CN109136240B

CN109136240B - Separated nucleic acid and application thereof

Info

Publication number: CN109136240B
Application number: CN201811078931.8A
Authority: CN
Inventors: 谌章舟; 慕婷; 赵萍; 董威
Original assignee: Bravovax Co ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2018-09-14
Filing date: 2018-09-14
Publication date: 2021-07-13
Anticipated expiration: 2038-09-14
Also published as: CN109136240A

Abstract

The invention discloses a separated nucleic acid and application thereof, relating to the technical field of biomolecules. The nucleotide sequence of the nucleic acid is as shown in any one of (1) to (3): (1) the base sequence is shown as SEQ ID NO. 3; (2) the base sequences at the 1 st to 225 th positions are shown in SEQ ID NO.1, the base sequences at the 226 th to 1737 th positions encode Env protein, and the amino acid sequence of the Env protein is shown in SEQ ID NO. 4; (3) the base sequences of 1 st to 225 th bits encode M protein, the base sequences of 226 th to 1737 th bits are shown as SEQ ID NO.2, and the amino acid sequence of the M protein is shown as SEQ ID NO. 5. The nucleic acid can express a large amount of immune antigens of the Zika virus, and is beneficial to research on Zika virus vaccines or Zika virus treatment medicines.

Description

Separated nucleic acid and application thereof

Technical Field

The invention relates to the technical field of biomolecules, in particular to separated nucleic acid and application thereof.

Background

Zika virus (Zika virus) was first discovered in 1947 in Wugan Dazhai Kasanlin Africa, and it parasitizes in rhesus monkeys, spreading mainly through mosquito bites. Currently, about 80% of individuals infected with Zika virus do not exhibit significant clinical symptoms, and a small number of clinically symptomatic infected individuals exhibit fever, rash, joint pain, muscle pain, headache, vomiting, and conjunctivitis, which typically last for more than 1 week and appear milder.

Zika virus transmission vectors are Aedes aegypti and Aedes albopictus, active throughout the year in many parts of the world, including most states in the coastal region of the southern United states and many vacation areas in Latin America and the Pacific ocean. The Zika virus disease has no specific treatment method except advanced prevention. The most effective protection against Zika virus infection is against mosquito bites.

However, Zika virus is able to be transmitted vertically from pregnant women to the fetus via the placental barrier and causes serious consequences to the fetus, such as structural abnormalities in the brain of the fetus, including hypoplasia of the brainstem and cerebellum, delayed myelination, enlargement of the ventricles, severe calcification of the parenchyma of the brain, and a few symptoms of anencephalia. Unlike other viruses that infect pregnant women and affect the development of multiple tissues and organs, Zika virus appears to affect only nervous tissue. The study demonstrated that 46 pregnant women, after infecting Zika virus at birth, directly caused microcephaly and severe brain damage during delivery. However, there are no commercially available Zika virus preventive vaccines and therapeutic drugs.

Disclosure of Invention

A first object of the present invention is to provide an isolated nucleic acid that can express a large amount of an immunizing antigen of Zika virus and is useful for the study of Zika virus vaccines or Zika virus therapeutic drugs.

It is a second object of the present invention to provide a vector comprising a nucleic acid as above.

The third purpose of the invention is to provide a construction method of the recombinant adenovirus.

The fourth object of the present invention is to provide a recombinant adenovirus, which is produced by the above construction method and is capable of expressing a large amount of replicated and expressed target genes.

The fifth object of the present invention is to provide a recombinant cell or a recombinant bacterium which can produce a high-quality immune antigen of Zika virus by culturing the recombinant cell or the recombinant bacterium and which is useful for the study of Zika virus vaccines or Zika virus therapeutic drugs.

A sixth object of the present invention is to provide a method for preparing Env protein, which can rapidly and efficiently obtain Env protein.

The seventh object of the present invention is to provide a Zika virus vaccine.

An eighth object of the present invention is to provide use of the isolated nucleic acid, the vector, or the recombinant adenovirus for the manufacture of a medicament for preventing or treating Zika virus infection.

The invention is realized by the following steps:

an isolated nucleic acid having a base sequence as defined in any one of (1) to (3):

(1) as shown in SEQ ID NO. 3;

(2) the base sequences of 1-225 are shown in SEQ ID NO.1, the base sequences of 226-1737 encode Env protein, and the amino acid sequence of the Env protein is shown in SEQ ID NO. 4;

(3) the base sequences of 1 st to 225 th bits encode M protein, the base sequences of 226 th to 1737 th bits are shown as SEQ ID NO.2, and the amino acid sequence of the M protein is shown as SEQ ID NO. 5.

In some embodiments of the invention, the isolated nucleic acid is linked to a PolyA sequence (PolyA) at the 3 'end and/or a signal peptide at the 5' end. The PolyA sequence may be an SV40PolyA sequence. The signal peptide may be signal peptide Ig κ.

In some embodiments of the invention, the base sequence of the isolated nucleic acid is as set forth in SEQ ID NO.6 or 7.

A vector comprising a nucleic acid as above.

In some embodiments of the invention, the vector is a recombinant adenoviral vector.

In a preferred embodiment of the present invention, the above-mentioned adenovirus vector may be adenovirus type 5 vector (rAd 5).

In a preferred embodiment of the invention, the adenovirus type 5 vector is a pAd5-CMV/V5-DEST plasmid vector.

A preparation method of a recombinant adenovirus vector comprises the steps of contacting a shuttle plasmid vector containing the nucleic acid with the adenovirus vector, and transferring the nucleic acid to the adenovirus vector through a homologous recombination mode to obtain the recombinant adenovirus vector containing the nucleic acid.

In some embodiments of the invention, the shuttle plasmid vector is pDONR 221.

In some embodiments of the invention, the preparation method comprises transforming a shuttle plasmid vector (pDONR221-M-Env-PolyA) containing the nucleic acid with correct sequencing into Escherichia coli TOP10 competent cells containing an adenovirus vector (pAd5-CMV/V5-DEST) for homologous recombination, and screening a recombinant adenovirus vector (pAd5-CMV-M-Env-PolyA) containing the nucleic acid with correct sequencing.

A method for constructing a recombinant adenovirus, which comprises transfecting a host cell with the adenovirus vector comprising the nucleic acid.

In a preferred embodiment of the present invention, the host cell is 293.

A recombinant adenovirus is prepared by the construction method of the recombinant adenovirus.

A recombinant cell or recombinant bacterium comprising the nucleic acid, or the vector or the recombinant adenovirus.

In some embodiments of the invention, the recombinant cell is a 293 cell.

A method of making an Env protein comprising: culturing the recombinant cell or the recombinant bacterium.

A zika virus vaccine comprising: the isolated nucleic acid, the vector, the recombinant adenovirus or the Env protein prepared by the preparation method.

The invention provides application of the isolated nucleic acid in preparing a medicament for preventing or treating Zika virus infection.

The invention provides an application of the vector in preparing a medicament for preventing or treating Zika virus infection.

The invention provides an application of the recombinant adenovirus in preparing a medicament for preventing or treating Zika virus infection.

The invention has the following beneficial effects:

the isolated nucleic acid provided by the invention comprises an optimized and recombined Zika virus gene sequence, has excellent stability, can express a large amount of immune antigens of Zika virus, and is beneficial to the research of Zika virus vaccines or Zika virus treatment medicines.

The recombinant adenovirus containing the isolated nucleic acid and the construction method thereof, which are provided by the invention, stain the recombinant adenovirus vector containing the isolated nucleic acid into 293 cells, and package the recombinant adenovirus vector, and the virus liquid of the recombinant adenovirus can stimulate an organism to generate very strong antibody titer after immunizing a mouse.

The recombinant cell or the recombinant bacterium provided by the invention can be cultured to express a large amount of high-quality immune antigens of the Zika virus, and is favorable for the research of Zika virus vaccines or Zika virus treatment medicines.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic representation of the Zika virus genome;

FIG. 2 is a diagram showing the result of colony PCR detection of the vector pDONR221-M-Env-PolyA in the first example;

FIG. 3 is a diagram showing the results of PCR for identifying the plasmid pAd5-CMV-M-Env-PolyA in the first example;

FIG. 4 is a graph showing the results of PCR amplification of M-Env fragments in the third example;

FIG. 5 is a graph showing the WB detection results of the recombinant adenovirus rAd5-M-Env-PolyA in the virus solutions before and after gene optimization in the third example;

FIG. 6 shows TEM examination of the purified recombinant adenovirus rAd5-M-Env-PolyA in the first comparative example;

FIG. 7 shows HPLC detection results of the purified recombinant adenovirus rAd5-M-Env-PolyA in the first comparative example;

FIG. 8 is a graph showing an antibody titer test for the recombinant adenovirus in the first comparative example;

FIG. 9 is an expression diagram of recombinant rAd5-M-Env virus Env protein before and after optimization of WB detection gene in the second comparative example.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The isolated nucleic acids provided by the embodiments of the present invention and their uses are described in detail below.

The full-length genome of Zika virus is a single-stranded positive-strand RNA molecule with the sequence length of about 11000 nucleotides, and the virus belongs to the genus Flaviviridae in the family Flaviviridae. The total genome of Zika virus encodes 3419 amino acids, and the whole length of the virus protein is divided into several functional structural domains: the nucleocapsid (Capsid region, region C), the viral membrane Precursor (prM region), the Envelope protein (region E) and the 7 non-structural protein (NS) regions, including: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 regions). The whole genome encodes only a single Open Reading Frame (ORF), which encodes a polyprotein of about 3400 amino acids and is then cleaved into mature viral proteins, and the open reading frame of the zika virus genome terminates in untranslated regions (UTRs) in the genome, as shown in fig. 1. The major immunizing antigen of the virus of the flavivirus genus is the Env protein.

An isolated nucleic acid provided in an embodiment of the present invention has a base sequence as set forth in any one of (1) to (3):

(1) the base sequence is shown as SEQ ID NO. 3;

(2) the base sequences of 1 st to 225 th sites are shown as SEQ ID NO.1, the 226 th to 1737 th sites are base sequences coding Env protein, and the amino acid sequence of the Env protein is shown as SEQ ID NO. 4;

(3) the base sequences of 1 st to 225 th positions encode M protein, the amino acid sequence of the M protein is shown as SEQ ID NO.5, and the base sequences of 226 th to 1737 th positions are shown as SEQ ID NO. 2.

Specifically, in the above (2), the nucleotide sequences 226 to 1737 are any sequences capable of encoding Env protein having an amino acid sequence shown in SEQ ID NO.4, and in the above (3), the nucleotide sequences 1 to 225 are any sequences capable of encoding M protein having an amino acid sequence shown in SEQ ID NO. 5.

The nucleic acid provided by the embodiment of the invention is a base sequence which can efficiently express main virus immune antigens of flavivirus after being optimized. The nucleic acid comprises a base sequence capable of coding M protein and/or a base sequence coding Env protein, the base sequence coding M protein and the base sequence coding Env protein are optimized sequences, and the optimized base sequence coding M protein (SEQ ID NO.1), the base sequence coding Env protein (SEQ ID NO.2) or a fusion sequence of the two (SEQ ID NO.3) can play a role of high-efficiency Env protein. The Env protein is the main immunizing antigen of the virus of the flavivirus genus, and the M protein can play a role in stabilizing the Env protein.

Further, a PolyA sequence (PolyA) is linked to the 3' -end of the nucleic acid. The addition to the 3' end of the nucleic acid serves to stabilize the mRNA state and thus increase the expression level of the Seavirus immune antigen. Specifically, the PolyA sequence can adopt an SV40PolyA sequence which is derived from a monkey vacuolating virus genome sequence, has the total length of about 255bp, contains a conserved sequence AATAAA, and has better stabilizing effect on the nucleic acid, and specifically, the nucleic acid sequence added with the SV40PolyA sequence is shown as SEQ ID NO. 6.

Further, a signal peptide is linked to the 5' end of the nucleic acid. The signal peptide can increase the secretion efficiency of Env protein. Specifically, the nucleotide sequence added with the signal peptide Ig kappa and the SV40PolyA sequence is shown as SEQ ID NO. 7.

The embodiment of the invention provides a vector, which comprises the nucleic acid.

The vector may be one or more of the following vectors: cloning vectors, expression vectors, sequencing vectors, transformation vectors, shuttle vectors, and multifunctional vectors.

Further, the vector is an adenovirus vector, preferably a recombinant adenovirus vector type 5 (rAd5), and further preferably a pAd5-CMV/V5-DEST plasmid vector.

Specifically, the above-mentioned adenovirus vector refers to a recombinant adenovirus vector comprising the nucleotide sequence of the above-mentioned nucleic acid.

The construction method of the adenovirus vector comprises the following steps: inserting the separated nucleic acid into a shuttle plasmid vector, screening the shuttle plasmid vector inserted with a target gene by adopting pDONR221, sequencing, contacting the shuttle plasmid vector pDONR221-M-Env-PolyA with correct sequencing with an adenovirus vector, specifically, transforming the shuttle plasmid vector into an escherichia coli TOP10 competent cell containing the adenovirus vector pAd5-CMV/V5-DEST for homologous recombination, and screening a recombinant adenovirus vector pAd5-CMV-M-Env-PolyA with correct sequencing.

The construction method of the recombinant adenovirus provided by the embodiment of the invention comprises the steps of transfecting the adenovirus vector (pAd5-CMV-M-Env-PolyA) into a host cell 293 cell, and packaging to obtain the recombinant adenovirus.

The recombinant cell or recombinant bacterium provided by the embodiment of the invention comprises the isolated nucleic acid, the vector or the recombinant adenovirus.

The method for preparing Env protein provided by the embodiment of the invention comprises the step of culturing the recombinant cell or the recombinant bacterium. Specifically, the method comprises the steps of culturing the recombinant cell or the recombinant bacterium, and then separating and purifying the culture to obtain the Env protein, namely the Zika virus antigen protein.

The Zika virus vaccine provided by the embodiment of the invention comprises the nucleic acid, the vector, the recombinant adenovirus or the Env protein prepared by the method for preparing the Env protein.

The features and properties of the present invention are described in further detail below with reference to examples.

First embodiment

Plasmid pAd5-CMV-M-Env-PolyA was prepared.

1. Construction of transfer plasmid (pDONR221-M-Env-PolyA)

1.1. Gene synthesis and primer design

The M-Env sequence was synthesized by Gene Synthesis according to the sequence shown in SEQ ID NO. 3.

M-Env sequence (shown as SEQ ID NO.3) is used as a template, and upstream and downstream primers (synthesized by Invitrogen company) are designed for PCR amplification.

Wherein the sequence of the upstream primer (M-Env-F) is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGCTGTGACGCTCCCCTCCCATTC-3' (shown in SEQ ID NO. 8);

the sequence of the downstream primer (SV40-R) is as follows: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGATGATAAGATACATTGATGAGT-3' (shown in SEQ ID NO. 9).

PCR amplification

An amplification system: 2. mu.L each of the upstream and downstream primers, 1. mu.L of the gene synthesis plasmid template, 25. mu.L of Primer STAR mix, plus ddH₂O to 50. mu.L.

The size of the amplified fragment M-Env fusion sequence is 1737bp, and the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 1min for 30s for 30 cycles; finally, the extension is carried out for 10min at 72 ℃.

The M-Env fragment amplified by PCR was digested simultaneously with SpeI and XhoI, respectively, with pDONR221-SV40Poly A plasmid (stored in the company), ligated with T4 ligase, and E.coli TOP10 competent cells were transformed and plated on solid LB plates containing kana antibiotics.

The next day, different clones were selected as PCR colonies with forward and reverse PCR primers M13-F/M13-R. The PCR conditions were as follows: 2 μ L of the bacterial suspension, 0.5 μ L (10. mu. mol/μ L) of each PCR forward and reverse primer (M13-F/M13-R), 5 μ L of 2 XTaq mix, ddH₂O2 mu L; circulation conditions are as follows: at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15s, 55 ℃ for 15s and 72 ℃ for 1min for 30 s; finally 5min at 72 ℃.

After the PCR is finished, the PCR result of agarose gel electrophoresis detection is shown in figure 2, wherein M is DL5000DNA marker, and 1-3 are PCR results of different colonies respectively of pDONR 221-M-Env-PolyA. A fragment M-Env-PolyA (SEQ ID NO.6) of about 2000bp, all of which correspond in size to the expected (2007bp), can be seen in FIG. 2; the PCR-identified positive bacteria were inoculated into 5mL LB liquid medium containing Kana antibiotic, respectively, and cultured overnight.

The next day, plasmids of different colonies were extracted and sent to the sequencing company for sequencing, with the upstream primer being M13-F (SEQ ID NO.10) and the downstream primer being M13-R (SEQ ID NO.11), and two-way sequencing. The sequencing result showed that the correct pDONR221-M-Env-PolyA plasmid was obtained.

2. Construction of adenovirus expression vector pAd5-CMV-M-Env-PolyA

2.1. The pDONR221-M-Env-PolyA plasmid 150ng sequenced correctly in step 1.2 was subjected to Gateway LR recombination with pAd5-CMV/V5-DEST plasmid 150ng, respectively (see the Invitrogen company Gateway recombination instruction manual for details), E.coli TOP10 competent cells (purchased from Invitrogen, USA), and plated on Amp antibiotic-containing solid LB plates.

2.2. The next day, different clones were selected for colony PCR with upstream primer T7-F (SEQ ID NO.12) and downstream primer V5-C-R (SEQ ID NO.13) under the following PCR conditions: 2 μ L of bacterial suspension, 0.5 μ L (10 μmol/μ L) of each of the upstream and downstream primers (T7-F/V5-C-R), 5 μ L of 2 XTaq mix, ddH₂O2 μ L, cycling conditions: one step at 95 ℃ for 2 min; 15s at 95 ℃, 15s at 45 ℃ and 30s at 72 ℃ for 1min for 30 cycles; finally 5min at 72 ℃. After the PCR is finished, the PCR result of agarose (purchased from BIOWEST corporation) gel electrophoresis detection is shown in figure 3, M is DL5000DNA marker, and 1-6 are different colony PCR results of pAd 5-CMV-M-Env-PolyA.

2.3. The PCR-identified positive bacteria were inoculated into 5mL LB liquid medium containing Kana antibiotic, respectively, and cultured overnight. The PCR-identified positive bacteria were inoculated into 5mL of LB liquid medium containing Amp antibiotics, respectively, and cultured overnight. The next day, plasmids of different colonies were extracted and sent to the sequencing company for sequencing, and the sequencing primers T7-F and V5-C-R were sequenced in both directions. The sequencing result showed that the correct pAd5-CMV-M-Env-PolyA plasmid was obtained.

Second embodiment

A linearized viral vector rAd5-M-Env-PolyA was prepared.

3. Amplification of adenovirus vectors

3.1. The correctly sequenced pAd5-CMV-M-Env-PolyA plasmid from step 2.3 was transformed into E.coli TOP10 competent cells and plated onto solid LB plates containing Amp antibiotics. Negative control is set in comparison: the pAd5-CMV/V5-Dest plasmid (purchased from Invitrogen, USA) was transformed into E.coli ccdBsuvival competent cells (purchased from Invitrogen, USA) and plated on solid LB plates containing Amp antibiotics.

3.2. The next day, single clones on LB plates were picked up and inoculated in a liquid medium containing 200mL of Amp, and after overnight culture, a large number of pAd5-CMV-M-Env-PolyA plasmids and pAd-CMV/V5-Dest plasmids were extracted separately using a large plasmid extraction kit.

4. Adenovirus vector linearization the pAd5-CMV-M-Env-PolyA plasmid and the pAd-CMV/V5-Dest plasmid obtained in step 3.2 were digested with PacI restriction enzyme (purchased from NEB, USA) at 37 ℃ for 3h, as follows: plasmid (pAd5-CMV-M-Env-PolyA or pAd-CMV/V5-Dest): 10 μ g, 10 xneb CutSmart buffer: 5 μ L, PacI enzyme: 5 μ L, plus ddH₂O to a final volume of 50. mu.L. After enzyme digestion, the linearized DNA fragment after enzyme digestion is recovered by a PCR product recovery kit (Tiangen company) to obtain a linearized viral vector rAd5-M-Env-PolyA, and the recovered DNA fragment is quantified by a trace nucleic acid quantifier.

5. Packaging of recombinant adenoviruses

5.1. One bottle of 293 cells in good growth state was taken (T75 cm)²) 293 cells were trypsinized the day before transfection and counted, 5 x 10 cells per well in 6-well plates⁵And (4) cells. 2mL of DMEM + 10% FBS + 1% Pen-strep (all from GIBCO, USA) was added to each well. On the day of transfection, the 6-well plate medium was discarded and replaced with 1.5mL of fresh DMEM + 10% FBS medium (without double antibody).

5.2. Preparation of DNA-lip2000 complexes (lip2000 from Invitrogen USA)

5.2.1. Mu.g of the pAd5-CMV-M-Env-PolyA and pAd-CMV/V5-Dest plasmids after PacI linearization treatment (step 4) were taken, respectively, and then diluted to 250. mu.L with DMEM serum-free medium, respectively, and gently mixed to obtain plasmid dilutions. Sucking 3 mu L of lip2000, adding 247 mu L of DMEM medium without serum, gently mixing uniformly, and standing at room temperature for 5min to obtain a lip2000 diluent. Mixing the plasmid diluent and lip2000 diluent, incubating at room temperature for 20min to form DNA-lip2000 complex (the solution may be turbid, and transfection is not affected), dripping the incubated DNA-lip2000 complex into 6-well plate cells, shaking back and forthMixing with 6-well plate, and placing 6-well plate at 37 deg.C and 5% CO₂The culture was carried out overnight in an incubator.

5.2.2. The next day, the cell lip 2000-containing medium in 6-well plates (purchased from Corning, usa) was aspirated and replaced with fresh DMEM + 10% FBS medium. The solution is changed every 2-3 days until CPE is seen (7-10 days after transfection). The 6-well plates were supplemented with medium and cultured until the CPE reached 80%, and the cells were pipetted off the 6-well plates to obtain virus fluid, which was transferred to a 15mL centrifuge tube (purchased from Corning, USA). Placing the collected virus liquid into a refrigerator at-80 deg.C for 30min, thawing the virus liquid in a water bath at 37 deg.C for not more than 15min, and repeating the freezing and thawing steps twice.

5.2.3. And (4) centrifuging the frozen and thawed virus solution at room temperature at 3000rpm for 15 min. The rAd5-M-Env-PolyA virus solution (pellet) and rAd5-Dest virus solution, which were obtained separately, were dispensed into a cryopreservation tube (purchased from Corning, USA) at 1mL per tube and stored in a refrigerator at-80 ℃.

Determination of recombinant adenovirus Titers by TCID50 method

6.1. One bottle of 293 cells in good growth state was taken (T75 cm)²) 293 cells were trypsinized the day before assay, pancreatin was discarded when digestion was terminated, and cells were resuspended in DMEM + 2% FBS and counted. 293 cells were placed in a 96-well cell plate (purchased from Corning, USA) and the cell concentration was adjusted to 8.3 x 10⁴mL, 100. mu.L cells per well, shaken well and placed at 37 ℃ in 5% CO₂The incubator lasts for 16-20 hours.

6.2. Diluting rAd5-M-Env-PolyA virus solution obtained in step 5.2.3: DMEM + 2% FBS medium is used as 10^-1～10^-10Ten times serial dilution. Yield 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9、10^-10And obtaining rAd5-M-Env-PolyA virus diluent and rAd5-Dest virus diluent by double dilution.

6.3. In 96-well cell plates, 100. mu.L of 10 obtained in step 6.2 was added per well^-3～10^-10Double-diluted rAd5-M-Env-PolyA virus dilutionRelease, repeat 10 wells for each virus dilution. Mixing, and standing at 37 deg.C for 5% CO₂An incubator. Meanwhile, 100. mu.L of DMEM + 2% FBS medium was added to the other two longitudinal rows as negative controls. Is placed in CO₂Culturing 7d, 10d and 14d (7d/10d/14d respectively observing cell and CPE conditions) in an incubator at 37 ℃, then observing under an inverted microscope, judging and recording cytopathic effect conditions of each row, and showing that the cytopathic rate is high, and the finally obtained virus has the average titer of 10⁷～10⁸Between pfu/mL, positive results were obtained only when a small number of cells had CPE, and when it could not be judged whether CPE or cell death occurred, the results were compared with the following negative control.

Third embodiment

The recombinant adenovirus rAd5-M-Env-PolyA of the second example was verified to be capable of expressing Env protein.

PCR identification of recombinant adenovirus Env Gene

7.1. The virus solution obtained in step 5.2.3 was used to extract viral genes using a viral RNA/DNA extraction kit (according to the protocol), and a positive control (M-Env-PolyA) and a negative control (rAd5-Dest) were set.

7.2. And (3) carrying out PCR amplification on the extracted virus DNA to identify the inserted target gene of the obtained recombinant adenovirus. The PCR positive and negative primers are as follows: T7-F/V5-C-R, PCR conditions: viral DNA 1. mu.L, forward and reverse primers 0.5. mu.L each, 2 XPrimerSTAR mix 5. mu.L, ddH₂O3 μ L, cycling conditions: one step at 95 ℃ for 2 min; 30 cycles of 95 ℃ for 15s, 45 ℃ for 15s, and 72 ℃ for 1min for 30 s; finally 5min at 72 ℃.

7.3 agarose gel electrophoresis after PCR was complete detects the PCR results, see FIG. 4. And the amplified band is sent to a sequencing company for sequencing. Sequencing proves that the amplified DNA fragment is the M-Env sequence of Zika virus. Thus, the M-Env sequence was included in the pAd5-CMV-M-Env-PolyA plasmid obtained in step 3.2.

WB (WesternBlot) detection of recombinant adenovirus Env Gene expression

8.1. One bottle of 293 cells in good growth state was taken (T75 cm)²) 293 cells were trypsinized the day before infection and counted, 5 x 10 cells per well in 6-well plates⁵And (4) cells. Adding 2mL of DMEM + into each hole10% FBS + 1% Pen-strep, cell confluence reached 90% after 24h, 200. mu.L of rAd5-M-Env-PolyA virus solution obtained in step 5.2.3 was added to one well and infected for 48 h, confirming that 70% of the cells had CPE, and if CPE did not reach 70%, infection was extended for another 24 h. The other well was set as a negative control.

8.2. The infected culture supernatant was discarded, the 6-well plate was placed on ice, 100. mu.L of cell lysate containing 1% PMSF (purchased from Biyuntian), was added to each of the infected wells and the negative wells, and gently shaken to rapidly lyse the cells, and the cells were lysed on ice for 15 min. The lysed cell fluid was aspirated into a 1.5mL EP tube. 7000rpm, 4 ℃ centrifugation for 5 minutes, collected supernatant for the next detection.

8.3. mu.L of the supernatant from step 8.2 was added to 20. mu.L of 4 XProtein loading loadingbuffer, rAd5-Dest and 293 cells as negative controls and boiled at 100 ℃ for 5 min. SDS-PAGE was run at 160V for 90 min.

SDS-PAGE Gel after the completion of electrophoresis was transferred onto a PVDF membrane by a wet transfer method (the transfer solution was prepared according to the PVDF membrane protocol). 90V, and the film is rotated for 1h in ice bath. After the membrane transfer is finished, the membrane is washed for 10min by 1 XPBST solution. 5% skimmed milk powder was prepared with 1 XPBST solution as blocking solution, and the transferred membrane was blocked overnight at 4 ℃ in blocking solution.

The following day, the membranes were washed with 1 XPBST solution for 10min × 2 times. Anti-zikaenvelope poly-antimibody (available from GeneTex, USA) is diluted with 1% skimmed milk powder-containing PBST solution at a ratio of 1:2000, and the membrane is incubated in primary antibody diluent at 37 deg.C for 2 h. After incubation, the membrane was washed with 1 XPBST solution for 10min X3 times. An anti-rabbitsecondary antibody (purchased from Jackson, USA) is diluted by PBST solution containing 1% skimmed milk powder according to the proportion of 1:2000, and after dilution, the membrane is placed in a secondary antibody diluent for incubation for 1h at 37 ℃. After incubation, the membrane was washed with 1 XPBST solution for 10min X3 times.

8.4. The chemiluminescence method comprises mixing A, B liquid color development liquid (purchased from Pierce company, USA) according to the kit specification, adding PVDF membrane (purchased from Millipore company, USA), developing color in dark for 2-3 min, and taking pictures with chemiluminescence membrane scanner. The WB results are shown in FIG. 5, wherein M is protein Marker, 1-2 are negative control 293 cells, 3 is 293 cells infected by rAd5-Dest virus, and 4 is 293 cells infected by rAd5-M-Env-PolyA virus.

Expression of the membrane protein M-Env of Zika virus was detected in 293 cells after rAd5-M-Env-PolyA infection, whereas expression of the M-Env protein was not detected in 293 and 293 negative control cells after rAd5-Dest infection. Thus, the pAd5-CMV-M-Env-PolyA plasmid in step 3.2 of the second example was able to express the M-Env fusion protein.

First comparative example

The immunogenicity of the recombinant adenovirus rAd5-M-Env-PolyA was verified.

9. Amplification of recombinant adenovirus rAd5-M-Env-PolyA

9.1. At four T75cm²293 cells were inoculated in square flasks (purchased from Corning, usa) in DMEM + 10% FBS + 1% Pen-strep to a density of 90%, one flask of T75 was taken, cells were resuspended and cell counted after trypsinization, and virus solutions of three recombinant adenoviruses were separately prepared according to the measured virus titers: the positive control M-Env genome (non-optimized M-Env genome), rAd5-M-Env-PolyA virus solution obtained in step 5.2.3, and rAd5-Dest virus solution were added to the remaining 3T 75 flasks of 293 cells, respectively, such that the final MOI values were all 0.1. After 3-4 days, the cells become almost round, and half of the cells float, and then all the cells are collected. Centrifuging at 500g, removing supernatant, freezing the collected cells (precipitate) in a refrigerator at-80 deg.C, and thawing the cells in 37 deg.C water bath. This operation was repeated for a total of three freeze-thaw cycles. Freezing the collected cells in a refrigerator at-80 deg.C, placing the cells in a water bath at 37 deg.C after the cells are completely frozen, and rapidly thawing. Repeating the operation, and repeatedly freezing and thawing for three times to obtain the second generation recombinant adenovirus.

9.2. The second generation recombinant adenovirus rAd5-M-Env-PolyA from step 9.1 was tested for TCID50 (as determined by TCID50 in the second example). Respectively inoculating 293 cells in a plurality of T225 bottles, wherein the culture medium is DMEM + 10% FBS + 1% Pen-strep until the density reaches 90%, taking one bottle of T225, carrying out trypsinization, then carrying out resuspension and cell counting on the cells, and adding the rest of the T225 bottles of 293 cells into the second generation harvest solution of the recombinant adenovirus according to the measured virus titer so that the final MOI values are all 0.1. After 3-4 days, the cells become almost round, and half of the cells float, and then all the cells are collected. Centrifuging at 500g, removing supernatant, freezing the collected cells (precipitate) in a refrigerator at-80 deg.C, and thawing the cells in 37 deg.C water bath. Repeating the operation, and repeatedly freezing and thawing for three times to obtain the virus preservation solution.

10. Purification of recombinant adenovirus rAd5-M-Env-PolyA

10.1. Discontinuous cesium chloride density gradient centrifugation is used for removing main cell pollutants and defective virus particles (remark: at least 3X 10 is needed for virus band amplification to ensure that virus bands are easily distinguished after cesium chloride (purchased from the national drug group) density gradient⁸Cell):

10.1.1. the rotor was centrifuged to 4 ℃ with precooling, and 12mL of 1.4g/mL cesium chloride (53g +87mL 10mM Trz-HCl, pH 7.9) were added slowly to the centrifuge tube, followed by 9mL of 1.2g/mL cesium chloride (26.8+92mL 10mM Tris-HCl, pH 7.9) very gently. The final purity of the virus depends on the quality of the density gradient. 13mL of the virus stock solution from step 9.2 (the amount of virus must be less than 10) was added to the top of the discontinuous gradient in a clean bench⁹The resulting cells, otherwise exceeding the density gradient load, were adjusted to 13mL with pH 7.910mM Tris-HCl if the volume of the storage solution was less than 13 mL).

10.1.2. The tubes were equilibrated and centrifuged at 100000 Xg (23000 rpw on a SW28 rotor) for 120 min at 4 ℃. Taking out the centrifugal tube from the super clean bench and vertically fixing the centrifugal tube by using a clamp. Most of the impurities were aspirated from the top of the gradient with a 10mL pipette. Puncture was performed with a 5mL syringe with an 18G needle from the centrifuge tube outer wall slightly below the viral band (the lowest band) (carefully aspirate the viral band to avoid aspiration of other bands and impurities). The virus-containing solution was transferred to a sterile 15mL centrifuge tube. 1 volume of 1 XTE was added to obtain a virus suspension.

10.2. Continuous cesium chloride density gradient centrifugation separates infectious and defective viral particles:

10.2.1. 20mL of 1.35g/mL cesium chloride was added to the centrifuge tube, and 15mL of the virus suspension from step 10.1.2 was added very slowly from the top of the centrifuge tube and centrifuged at 100000 Xg for 18 hours at 4 ℃. After ultracentrifugation, a 10mL pipette is used to aspirate most of the gradient solution and impurities from the top of the centrifuge tube (avoiding aspiration into the bottom of the blue-white band containing virus, which helps to reduce the solution outflow rate when collecting virus). Bottom blue-white viral bands were collected by bottom puncture with a 20G needle.

10.3. Dialysis for removal of cesium chloride

10.3.1. The viral bands collected in step 10.2.1 were dialyzed against 10000 daltons cellulose ester membrane (purchased from BD, usa) at 4 ℃ to remove cesium chloride salt. The dialysis solution contained 20mM Tris (pH8.0), 25mM NaCl, 2.5% (w/v) glycerol, and the volume of the dialysis solution was 200 times that of the virus solution, and the solution was dialyzed for one hour each time, and then the buffer was changed, and no trace of cesium chloride was left after 3 times of buffer change. The virus solution after dialysis is frozen and stored in a refrigerator at minus 80 ℃.

10.4. The virus solution dialyzed in step 10.3.1 was subjected to PCR, western-blot (WB), TCID50 (see the second example for the detection procedure), TEM (see FIG. 6 for TEM) and HPLC (see FIG. 7 for HPLC). The result proves that the purified recombinant adenovirus rAd5-M-Env contains an M-Env fusion sequence, the size of 2000bp is in line with the expectation, and the recombinant adenovirus rAd5-M-Env can express Env protein.

11. Verification of immunogenicity of recombinant adenovirus rAd5-M-Env-PolyA

11.1. Selecting 40 BALB/c mice (female/male half) with the age of 6-8 weeks and the weight of 18-22 g, randomly grouping the animals according to the weight after inspection and quarantine into a blank control group, an rAd5-empty group (negative control), an rAd5-Env-PolyA group (positive control) and an rAd5-M-Env-PolyA group (target group), wherein all samples are virus liquid for encapsulating success, and adopting an intramuscular injection mode. Specific information of each experimental group is shown in table 1.

Table 1 shows the immunization information of the experimental groups

Remarking: the target gene in the positive control group adopts an unoptimized M-Env gene sequence.

The blank control group (10) is not injected with immunity, blood is collected at 28 days and 56 days after the experiment is started, serum is separated for detection, and all the sample serum is stored in a low-temperature refrigerator with the temperature of less than or equal to-60 ℃. Remarking: the animal experiments are completed by drug tests in Wuhan City of third-party detection institutions, and all the serum of the experimental samples is taken according to contract agreement. All serum antibody titers of the experimental samples were strictly performed according to the Kit instructions (Recombivirus (TM) Mouse Anti-Zika Virus Envelope (ENV) Protein IgG ELISA Kit) (purchased from ADI, USA), and the required reagents and consumables were provided in the Kit, as follows:

and respectively adding 100 mu L of Anti-ZIKV-E calibrator, sample serum and Anti-ZIKV-E positive control mixed liquor into the pretreated ZIKV-E coated pore plate. The coated well plate was shaken to mix the mixture well and incubated for 60min at rest. The well plate was decanted, the plate was washed repeatedly 4 times with 1 x washes (ensuring no sample reagent remained attached to the plate), and the residual wash solution in the plate was then blotted dry by inverting the plate and drying on plain paper.

Add 100. mu.L of diluted Anti-Mouse IgG HRP conjugate to the test well plate, incubate for 30min, pour off the well plate, and wash the plate 5 times with 1 Xwash. Add 100. mu.L of TMB substrate to the test well plate, which gradually turns blue, and then place the plate in the dark for 15 min. Adding 100 mu L of stop solution into the pore plate to be detected, slightly shaking to fully and uniformly mix the solution in the pore plate, and gradually changing the solution into yellow after uniform mixing.

And (3) adjusting the wavelength of the spectrophotometer to 450nm, then putting the pore plate to be detected into the spectrophotometer for reading, calculating the antibody titer values in all serum samples after obtaining the data, and drawing a graph, referring to the attached drawing 8.

According to result analysis, the serum titer of the target group and the positive control group obviously exceeds the serum titer of the negative control group and the blank group, and the target group and the positive control group do not belong to false positive. The serum titer after injection of rAd5-M-Env-PolyA group virus solution (target group) was higher than that after injection of rAd5-Env-PolyA group virus solution (positive control).

Second comparative example

The M-Env nucleic acid sequence provided by the invention is compared with other existing M-Env fusion sequences in Env expression level.

See the third example for specific experimental procedures, comparative setup 1 set of comparative examples. Referring to FIG. 9, in FIG. 9, M is protein marker, 1 is a negative control 293 cell, 2 is 293 cell infected by rAd5-M-Env-PolyA virus (viral gene sequence see GenBank: KU963796.1) without codon optimization (comparative example), 3 is 293 cell infected by rAd5-M-Env-PolyA with codon optimization (third example), and 4 is 293 cell infected by rAd5-Dest virus. The result shows that the expression quantity of the M-Env nucleic acid sequence provided by the invention is higher than that of other existing M-Env fusion sequences.

In conclusion, the invention provides an isolated nucleic acid, which comprises an optimized and recombined Zika virus gene sequence, has excellent stability, can efficiently express a large amount of immune antigens of Zika virus, and is beneficial to the research of Zika virus vaccines or Zika virus therapeutic drugs.

After mice are immunized by the virus liquid of the recombinant adenovirus provided by the embodiment of the invention, the body can be stimulated to generate very strong antibody titer. In addition, the recombinant adenovirus can conveniently carry out immunization through mucous membrane and can induce the organism to generate mucous membrane and system immune response; the inactivated vaccine has the advantages of high immune effect, low cost, high safety and the like.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan Bowo Biotechnology Ltd

<120> an isolated nucleic acid and uses thereof

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 225

<212> DNA

<213> Artificial sequence

<400> 1

gccgtgacct tgccctccca ctccacccgg aagctccaga ccaggagcca gacctggctg 60

gagtccaggg agtacaccaa gcacctgatc cgggtggaga actggatctt tcggaaccca 120

ggctttgccc tggccgccgc cgccatcgcc tggctgctcg gcagctccac cagccagaag 180

gtcatctacc ttgtgatgat cctgctcatc gcccccgcct actcc 225

<210> 2

<211> 1512

<212> DNA

<213> Artificial sequence

<400> 2

atcaggtgca tcggcgtcag caacagggac tttgtggagg ggatgagcgg aggcacctgg 60

gtcgatgtgg tgctggagca cggggggtgc gtgacagtga tggcccagga caagcccacc 120

gtggacattg agctcgtgac tactacagtg tccaacatgg ccgaggtcag atcctactgc 180

tacgaggcct ccatctccga tatggctagc gactccagat gcccaacaca gggcgaggcc 240

tacctcgata agcagagcga cacccagtac gtgtgcaagc ggacactggt ggacaggggg 300

tgggggaacg ggtgcgggct gtttgggaag gggagcctcg tcacctgcgc caagttcgct 360

tgctccaaga agatgacagg caagagcatc cagcctgaga acctggagta ccggatcatg 420

ctgagcgtgc atggaagcca gcactccggc atgatcgtga acgatacagg gcacgaaacc 480

gatgagaacc gggccaaggt ggagatcaca cccaactccc ctagggctga ggccacactg 540

gggggcttcg gctccctggg gctggattgc gagccccgca ccggcctgga cttcagcgac 600

ctgtactacc tgaccatgaa caacaagcac tggctggtgc acaaggagtg gtttcacgat 660

atccccttgc cttggcacgc cggggccgac acaggcaccc cccactggaa caacaaggag 720

gccctcgtgg agttcaagga cgcccacgcc aagaggcaga ctgtggtggt gttgggatcc 780

caggaggggg ccgtccacac cgccctcgct ggcgccctgg aggccgagat ggatggcgcc 840

aaggggaggc tgagtagcgg gcacctgaag tgccggctca agatggataa gctgaggctg 900

aagggggtga gctactccct ctgcacagcc gccttcacct tcacaaagat ccctgccgag 960

acactgcacg gcaccgtgac agtcgaagtg cagtacgccg gcaccgacgg cccatgcaag 1020

gtgcccgccc agatggccgt ggacatgcag acactcaccc ccgtgggcag actgatcacc 1080

gccaaccccg tgattacaga gtccacagag aacagcaaga tgatgctgga gcttgatccc 1140

ccatttgggg atagctacat tgtgatcggc gtcggcgaga agaagatcac acatcactgg 1200

catcggtccg gcagcaccat cggcaaggcc ttcgaggcca cagtgagagg cgcccggagg 1260

atggccgtcc tgggcgatac agcctgggac ttcgggagcg tggggggcgc cctgaacagc 1320

ctcgggaagg gcatccacca gatcttcggc gccgccttta agtccctctt tgggggcatg 1380

agctggttta gccagatcct gatcggcacc ctgctgatgt ggctggggct gaacaccaag 1440

aacgggagca ttagcctgat gtgcctggcc ctgggcgggg tcctgatctt cctgagcaca 1500

gccgtgagcg cc 1512

<210> 3

<211> 1737

<212> DNA

<213> Artificial sequence

<400> 3

gccgtgacct tgccctccca ctccacccgg aagctccaga ccaggagcca gacctggctg 60

gagtccaggg agtacaccaa gcacctgatc cgggtggaga actggatctt tcggaaccca 120

ggctttgccc tggccgccgc cgccatcgcc tggctgctcg gcagctccac cagccagaag 180

gtcatctacc ttgtgatgat cctgctcatc gcccccgcct actccatcag gtgcatcggc 240

gtcagcaaca gggactttgt ggaggggatg agcggaggca cctgggtcga tgtggtgctg 300

gagcacgggg ggtgcgtgac agtgatggcc caggacaagc ccaccgtgga cattgagctc 360

gtgactacta cagtgtccaa catggccgag gtcagatcct actgctacga ggcctccatc 420

tccgatatgg ctagcgactc cagatgccca acacagggcg aggcctacct cgataagcag 480

agcgacaccc agtacgtgtg caagcggaca ctggtggaca gggggtgggg gaacgggtgc 540

gggctgtttg ggaaggggag cctcgtcacc tgcgccaagt tcgcttgctc caagaagatg 600

acaggcaaga gcatccagcc tgagaacctg gagtaccgga tcatgctgag cgtgcatgga 660

agccagcact ccggcatgat cgtgaacgat acagggcacg aaaccgatga gaaccgggcc 720

aaggtggaga tcacacccaa ctcccctagg gctgaggcca cactgggggg cttcggctcc 780

ctggggctgg attgcgagcc ccgcaccggc ctggacttca gcgacctgta ctacctgacc 840

atgaacaaca agcactggct ggtgcacaag gagtggtttc acgatatccc cttgccttgg 900

cacgccgggg ccgacacagg caccccccac tggaacaaca aggaggccct cgtggagttc 960

aaggacgccc acgccaagag gcagactgtg gtggtgttgg gatcccagga gggggccgtc 1020

cacaccgccc tcgctggcgc cctggaggcc gagatggatg gcgccaaggg gaggctgagt 1080

agcgggcacc tgaagtgccg gctcaagatg gataagctga ggctgaaggg ggtgagctac 1140

tccctctgca cagccgcctt caccttcaca aagatccctg ccgagacact gcacggcacc 1200

gtgacagtcg aagtgcagta cgccggcacc gacggcccat gcaaggtgcc cgcccagatg 1260

gccgtggaca tgcagacact cacccccgtg ggcagactga tcaccgccaa ccccgtgatt 1320

acagagtcca cagagaacag caagatgatg ctggagcttg atcccccatt tggggatagc 1380

tacattgtga tcggcgtcgg cgagaagaag atcacacatc actggcatcg gtccggcagc 1440

accatcggca aggccttcga ggccacagtg agaggcgccc ggaggatggc cgtcctgggc 1500

gatacagcct gggacttcgg gagcgtgggg ggcgccctga acagcctcgg gaagggcatc 1560

caccagatct tcggcgccgc ctttaagtcc ctctttgggg gcatgagctg gtttagccag 1620

atcctgatcg gcaccctgct gatgtggctg gggctgaaca ccaagaacgg gagcattagc 1680

ctgatgtgcc tggccctggg cggggtcctg atcttcctga gcacagccgt gagcgcc 1737

<210> 4

<211> 480

<212> PRT

<213> amino acid sequence of Env protein

<400> 4

Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser

1 5 10 15

Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr

20 25 30

Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr

35 40 45

Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser

50 55 60

Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala

65 70 75 80

Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu

85 90 95

Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser

100 105 110

Leu Val Thr Cys Ala Lys Phe Ala Cys Ser Lys Lys Met Thr Gly Lys

115 120 125

Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His

130 135 140

Gly Ser Gln His Ser Gly Met Ile Val Asn Asp Thr Gly His Glu Thr

145 150 155 160

Asp Glu Asn Arg Ala Lys Val Glu Ile Thr Pro Asn Ser Pro Arg Ala

165 170 175

Glu Ala Thr Leu Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro

180 185 190

Arg Thr Gly Leu Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn

195 200 205

Lys His Trp Leu Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro

210 215 220

Trp His Ala Gly Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu

225 230 235 240

Ala Leu Val Glu Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val

245 250 255

Val Leu Gly Ser Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala

260 265 270

Leu Glu Ala Glu Met Asp Gly Ala Lys Gly Arg Leu Ser Ser Gly His

275 280 285

Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser

290 295 300

Tyr Ser Leu Cys Thr Ala Ala Phe Thr Phe Thr Lys Ile Pro Ala Glu

305 310 315 320

Thr Leu His Gly Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp

325 330 335

Gly Pro Cys Lys Val Pro Ala Gln Met Ala Val Asp Met Gln Thr Leu

340 345 350

Thr Pro Val Gly Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser

355 360 365

Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp

370 375 380

Ser Tyr Ile Val Ile Gly Val Gly Glu Lys Lys Ile Thr His His Trp

385 390 395 400

His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg

405 410 415

Gly Ala Arg Arg Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly

420 425 430

Ser Val Gly Gly Ala Leu Asn Ser Leu Gly Lys Gly Ile His Gln Ile

435 440 445

Phe Gly Ala Ala Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser

450 455 460

Gln Ile Leu Ile Gly Thr Leu Leu Met Trp Leu Gly Leu Asn Thr Lys

465 470 475 480

<210> 5

<211> 75

<212> PRT

<213> amino acid sequence of M protein

<400> 5

Ala Val Thr Leu Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser

1 5 10 15

Gln Thr Trp Leu Glu Ser Arg Glu Tyr Thr Lys His Leu Ile Arg Val

20 25 30

Glu Asn Trp Ile Phe Arg Asn Pro Gly Phe Ala Leu Ala Ala Ala Ala

35 40 45

Ile Ala Trp Leu Leu Gly Ser Ser Thr Ser Gln Lys Val Ile Tyr Leu

50 55 60

Val Met Ile Leu Leu Ile Ala Pro Ala Tyr Ser

65 70 75

<210> 6

<211> 2007

<212> DNA

<213> Artificial sequence

<400> 6

gccgtgacct tgccctccca ctccacccgg aagctccaga ccaggagcca gacctggctg 60

gagtccaggg agtacaccaa gcacctgatc cgggtggaga actggatctt tcggaaccca 120

ggctttgccc tggccgccgc cgccatcgcc tggctgctcg gcagctccac cagccagaag 180

gtcatctacc ttgtgatgat cctgctcatc gcccccgcct actccatcag gtgcatcggc 240

gtcagcaaca gggactttgt ggaggggatg agcggaggca cctgggtcga tgtggtgctg 300

gagcacgggg ggtgcgtgac agtgatggcc caggacaagc ccaccgtgga cattgagctc 360

gtgactacta cagtgtccaa catggccgag gtcagatcct actgctacga ggcctccatc 420

tccgatatgg ctagcgactc cagatgccca acacagggcg aggcctacct cgataagcag 480

agcgacaccc agtacgtgtg caagcggaca ctggtggaca gggggtgggg gaacgggtgc 540

gggctgtttg ggaaggggag cctcgtcacc tgcgccaagt tcgcttgctc caagaagatg 600

acaggcaaga gcatccagcc tgagaacctg gagtaccgga tcatgctgag cgtgcatgga 660

agccagcact ccggcatgat cgtgaacgat acagggcacg aaaccgatga gaaccgggcc 720

aaggtggaga tcacacccaa ctcccctagg gctgaggcca cactgggggg cttcggctcc 780

ctggggctgg attgcgagcc ccgcaccggc ctggacttca gcgacctgta ctacctgacc 840

atgaacaaca agcactggct ggtgcacaag gagtggtttc acgatatccc cttgccttgg 900

cacgccgggg ccgacacagg caccccccac tggaacaaca aggaggccct cgtggagttc 960

aaggacgccc acgccaagag gcagactgtg gtggtgttgg gatcccagga gggggccgtc 1020

cacaccgccc tcgctggcgc cctggaggcc gagatggatg gcgccaaggg gaggctgagt 1080

agcgggcacc tgaagtgccg gctcaagatg gataagctga ggctgaaggg ggtgagctac 1140

tccctctgca cagccgcctt caccttcaca aagatccctg ccgagacact gcacggcacc 1200

gtgacagtcg aagtgcagta cgccggcacc gacggcccat gcaaggtgcc cgcccagatg 1260

gccgtggaca tgcagacact cacccccgtg ggcagactga tcaccgccaa ccccgtgatt 1320

acagagtcca cagagaacag caagatgatg ctggagcttg atcccccatt tggggatagc 1380

tacattgtga tcggcgtcgg cgagaagaag atcacacatc actggcatcg gtccggcagc 1440

accatcggca aggccttcga ggccacagtg agaggcgccc ggaggatggc cgtcctgggc 1500

gatacagcct gggacttcgg gagcgtgggg ggcgccctga acagcctcgg gaagggcatc 1560

caccagatct tcggcgccgc ctttaagtcc ctctttgggg gcatgagctg gtttagccag 1620

atcctgatcg gcaccctgct gatgtggctg gggctgaaca ccaagaacgg gagcattagc 1680

ctgatgtgcc tggccctggg cggggtcctg atcttcctga gcacagccgt gagcgcctaa 1740

taatgactcg aggaattcaa gcttgggatc tttgtgaagg aaccttactt ctgtggtgtg 1800

acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta 1860

agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat tcacagtccc 1920

aaggctcatt tcaggcccct cagtcctcac agtctgttca tgatcataat cagccatacc 1980

acatttgtag aggttttact tgcttta 2007

<210> 7

<211> 2076

<212> DNA

<213> Artificial sequence

<400> 7

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gacactagtg ccgtgacctt gccctcccac tccacccgga agctccagac caggagccag 120

acctggctgg agtccaggga gtacaccaag cacctgatcc gggtggagaa ctggatcttt 180

cggaacccag gctttgccct ggccgccgcc gccatcgcct ggctgctcgg cagctccacc 240

agccagaagg tcatctacct tgtgatgatc ctgctcatcg cccccgccta ctccatcagg 300

tgcatcggcg tcagcaacag ggactttgtg gaggggatga gcggaggcac ctgggtcgat 360

gtggtgctgg agcacggggg gtgcgtgaca gtgatggccc aggacaagcc caccgtggac 420

attgagctcg tgactactac agtgtccaac atggccgagg tcagatccta ctgctacgag 480

gcctccatct ccgatatggc tagcgactcc agatgcccaa cacagggcga ggcctacctc 540

gataagcaga gcgacaccca gtacgtgtgc aagcggacac tggtggacag ggggtggggg 600

aacgggtgcg ggctgtttgg gaaggggagc ctcgtcacct gcgccaagtt cgcttgctcc 660

aagaagatga caggcaagag catccagcct gagaacctgg agtaccggat catgctgagc 720

gtgcatggaa gccagcactc cggcatgatc gtgaacgata cagggcacga aaccgatgag 780

aaccgggcca aggtggagat cacacccaac tcccctaggg ctgaggccac actggggggc 840

ttcggctccc tggggctgga ttgcgagccc cgcaccggcc tggacttcag cgacctgtac 900

tacctgacca tgaacaacaa gcactggctg gtgcacaagg agtggtttca cgatatcccc 960

ttgccttggc acgccggggc cgacacaggc accccccact ggaacaacaa ggaggccctc 1020

gtggagttca aggacgccca cgccaagagg cagactgtgg tggtgttggg atcccaggag 1080

ggggccgtcc acaccgccct cgctggcgcc ctggaggccg agatggatgg cgccaagggg 1140

aggctgagta gcgggcacct gaagtgccgg ctcaagatgg ataagctgag gctgaagggg 1200

gtgagctact ccctctgcac agccgccttc accttcacaa agatccctgc cgagacactg 1260

cacggcaccg tgacagtcga agtgcagtac gccggcaccg acggcccatg caaggtgccc 1320

gcccagatgg ccgtggacat gcagacactc acccccgtgg gcagactgat caccgccaac 1380

cccgtgatta cagagtccac agagaacagc aagatgatgc tggagcttga tcccccattt 1440

ggggatagct acattgtgat cggcgtcggc gagaagaaga tcacacatca ctggcatcgg 1500

tccggcagca ccatcggcaa ggccttcgag gccacagtga gaggcgcccg gaggatggcc 1560

gtcctgggcg atacagcctg ggacttcggg agcgtggggg gcgccctgaa cagcctcggg 1620

aagggcatcc accagatctt cggcgccgcc tttaagtccc tctttggggg catgagctgg 1680

tttagccaga tcctgatcgg caccctgctg atgtggctgg ggctgaacac caagaacggg 1740

agcattagcc tgatgtgcct ggccctgggc ggggtcctga tcttcctgag cacagccgtg 1800

agcgcctaat aatgactcga ggaattcaag cttgggatct ttgtgaagga accttacttc 1860

tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa ggtaaatata 1920

aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt 1980

cacagtccca aggctcattt caggcccctc agtcctcaca gtctgttcat gatcataatc 2040

agccatacca catttgtaga ggttttactt gcttta 2076

<210> 8

<211> 72

<212> DNA

<213> Artificial sequence

<400> 8

ggggacaagt ttgtacaaaa aagcaggctt cgaaggagat agaaccatgg ctgtgacgct 60

cccctcccat tc 72

<210> 9

<211> 54

<212> DNA

<213> Artificial sequence

<400> 9

ggggaccact ttgtacaaga aagctgggtc agatgataag atacattgat gagt 54

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

tgtaaaacga cggccagt 18

<210> 11

<211> 17

<212> DNA

<213> Artificial sequence

<400> 11

caggaaacag ctatgac 17

<210> 12

<211> 17

<212> DNA

<213> Artificial sequence

<400> 12

taatacgact cactata 17

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence

<400> 13

accgaggaga gggttaggga t 21

Claims

1. An isolated nucleic acid having a base sequence as set forth in SEQ ID No. 3.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid is linked to a PolyA sequence at the 3 'end and/or a signal peptide at the 5' end.

3. The isolated nucleic acid of claim 2, wherein said PolyA sequence is the SV40PolyA sequence.

4. The isolated nucleic acid of claim 2, wherein the signal peptide is signal peptide Ig κ.

5. The isolated nucleic acid of claim 2, wherein the base sequence of the isolated nucleic acid is as set forth in SEQ ID No.6 or 7.

6. A vector comprising the base sequence of the nucleic acid according to any one of claims 1 to 5.

7. The vector of claim 6, wherein the vector is an adenoviral vector.

8. The vector of claim 7, wherein the adenoviral vector is a type 5 adenoviral vector.

9. The vector of claim 8, wherein the adenovirus type 5 vector is a pAd5-CMV/V5-DEST plasmid vector.

10. A method for constructing a recombinant adenovirus, comprising: transfecting a host cell with an adenoviral vector according to any one of claims 7 to 9.

11. The method for constructing a recombinant adenovirus according to claim 10, wherein the host cell is 293 cell.

12. A recombinant adenovirus constructed by the construction method according to claim 10 or 11.

13. A recombinant cell or recombinant bacterium comprising an isolated nucleic acid according to any one of claims 1 to 5, a vector according to any one of claims 6 to 9 or a recombinant adenovirus according to claim 12.

14. The recombinant cell or recombinant bacterium of claim 13, wherein the recombinant cell is 293.

15. A method of producing an Env protein, comprising: culturing the recombinant cell or recombinant bacterium of claim 13 or 14.

16. A Zika virus vaccine, comprising: an isolated nucleic acid according to any one of claims 1 to 5, a vector according to any one of claims 6 to 9, a recombinant adenovirus according to claim 12 or an Env protein produced by the method of claim 15.

17. Use of an isolated nucleic acid according to any one of claims 1 to 5, a vector according to any one of claims 6 to 9 or a recombinant adenovirus according to claim 12 or a recombinant cell or recombinant bacterium according to claim 13 or 14 in the manufacture of a medicament for the prevention or treatment of Zika virus infection.