg atatagcaag aaaattgaaa acagaaaaag 5160

ttagggtaag caatggcata taaccataac tgtataactt gtaacaaagc gcaacaagac 5220

ctgcgcaatt ggccccgtgg tccgcctcac ggaaactcgg ggcaactcat attgacacat 5280

taattggcaa taattggaag cttacataag cttaattcga cgaataattg gatttttatt 5340

ttattttgca attggttttt aatatttcca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5400

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac tagtctgcat taatgaatcg 5460

gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 5520

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 5580

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 5640

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 5700

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 5760

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 5820

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 5880

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 5940

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6000

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6060

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6120

gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6180

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6240

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 6300

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 6360

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 6420

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 6480

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 6540

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 6600

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 6660

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 6720

cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 6780

cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 6840

ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 6900

tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 6960

catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7020

gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 7080

gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7140

tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7200

catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7260

aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 7320

attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 7380

aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 7440

aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 7500

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 7560

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 7620

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 7680

accattcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg 7740

aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt 7800

cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 7860

tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 7920

gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatctggct agcgatgacc 7980

ctgctgattg gttcgctgac catttccggg tgcgggacgg cgttaccaga aactcagaag 8040

gttcgtccaa ccaaaccgac tctgacggca gtttacgaga gagatgatag ggtctgcttc 8100

agtaagccag atgctacaca attaggcttg tacatattgt cgttagaacg cggctacaat 8160

taatacataa ccttatgtat catacacata cg 8192

<210> 4

<211> 31

<212> DNA

<213> Artificial Synthesis

<400> 4

gtattgggcg ctcttccgct tcctcgctca c 31

<210> 5

<211> 32

<212> DNA

<213> Artificial Synthesis

<400> 5

gtttcatgtc gacatcattc tcctggaagg cg 32

<210> 6

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 6

gatgtcgaca tgaaacgaga tgtcaaagtc 30

<210> 7

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 7

ccgctcctgt cggtatagtg aacctacctc 30

Claims

1. A method for preparing pSFVCs-LacZ virus-like particles, comprising:

(1) constructing SFV-helper plasmid;

2. The method of preparing pSFVCs-LacZ virus-like particles of claim 1, wherein step (1) comprises:

3. The method of claim 2, wherein step (1.3) comprises:

4. The method of claim 2, wherein step (1.1) comprises:

using pSFVCs-lacz as a template, respectively using a first section-F and a first section-R as primers to amplify a fragment 1, and using a second section-F and a second section-R as primers to amplify a fragment 2, so as to obtain a vector fragment 1 and a vector fragment 2;

extracting plasmids from the identified recombinant product;

and carrying out enzyme digestion identification and sequencing on the plasmid.

5. The method of claim 3, wherein the primers are designed to have the following sequences:

the sequence of the first section-F is SEQ ID NO. 4;

the sequence of the first section-R is SEQ ID NO. 5;

the sequence of the second section-F is SEQ ID NO. 6;

the sequence of the second segment-R is SEQ ID NO. 7.

6. The method of claim 1, wherein step (2) comprises:

linearizing the pSFVCs-Lacz and SFV-helper;

performing in vitro transcription on the linearized pSFVCs-Lacz and SFV-helper;

the in vitro transcribed mRNA of pSFVCs-Lacz and pSFVCs-helper plasmids were co-transfected into BHK cells to obtain pSFVCs-Lacz virus-like particles.

7. An SFV-helper plasmid is characterized in that the SFV-helper plasmid is obtained by constructing a recombinant vector shown as SEQ ID NO.1, synthesizing an insert gene shown as SEQ ID NO.2 and cloning the insert gene into the recombinant vector.

8. pSFVCs-LacZ virus-like particles produced by the production method according to any one of claims 1 to 6.

9. The method of any one of claims 1-6 for preparing pSFVCs-LacZ virus-like particles, the SFV-helper plasmid of claim 7, and the use of pSFVCs-LacZ virus-like particles of claim 8 for the expression of β -galactosidase.

10. The method of any one of claims 1-6 for preparing pSFVCs-LacZ virus-like particles, the SFV-helper plasmid of claim 7, and the use of pSFVCs-LacZ virus-like particles of claim 8 in food, therapeutic drugs, animal models.