阅读说明：本技术 一种基于腺病毒HAd49的重组腺病毒表达载体及其构建方法 (Recombinant adenovirus expression vector based on adenovirus HAd49 and construction method thereof ) 是由 黎诚耀 罗升学 刘博超 张攀丽 于 2019-08-08 设计创作，主要内容包括：本发明公开了一种基于人稀有血清型腺病毒HAd49的重组腺病毒表达载体及其构建方法,其是以野生型腺病毒HAd49的基因组为基础,通过基因组直接克隆方法,删除E1编码区和E3编码区,在E1删除区插入I-CeuI和PI-SceI酶切位点,同时以人源腺病毒Ad5的E4orf6替换HAd49的E4orf6,提高了在人源肾胚细胞(HEK293)中包装重组病毒的成功率和包装出重组病毒的滴度,获得复制缺陷性的重组腺病毒表达载体Ad49。此表达载体可以用于表达多种不同种类的抗原,为疫苗和生物医药研发提供基础。(The invention discloses a recombinant adenovirus expression vector based on human rare serotype adenovirus HAd49 and a construction method thereof, which is based on the genome of wild type adenovirus HAd49, deletes the coding region of E1 and the coding region of E3 by a direct genome cloning method, inserts I-CeuI and PI-SceI enzyme cutting sites in the deletion region of E1, and replaces E4orf6 of HAd49 with E4orf6 of human adenovirus Ad5, thereby improving the success rate of packaging recombinant virus in human kidney embryo cells (HEK293) and the titer of the packaged recombinant virus, and obtaining the replication-defective recombinant adenovirus expression vector Ad 49. The expression vector can be used for expressing various antigens of different types, and provides a basis for research and development of vaccines and biological medicines.)

1. A recombinant adenovirus expression vector based on adenovirus HAd49 is characterized in that based on the genome of human rare serotype adenovirus HAd49, the E1 coding region and the E3 coding region are deleted by a direct cloning method, I-CeuI and PI-SceI enzyme cutting sites are inserted into the E1 deletion region, and simultaneously, the open reading frame 6 of the E4 coding region of human serum adenovirus Ad5 is used for replacing the corresponding reading frame of the genome HAd49, so that the recombinant adenovirus expression vector with replication defect is obtained; wherein the GenBank accession number of the genome of the human rare serotype adenovirus HAd49 is DQ393829.1, and the GenBank accession number of the genome of the human serum adenovirus Ad5 is AY 601635.1.

2. The recombinant adenoviral expression vector of claim 1 wherein the deletion of the E1 coding region is a deletion of the base sequence from 462-3362 of the genome of human rare serotype adenovirus HAd 49.

3. The recombinant adenoviral expression vector of claim 1 wherein the deletion of the E3 coding region is a deletion of the base sequence from positions 26655 to 30736 in the genome of human rare serotype adenovirus HAd 49.

4. The recombinant adenoviral expression vector of claim 1 wherein the corresponding reading frame of the genome of HAd49 is open reading frame 6 of the E4 coding region of the genome of human rare serotype adenovirus HAd49, in particular the base sequence of positions 32257-33389 of the genome of human rare serotype adenovirus HAd 49.

5. The recombinant adenovirus expression vector of claim 1, wherein the open reading frame 6 of the E4 coding region of human serotype adenovirus Ad5 is the base sequence of 33193-34077 in the genome of human serotype adenovirus Ad 5.

6. The recombinant adenoviral expression vector of claim 1, wherein the nucleotide sequence of the recombinant adenoviral expression vector is as set forth in SEQ ID NO: 1 is shown.

7. The recombinant adenovirus expression vector of claim 1, further comprising a foreign antigen encoding gene between cleavage sites I-CeuI and PI-SceI of the recombinant adenovirus expression vector.

8. The recombinant adenovirus expression vector of claim 7, wherein the exogenous antigen is the envelope protein prM-E antigen of Zika virus.

9. The method of claim 1, wherein the recombinant adenoviral expression vector of adenovirus HAd49 is constructed comprising:

(1) Carrying out PCR amplification on an LITR fragment of a genome of HAd49, and carrying out PCR amplification on a Linker sequence by using a pUC57-Linker plasmid as a template;

(2) Taking LITR and Linker as templates, performing OverlappingPCR amplification to obtain a sequence LITR-Linker, performing double enzyme digestion on a target gene and a vector by using PmeI and SpeI respectively, and inserting a target fragment after enzyme digestion into a plasmid pNEB193 to obtain a plasmid pNEB193-LITR + Linker;

(3) The segment from position 30736 to 32257 of the genome of HAd49 was PCR amplified to be 3; the fragment of E4orf6 of the genome of Ad5 was PCR amplified to 4; PCR amplifying a fragment from position 33389 to 35215 of the genome of Ad49 to 5; using DNA fragments 3,4 and 5 as templates, carrying out overlappingPCR amplification to obtain a fragment 345, carrying out double digestion on a target gene and a vector by using SwaI and PacI respectively, and inserting the digested target fragment into a plasmid pNEB193-LITR + linker to obtain a plasmid pNEB193-LITR + linker +345;

(4) Carrying out PCR amplification on a DNA fragment from 3363 to 15439 of HAd49 genome to obtain 1, and connecting the DNA fragment to a plasmid pNEB193-LITR + linker +345 through SpeI and SrfI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker + 1345;

(5) The position 15439-26665 fragment of the genome of the HAd49 amplified by PCR is 2, and is connected with a plasmid pNEB193-LITR + linker +1345 through SrfI and SwaI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker +12345, namely the recombinant adenovirus expression vector.

10. A method of making a vaccine, the method comprising:

(1) Providing a recombinant adenoviral expression vector according to claim 1;

(2) inserting the exogenous antigen encoding gene between enzyme cutting sites I-CeuI and PI-SceI of the recombinant adenovirus expression vector in the step (1);

(3) and (3) transfecting the recombinant expression vector in the step (2) to package virus cells, and completing virus packaging in the cells, thereby obtaining the vaccine with immunogenicity.

Technical Field

The invention belongs to the field of biotechnology, in particular to a recombinant adenovirus vector for gene therapy and vaccine vector development; in particular to a recombinant adenovirus expression vector based on adenovirus HAd49 and a construction method thereof.

background

Since the beginning of the 20 th century 60 th era, the development of adenovirus (Ad) as a gene expression vector began, and scientists found that the adenovirus genome could be hybridized with the simian virus 40 (SV 40) genome, indicating that the adenovirus genome could be accessed into heterologous genes. Thereafter, adenovirus has evolved into an important vector system. The existing research proves that the adenovirus vector vaccine can stimulate and induce specific T cell and B cell immune responses in different animals, but the infection and stimulation abilities of different subtypes of adenovirus are different. Adenovirus in subgroup C induced the strongest responses to specific T-cell and B-cell immunity against foreign genes. Expression vectors based on human adenovirus serotype 5 (Ad 5) and 2 (Ad 2) are widely used in vaccine research and treatment, but because of immune response to Ad5 and Ad2 adenoviruses prestored in human bodies, the adenovirus is inhibited from infecting organism cells, so that target genes carried by the adenovirus cannot be expressed at an expected level or even not expressed at all, and in addition, cellular immune CTL response to the adenovirus vectors with cross property can kill target cells infected with the adenovirus vectors so as to block the continuous expression of foreign genes. In order to overcome the effect of pre-existing immune responses on adenoviral vectors, it is necessary to develop new recombinant adenoviral vectors from other human adenoviral serotypes and even other animal species. Therefore, the vaccine vector based on the human rare serotype adenovirus 49 (HAd 49) has remarkable immune effect and can overcome the pre-existing immune response against Ad5 adenovirus.

Disclosure of Invention

in order to make up for the defects of the existing adenovirus expression vector, the invention provides a recombinant adenovirus expression vector based on adenovirus HAd49 and a construction method thereof, which are more favorable for packaging recombinant viruses with higher purity and titer.

The technical problem to be solved by the invention is realized by the following technical scheme:

A recombinant adenovirus expression vector based on adenovirus HAd49 is prepared by deleting the coding region of E1 and the coding region of E3 by a direct cloning method based on the genome of human rare serotype adenovirus HAd49, inserting I-CeuI and PI-SceI restriction enzyme cutting sites into the deleted region of E1, and replacing the corresponding reading frame of the genome HAd49 with the open reading frame 6 of the E4 coding region of human serum adenovirus Ad5 to obtain a replication-defective recombinant adenovirus expression vector; wherein the GenBank accession number of the genome of the human rare serotype adenovirus HAd49 is DQ393829.1, and the GenBank accession number of the genome of the human serum adenovirus Ad5 is AY 601635.1.

Further, the deletion of the coding region of E1 refers to the deletion of the base sequence from 462 to 3362 in the genome of human rare serotype adenovirus HAd 49.

Further, the deletion of the coding region of E3 refers to the deletion of the base sequence from 26655 to 30736 th in the genome of human rare serotype adenovirus HAd 49.

Further, the corresponding reading frame of the genome of HAd49 is open reading frame 6 (E4 orf 6) of the E4 coding region of the genome of human rare serotype adenovirus HAd49, specifically, the base sequence from 32257 to 33389 in the genome of human rare serotype adenovirus HAd 49.

Furthermore, the open reading frame 6 (E4 orf 6) of the E4 coding region of the human serum type adenovirus Ad5 refers to a base sequence from 33193 to 34077 in the genome of the human serum type adenovirus Ad 5.

Further, the construction method of the recombinant adenovirus expression vector based on the adenovirus HAd49 comprises the following steps:

(1) PCR-amplifying a LITR fragment (1-462) of HAd49 genome (sequence sites are calculated based on the sequence of genbank accession number DQ 393829.1), and PCR-amplifying a Linker sequence by taking a pUC57-Linker plasmid as a template;

(2) Taking LITR and Linker as templates, performing OverlappingPCR amplification to obtain a sequence LITR-Linker, performing double enzyme digestion on a target gene and a vector by using PmeI and SpeI respectively, and inserting a target fragment after enzyme digestion into a plasmid pNEB193 to obtain a plasmid pNEB193-LITR + Linker;

(3) The segment from position 30736 to 32257 of the genome of HAd49 was PCR amplified to be 3; the fragment of E4orf6 of the genome of Ad5 was PCR amplified to 4; PCR amplifying a fragment from position 33389 to 35215 of the genome of Ad49 to 5; using DNA fragments 3,4 and 5 as templates, carrying out overlappingPCR amplification to obtain a fragment 345, carrying out double digestion on a target gene and a vector by using SwaI and PacI respectively, and inserting the digested target fragment into a plasmid pNEB193-LITR + linker to obtain a plasmid pNEB193-LITR + linker +345;

(4) Carrying out PCR amplification on a DNA fragment from 3363 to 15439 of HAd49 genome to obtain 1, and connecting the DNA fragment to a plasmid pNEB193-LITR + linker +345 through SpeI and SrfI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker + 1345;

(5) The position 15439-26665 fragment of the genome of the HAd49 amplified by PCR is 2, and is connected with a plasmid pNEB193-LITR + linker +1345 through SrfI and SwaI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker +12345, namely the recombinant adenovirus expression vector.

Further, the nucleotide sequence of the recombinant adenovirus expression vector is shown as SEQ ID NO: 1 is shown.

In another alternative, the recombinant adenovirus expression vector also comprises a foreign antigen coding gene between the enzyme cutting sites I-CeuI and PI-SceI.

Further, the foreign antigen is an envelope protein prM-E antigen of Zika virus.

In another aspect of the present invention, there is provided a method of preparing a vaccine, the method comprising:

(1) Providing said recombinant adenoviral expression vector;

(2) Inserting the exogenous antigen encoding gene between enzyme cutting sites I-CeuI and PI-SceI of the recombinant adenovirus expression vector in the step (1);

(3) And (3) transfecting the recombinant expression vector in the step (2) to package virus cells, and completing virus packaging in the cells, thereby obtaining the vaccine with immunogenicity.

The invention has the following beneficial effects:

(1) the inventor successfully constructs a novel expression vector Ad49 based on the human rare serotype adenovirus 49 (HAd 49), and the vaccine vector can be applied to preparation of virus vaccines with high expression and good immunogenicity.

(2) the recombinant adenovirus expression vector of the invention replaces E4orf6 of HAd49 with the E4orf6 coding region of Ad5, thus improving the success rate of packaging virus in human kidney embryo cells and the titer of the packaged virus.

(3) The recombinant adenovirus vector of the invention can be widely applied to the fields of vaccines and gene therapy.

Drawings

FIG. 1 shows the results of the purification and enzymatic identification of the genome of human rare serotype adenovirus HAd49, in which,

A: results plot of cesium chloride purified human rare serotype adenovirus HAd 49;

B: the result of the genome enzyme digestion identification of the human rare serotype adenovirus HAd49 is shown.

FIG. 2 shows the restriction enzyme digestion identification result of constructing replication-defective recombinant adenovirus expression vector Ad 49.

FIG. 3 shows the result of the identification of Ad49-eGFP constructed as a recombinant adenovirus

A: the enzyme digestion identification result of the recombinant adenovirus vector Ad 49-eGFP;

B: viral plaque formation results for recombinant adenovirus vector Ad 49-eGFP.

FIG. 4 shows the result of identifying the construction of recombinant adenovirus Ad49-prM-E, in which,

A, enzyme digestion identification result of Ad 49-prM-E;

B, virus plaque results of Ad 49-prM-E;

WB detection of envelope E protein antigen expression after Ad49-prM-E virus infection of cells.

FIG. 5 is a flow chart of construction of a replication-defective recombinant adenovirus expression vector Ad 49.

Detailed Description

the invention provides a recombinant adenovirus expression vector, which comprises: based on the genome of the human rare serotype adenovirus HAd49, an E1 coding region and an E3 coding region are deleted by a direct cloning method, enzyme cutting sites I-CeuI and PI-SceI are inserted into the E1 deletion region, and meanwhile, the E4orf6 coding region of Ad5 replaces the E4orf6 coding region of the human rare serotype adenovirus HAd49, so that the success rate of packaging the recombinant virus in human kidney embryo cells (HEK293) is improved, and the titer of the packaged recombinant virus is increased. Wherein, the nucleotide sequence of the E1 coding region of the human rare serotype adenovirus HAd49 is shown as SEQ ID NO: 2, the nucleotide sequence of the E3 coding region of the human rare serotype adenovirus HAd49 is shown as SEQ ID NO: 3, the nucleotide sequence of the E4orf6 coding region of the human serum adenovirus Ad5 is shown as SEQ ID NO: 4, the nucleotide sequence of the E4orf6 coding region of the human rare serotype adenovirus HAd49 is shown as SEQ ID NO: 5.

HAd49, the restriction sites I-CeuI and PI-SceI were inserted into the deletion site of the E1 coding region as the connection site of the foreign gene, and the restriction sites were not present in other positions of the adenovirus expression vector.

the human rare serotype adenovirus HAd49, isolated from human body, has a very low population prevalence. The invention is based on wild HAd49 genome, deletes the coding regions of E1 and E3 by direct cloning method, inserts restriction enzyme cutting sites I-CeuI and PI-SceI in the deletion region of E1, and replaces the E4orf6 coding region of HAd49 with the E4orf6 coding region of Ad5, finally obtains the replication-defective Ad49 recombinant adenovirus expression vector.

as a preferred mode of the present invention, the cloning step comprises:PCR amplifying the LITR fragment (1-462) of HAd49 genome, PCR amplifying the Linker fragment with pUC57-Linker plasmid as template, PCR amplifying the Linker fragment with DNA fragments of LITR and Linker as template, OverlappingPCR amplifying to obtain the sequence LITR-Linker, double enzyme cutting the target gene and vector with PmeI and SpeI, inserting the cut target fragment into the vectorObtaining a plasmid pNEB193-LITR + linker by adding the plasmid pNEB 193;The segment from position 30736 to 32257 of the genome of HAd49 was PCR amplified to be 3; the fragment of E4orf6 of the genome of Ad5 was PCR amplified to 4; PCR amplifying a fragment from position 33389 to 35215 of the genome of Ad49 to 5; using DNA fragments 3,4 and 5 as templates, carrying out overlappingPCR amplification to obtain a fragment 345, carrying out double digestion on a target gene and a vector by using SwaI and PacI respectively, and inserting the digested target fragment into a plasmid pNEB193-LITR + linker to obtain a plasmid pNEB193-LITR + linker +345;The gene group of HAd49 is amplified by PCR, the fragment from position 3363 to 15439 is 1, and is connected with a plasmid pNEB193-LITR + linker +345 through SpeI and SrfI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker + 1345;The position 15439-26665 fragment of the genome of HAd49 amplified by PCR is 2, and is connected with a plasmid pNEB193-LITR + linker +1345 through SrfI and SwaI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker +12345, namely the recombinant adenovirus expression vector Ad 49.

After obtaining the recombinant adenovirus expression vector, transfecting the recombinant adenovirus expression vector into a packaging virus cell after linearization, and packaging the virus; after a period of transfection, the virus can be harvested; the harvested virus was repeatedly infected with HEK293 and passaged continuously.

The replication-defective recombinant adenovirus expression vector is used as an expression vector platform and is suitable for expressing various antigens so as to prepare virus vaccines.

In order to verify the effectiveness of Ad49, the green fluorescent protein and envelope protein genes of Zika virus are cloned into an Ad49 vector, and research shows that the Ad49 vector can highly express antigen genes, so that the invention proves that the novel adenovirus vector is obtained, can be used for vaccine research and development and other biomedical basic research, and provides a novel vector platform for novel vaccine research and development.

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not specified in the following examples, are generally carried out under conventional conditions such as those described in molecular cloning guidelines, third edition, published by science publishers, 2002, written by J. SammBruker et al, or under the instructions for reagents.

Material

1.1 Virus strains and cells

human rare serotype adenovirus HAd49 was purchased from ATCC (VR-1047); the HEK293 cell line was stored in the laboratory in DMEM with 10% Fetal Bovine Serum (FBS).

restriction enzymes, strains and plasmids

Restriction enzymes were purchased from New England Biolabs; DH 5. alpha. was obtained from Tiangen Biochemical technology Ltd.

The pUC19-eGFP plasmid was stored in the laboratory.

the pBHGlox (delta) E1,3Cre plasmid was stored in the laboratory.

The pDC315-prM-E (ZIKV) plasmid was stored in the laboratory.

The pNEB193 plasmid was stored in the laboratory.

pShuttle2-CMV-Flag vector was purchased from Addgene.

pUC57-linker plasmid: the linker sequence (SEQ ID NO: 6) was synthesized from the Beijing Huada gene and cloned into a pUC57 vector to obtain pUC 57-linker.

Method of producing a composite material

2.1 amplification and purification of adenovirus

HEK293 cells were cultured in DMEM medium containing 10% FBS, HAd49 adenovirus was proliferated in HEK293 cells, infected cells were collected after all cells showed viral lesions (CPE), centrifuged, resuspended in DMEM, and cells were repeatedly frozen and thawed three times to lyse the cells to release the virus. The virus is purified by cesium chloride density gradient centrifugation, added with glycerol and stored in a liquid nitrogen tank.

Construction of replication-defective recombinant adenovirus vectors

2.2.1 construction of plasmid pNEB193-LITR + linker

Using HAd49 genome as template, the primers LITR-F and LITR-R were used to obtain about 462bp from 1 to 462 by PCR amplification. Linker fragments were PCR amplified using primers Linker-F and Linker-R, and pUC57-Linker plasmid as template. DNA fragments of the LITR and the Linker are used as templates, primers LITR-F and Linker-R are used, overlappinging PCR is used for amplification to obtain a sequence LITR-Linker, a target gene and a vector pNEB193 are respectively subjected to double enzyme digestion by PmeI and SpeI, and the plasmid pNEB193-LITR + Linker is obtained by connection.

construction of plasmid pNEB193-LITR + linker +345

using the genome of HAd49 as a template, the 3-position 30736 to 32257 fragment of HAd49 was PCR amplified with primers 3-F and 3-R as 3. A fragment of E4orf6 of the genome of Ad5 was PCR amplified with primers 4-F and 4-R using pBHGlox (delta) E1,3Cre plasmid as template to be 4. A fragment of 33389 to 35215 of the genome of Ad49 was PCR amplified to 5 with primers 5-F and 5-R using the genome of HAd49 as template. DNA fragments 3,4 and 5 are taken as templates, primers 3-F and 5-R are used, overlappingPCR is carried out to obtain a fragment 345, SwaI and PacI are respectively used for double digestion of a target gene and a vector, the digested target fragment is inserted into a plasmid pNEB193-LITR + linker, and the plasmid pNEB193-LITR + linker +345 is obtained.

the framework plasmid pNEB193-LITR + linker +1345

A gene group of HAd49 is used as a template, a 3363-15439 fragment of HAd49 gene group is amplified by PCR to be 1 by using primers 1-F and 1-R, and the fragment is connected with a plasmid pNEB193-LITR + linker +345 through a SpeI and SrfI enzyme cutting site to obtain a plasmid pNEB193-LITR + linker + 1345.

Construction of plasmid pNEB193-LITR + linker +12345 (Ad 49)

A genome of HAd49 is used as a template, primers 2-F and 2-R are used, a position 15439-26665 fragment of the genome of HAd49 is amplified by PCR to be 2, and the amplified genome is connected to a plasmid pNEB193-LITR + linker +1345 through SrfI and SwaI enzyme cutting sites to obtain a plasmid pNEB193-LITR + linker +12345, namely a recombinant adenovirus vector Ad 49.

2.3 construction of recombinant adenovirus Ad49-eGFP

2.3.1 cloning of the eGFP Gene into the Ad49 vector

EcoRI and BamHI are respectively used for double digestion of pUC19-eGFP and pShuttle2-CMV-Flag plasmids, target fragments are recovered by agarose gel, and the target fragments are connected to obtain a vector pShuttle 2-CMV-eGFP; and carrying out double digestion on pShuttle2-CMV-eGFP and Ad49 by using I-CeuI and PI-SceI respectively, recovering DNA fragments by agarose gel electrophoresis, connecting, transforming and identifying to obtain Ad 49-eGFP.

Packaged Ad49-eGFP recombinant adenovirus

The linearized Ad49-eGFP plasmid (eGFP nucleotide sequence SEQ ID NO: 21) was digested with AsiSI; hexawell plates were plated with HEK293 cells, and about 2.5ug of linearized Ad49-eGFP plasmid was transfected into HEK293 cells with X-tremeGENE (Roche) when the cell confluence was 80% -90%, and the recombinant adenovirus was packaged. Fluorescence and cell status were observed daily until cytopathic effect (CPE) appeared around the fifth day.

Construction of recombinant adenovirus Ad49-prM-E

2.4.1 cloning of the prM-E Gene of Zika Virus (ZIKV) into Ad49

The pShuttle2-CMV-Flag plasmid and the pDC315-prM-E (ZIKV) plasmid were double-digested with KpnI and BamHI, respectively, and the vector and the target gene fragment were recovered with gelatin, ligated, transformed, and identified to obtain pShuttle2-CMV-prM-E (ZIKV) (Zika virus prM-E antigenic nucleotide sequence SEQ ID NO: 22). The plasmid pShuttle2-CMV-prM-E and the plasmid Ad49 are respectively cut by I-CeuI and PI-SceI in a double-enzyme way, DNA fragments are recovered by agarose gel electrophoresis, and the vector Ad49-prM-E is obtained by connection, transformation and identification.

Packaged Ad49-prM-E recombinant adenovirus

Digesting the linearized Ad49-prM-E plasmid by AsiSI; six-well plates were plated with HEK293 cells, and when the degree of cell fusion in the six-well plates reached 80% -90%, about 2.5ug of the linearized Ad49-prM-E plasmid was transfected into HEK293 cells using X-tremeGENE (Roche), and the recombinant adenovirus was packaged. The cell status was observed daily until the appearance of cytopathic effect (CPE) around the fifth day.

Amplification and purification of recombinant adenovirus Ad49-prM-E

HEK293 cells were cultured in DMEM medium containing 10% FBS, and Ad49-prM-E adenovirus was expanded in HEK293 cells. After the HEK293 cell is infected by the recombinant adenovirus, when the cell has cytopathic effect (CPE), the infected cell is collected, centrifuged, the cell is resuspended by DMEM, and the cell is repeatedly frozen and thawed for three times, so that the cell is cracked, and the virus is released. The virus-containing supernatant was purified by cesium chloride density gradient centrifugation and then frozen in a freezer at-80 ℃ using a virus stock solution.

Detection of E protein expression of Zika Virus

Spreading a six-hole plate by using HEK293 cells; when the cell fusion degree reaches 80-90%, the cells are deinfected by Ad49-prM-E virus, and are deinfected by Ad49-eGFP virus to be used as a control group, the cells are harvested after 24 hours, and the expression of E protein is detected by western blot.