CN106085974B - A kind of Zika virus pseudovirion particle and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of zika virus pseudovirions and preparation method thereof.The present invention provides zika virus pseudovirions, wrap up the pseudovirion that zika virus NS5 gene is formed for coat protein；The coat protein is made of bacteriophage MS2 maturase and bacteriophage MS2 capsid protein；The nucleotides sequence of the zika virus NS5 gene is classified as sequence 2 19-2727.The experiment proves that, the present invention imports host strain with the recombinant vector of expression phage capsid protein and maturase Protein reconstitution carrier and expression zika virus genome portion segment, culture, is packaged to be zika virus pseudovirion, to obtain zika virus pseudovirion for the first time.

Description

A kind of Zika virus pseudovirion particle and preparation method thereof

technical field

The invention relates to a Zika virus pseudovirion particle and a preparation method thereof, belonging to the field of biotechnology.

Background technique

Zika virus, a mosquito-borne virus, can cause "microcephaly" in babies. The virus belongs to the Flavivirus genus of the Flaviviridae family, a single-stranded RNA virus, and is homologous to the dengue virus. In 2015, there were 2,700 babies suspected of having microcephaly in Brazil, and 29 of them died, mainly in the northeast. In 2014, there were only 147 cases of microcephaly in Brazil. The Zika virus was first discovered in Uganda in the 1940s and was once endemic in Africa. Zika has since spread to the South Pacific and Asia, and has just recently spread to Latin America. In Brazil, the first case of Zika virus appeared in April 2015, and it quickly spread to 18 provinces. The development of Zika virus diagnostic reagents is imminent, especially the development of nucleic acid diagnostic reagents, which requires the use of internal control reference materials. Although using real viruses as reference products can truly reflect the performance of nucleic acid diagnostic reagents, the real Zika virus is infectious to a certain extent, which can easily cause biosafety problems, and is not easy to store and transport. If naked RNA is used as its reference product, the naked RNA is unstable and easily degraded due to the presence of a large number of RNases in the environment. This requires an RNA with RNase-resistant properties, and armored RNA pseudovirus is one of them. Armored RNA can wrap RNA in the capsid protein of MS2 bacteriophage, making RNA resistant to RNase, and can be used to simulate the extraction process of real samples and achieve large-scale production.

Armored RNA is a type of pseudovirion packaged with exogenous RNA, and the MS2 phage packaging system is generally used. The system prepares pseudovirion particles simply, safely, and has a short preparation period. It mainly uses the capsid protein and mature enzyme protein of the phage to recognize and package the foreign RNA containing the MS2 phage recognition sequence and assemble it to form mature pseudovirion particles.

Contents of the invention

One object of the present invention is to provide a Zika virus pseudovirion.

The Zika virus pseudovirus particle provided by the invention is a pseudovirus particle formed by encapsulating the Zika virus NS5 gene;

The coat protein is composed of phage MS2 maturation enzyme and phage MS2 capsid protein;

The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2.

Another object of the present invention is to provide a method for preparing the above Zika virus pseudovirion.

The method provided by the invention is to use the MS2 phage packaging system to package the Zika virus NS5 gene to obtain Zika virus pseudovirus particles;

The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2.

In the above method, said adopting the MS2 phage packaging system to package the Zika virus NS5 gene is to express the Zika virus NS5 gene, the phage MS2 maturation enzyme coding gene and the phage MS2 capsid protein coding gene in the host bacterium, and the expressed phage MS2 capsid The protein will automatically recognize the MS2 packaging site on the NS5 gene, and the virus particles will be packaged under the action of the mature enzyme protein, so as to obtain Zika virus pseudovirion particles after packaging.

The Zika virus NS5 gene is expressed in the host bacteria in the form of a fragment containing the Zika virus NS5 gene; the fragment containing the Zika virus NS5 gene is composed of an MS2 phage packaging site recognition fragment and the Zika virus NS5 gene.

The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2.

Above-mentioned method comprises the steps:

1) Introducing a fragment containing the Zika virus NS5 gene, a phage MS2 maturation enzyme encoding gene and a phage MS2 capsid protein encoding gene into a host bacterium to obtain a recombinant bacterium;

The fragment containing the Zika virus NS5 gene is composed of an MS2 phage packaging site recognition fragment and the Zika virus NS5 gene;

2) Inducing and culturing the recombinant bacteria to obtain Zika virus pseudovirion particles.

In the above method,

The fragment containing the Zika virus NS5 gene is introduced into the host bacterium through the recombinant vector A,

The recombinant vector A is a vector obtained by inserting a fragment containing the Zika virus NS5 gene into an expression vector;

The fragment containing the Zika virus NS5 gene is any of the following 1)-3):

1) the coding region is the DNA molecule shown in sequence 2 in the sequence listing;

2) A DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes a protein with the same function;

3) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least DNA molecules having 98% or at least 99% homology and encoding proteins with the same function;

The above-mentioned stringent conditions can be hybridization at 65° C. in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

The phage MS2 maturation enzyme coding gene and the phage MS2 capsid protein coding gene are introduced into the host bacterium through the recombinant vector B,

The recombinant vector B is a vector obtained by inserting a fragment containing a phage MS2 maturation enzyme coding gene and a phage MS2 capsid protein coding gene into an expression vector;

The fragment containing the phage MS2 maturation enzyme coding gene and the phage MS2 capsid protein coding gene is any one of the following 1)-3):

1) The coding region is the DNA molecule shown in sequence 1 in the sequence listing;

2) A DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes a protein with the same function;

3) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least DNA molecules having 98% or at least 99% homology and encoding proteins with the same function.

In the above method,

The expression vector in the recombinant vector A is the same as or different from the expression vector in the recombinant vector B.

In the above method,

The expression vector in the recombinant vector B is pET-30a+;

The expression vector in the recombinant vector A is pACYCDuet-1;

The host bacteria is Escherichia coli.

In the above method,

In the step 2), after the induction culture, the following steps are further included: crushing the cells after the induction culture, collecting the supernatant of the crushed product, and obtaining Zika virus pseudovirion particles.

The application of the above-mentioned Zika virus pseudovirus particles or the Zika virus pseudovirus particles prepared by the above method as a reference product for Zika virus nucleic acid detection reagents is also within the protection scope of the present invention;

Or the application of the above-mentioned Zika virus pseudovirus particles or the Zika virus pseudovirus particles prepared by the above method in the preparation of Zika virus nucleic acid detection reagents is also within the protection scope of the present invention.

Zika virus pseudovirus particles prepared by the above method are also within the protection scope of the present invention.

Experiments of the present invention prove that the present invention uses recombinant vectors expressing phage capsid protein and maturation enzyme protein and recombinant vectors expressing Zika virus genome partial fragments to introduce host bacteria, cultivate and package Zika virus pseudovirions, which is the first time to obtain Zika virus particles. Cavirus pseudovirion particles.

Description of drawings

Figure 1 shows the identification of positive clones of the recombinant plasmid pET-30a-MS2.

Figure 2 is the positive clone identification of the recombinant plasmid pACYCD-NS5.

Figure 3 is the RT-PCR and PCR real-time fluorescent quantitative PCR of Zika virus armored RNA pseudovirion particles.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The strains, plasmids and reagents used in the following examples are as follows:

Plasmid pACYCDuet-1 was purchased from Novagen.

Plasmid pET-30a+ was purchased from Novagen.

Product strain BL21(DE3), Top10 were purchased from Tiangen Company.

Restriction endonuclease and DNA polymerase were purchased from NEB Company.

T4 DNA ligase and AMV reverse transcriptase were purchased from Promega.

The plasmid mini-extraction kit and viral RNA extraction kit were purchased from Tiangen Company.

Embodiment 1, the construction of the recombinant vector pET-30a-MS2 expressing MS2 bacteriophage capsid protein gene and mature enzyme protein gene

1. Amplification of MS2 bacteriophage capsid protein and mature enzyme protein gene sequences

Using the plasmid pMS27 (the plasmid was purchased from the Department of Biomedical Molecular Biology, GhentUniversity, Cat. No.: LMBP05349) as a template, use the following MS2 upstream primers and MS2 downstream primers to perform PCR amplification to obtain PCR products and obtain capsid proteins containing MS2 phage DNA fragments of gene and mature enzyme protein gene.

MS2 upstream primer: 5'- catg CCATGG tgcgagcttttagtacccttgatag-3'

MS2 downstream primer: 5'-gcgc AAGCTT tggccggcgtctattagtagatgc-3'

Amplification system: 4 tubes of 50 μl system amplification, 10 μl of 5*Buffer, 0.5 μl of DNA polymerase, 1 μl of dNTP, 1 μl of upstream primer, 1 μl of downstream primer, 1 μl of template pMS27, 35.5 μl of ddH2O

Amplification program: 98°C for 5min; 98°C for 30s, 58°C for 30s, 72°C for 60s; repeat steps 2-440 cycles, 72°C for 10min, 12°C for 5min.

The PCR product was subjected to agarose gel electrophoresis, and the size was 1611bp, which was consistent with the expectation. Recycle the destination band.

The PCR product was sent for sequencing, and the PCR product had the nucleotide shown in sequence 1 in the sequence listing.

The DNA molecule shown in sequence 1 in the sequence listing consists of the MS2 phage maturation enzyme protein coding gene (1-1182 nucleotides) and the MS2 phage capsid protein coding gene (1183-1611 nucleotides).

2. Construction of recombinant vector pET-30a-MS2

Amplify the above-prepared PCR product and vector pET-30a+ with restriction endonucleases BamH Ⅰ and Hind Ⅲ, and digest at 37°C for 2 hours. Reaction system: 2 μl of BamH Ⅰ, 2 μl of HindⅢ, 4 μl of Buffer, the PCR product prepared above (or vector pET-30a+) 32 μl.

The digested product was purified, and the purified target fragment and vector were subjected to enzyme ligation reaction in the following system: T4DNA Ligase 0.5μl, T4DNA Ligase Buffer 1μl, target fragment 5.5μl, pET-30a+3μl; ligation at 22°C for 2h. Take 5 μl of the ligation product and 50 μl of Top10 competent cells and mix gently on ice, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, add 800 μl of non-resistant LB medium, incubate at 37°C, 150 rpm After 60 minutes, the incubated product was centrifuged at 5000 rpm for 4 minutes, 600 μl of supernatant was discarded, the remaining supernatant was mixed with the cell pellet, and LB solid plate containing kan resistance was coated, and incubated overnight at 37°C upside down. Results: A single colony of bacteria grew on the plate, as expected.

Pick a single colony on the plate, resuspend it in 20 μl of normal saline, and perform colony PCR to verify positive clones;

Amplification system: 8 tubes for 20μl system amplification, 1μl of 10*Buffer, 0.5μl of Taq DNA polymerase, 1.2μl of MgCl2, 1μl of dNTP, 0.5μl of upstream primer, 0.5μl of downstream primer, 1μl of single colony suspension, 13.3μl of ddH2O

Amplification program: 95°C for 5min; 95°C for 30s, 58°C for 30s, 72°C for 60s; repeat steps 2-4 for 40 cycles, 72°C for 10min, 12°C for 5min.

The amplified product was subjected to 0.8% agarose gel electrophoresis, and the result is shown in Figure 1. The amplified target band was sent to Qingke Company for sequencing, and the sequence was correct for the successful construction For cloning, the single colony was inoculated in 10 ml of Kan-resistant LB liquid medium, and cultivated overnight at 37° C. and 220 rpm. Collect the cultured bacterial solution into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 2min, discard the supernatant, collect the bacterial pellet, and extract the plasmid according to the plasmid mini-extraction kit from Tiangen Company to obtain the recombinant vector pET-30a-MS2. The recombinant plasmids were stored at -20°C, and the strains were stored at -80°C.

The recombinant vector pET-30a-MS2 was sent for sequencing, which is a vector obtained by replacing the DNA fragment between the BamH I and Hind III restriction sites of the vector pET-30a+ with the DNA molecule shown in Sequence 1 in the sequence listing.

Example 2, Construction of recombinant vector pACYCD-NS5 vector expressing Zika virus NS5 gene

1. Amplification of the intestinal matrix protein gene sequence

Using the cloning vector pUC-NS5 containing intestinal matrix protein (the plasmid was synthesized by Sangon Biotechnology Co., Ltd., and the sequence gi|226377833| was selected) as a template, the following NS5 upstream primers and NS5 downstream primers were used for PCR amplification to obtain A fragment of the Cavirus NS5 gene.

NS5 upstream primer: 5'-ccc AAGCTT ccggaggatcaccacgggAGGTGGGACGGGAGAGACTCTG-3'

NS5 downstream primer: 5'-ccc CTCGAG ccggaggatcaccacgggCTCGTCTGAATCAGATGTCGGCC-3'

Amplification system: 4 tubes of 50μl system amplification, 5*Buffer 10μl, DNA polymerase 0.5μl, dNTP 1μl, upstream primer 1μl, downstream primer 1μl, template 1μl, ddH2O 35.5μl

Amplification program: 98°C for 5min; 98°C for 30s, 58°C for 30s, 72°C for 60s; repeat steps 2-440 cycles, 72°C for 10min, 12°C for 5min.

The PCR product was subjected to agarose gel electrophoresis, and the size was 2727bp, which was consistent with the expectation. Recycle the destination band.

The PCR product was sent for sequencing, and the PCR product had the nucleotide shown in sequence 2 in the sequence listing.

The fragment containing the Zika virus NS5 gene shown in sequence 2 is recognized by the MS2 phage packaging site (position 1-18 in sequence 2) and the Zika virus NS5 gene (position 19-2727 in sequence 2).

2. Construction of recombinant vector pACYCD-NS5

The PCR product prepared above (containing the Zika virus NS5 gene fragment) and the vector pACYCDuet-1 (purchased from Novagen, catalog number: 71147-3) were amplified by restriction endonucleases HindⅢ and Xho I. , digestion at 37°C for 2 hours, reaction system: HindⅢ 2 μl, Xho Ⅰ 2 μl, Buffer 4 μl, PCR product (or vector pACYCDuet-1) 32 μl.

The digested product was purified, and the purified target fragment and vector were subjected to enzyme ligation reaction in the following system: T4DNA Ligase 0.5 μl, T4DNA Ligase Buffer 1 μl, target fragment 5.5 μl, pACYCDuet-1 3 μl; ligation at 22°C for 2 hours. Take 5 μl of the ligation product and 50 μl of Top10 competent cells and mix gently on ice, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, add 800 μl of non-resistant LB medium, incubate at 37°C, 150 rpm After 60 minutes, centrifuge the incubated product at 5000 rpm for 4 minutes, discard 600 μl of supernatant, mix the remaining supernatant with the cell pellet, spread the LB solid plate containing Cm resistance, and incubate overnight at 37°C upside down. Results: A single colony of bacteria grew on the plate, as expected.

Pick a single colony on the plate, resuspend it in 20 μl of normal saline, and perform colony PCR to verify positive clones; use segmented identification, and use two pairs of primers for colony PCR for the same strain.

Amplification system: 8 tubes for 20μl system amplification, 2μl 10*Buffer, 0.5μl Taq DNA polymerase, 1.2μl MgCl2, 1μl dNTP, 0.5μl upstream primer, 0.5μl downstream primer, 1μl single colony suspension, 13.3μl ddH2O

Amplification program: 95°C for 5min; 95°C for 30s, 58°C for 30s, 72°C for 60s; repeat steps 2-440 cycles, 72°C for 10min, 12°C for 5min.

The amplified products were subjected to 0.8% agarose gel electrophoresis, and the results are shown in Figure 2. The sizes of the amplified bands of the two pairs of primers were 1700bp and 1200bp respectively, and the results were in line with expectations. The amplified target band was sent to Qingke Company for sequencing. The correct sequenced clone was successfully constructed. The single colony was inoculated in 10ml of Cm-resistant LB liquid medium and cultivated overnight at 37°C and 220rpm. Collect the cultured bacterial solution into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 2min, discard the supernatant, collect the bacterial pellet, and extract the plasmid according to the plasmid mini-extraction kit from Tiangen Company to obtain the recombinant vector pACYCD-NS5. The recombinant plasmids were stored at -20°C, and the strains were stored at -80°C.

The recombinant vector pACYCD-NS5 was sent for sequencing, which is a vector obtained by replacing the DNA fragment between the Hind III and Xho I restriction sites of the vector pACYCDuet-1 with the fragment containing the Zika virus NS5 gene shown in Sequence 2 in the sequence listing .

Embodiment 3, packing Zika virus pseudovirus

1. Construction of recombinant bacteria

Take the recombinant plasmids pET-30a-MS2 and pACYCD-NS5 constructed in Example 1 and Example 2 and mix them according to the volume of 5 μl+5 μl, take 5 μl and add it to 50 μl of BL21 (DE3) competent cells, use Gently blow and mix with the gun, place on ice, ice bath for 30min, heat shock at 42°C for 90s, immediately place on ice for 3min, add 800μl non-resistant LB medium, incubate at 37°C in a constant temperature shaking incubator, 150rpm for 60min, Centrifuge the incubated product at 5000rpm for 4min, discard 600μl supernatant, mix the remaining supernatant with the cell pellet, spread the LB solid plate containing Cm and Kan resistance, and incubate overnight at 37°C upside down. Results: A single colony of bacteria grew on the plate, as expected.

Pick a single colony on the plate, resuspend it in 20 μl of physiological saline, and carry out colony PCR to verify positive clones. At the same time, MS2 gene (primers are MS2 upstream primer and MS2 downstream primer, and the size of the amplified product is 1611bp) and NS5 gene ( The primers are NS5 upstream primer and NS5 downstream primer, and the size of the amplified product is 2727bp) sequence for amplification.

Amplification system: 8 tubes for 20μl system amplification, 2μl 10*Buffer, 0.5μl Taq DNA polymerase, 1.2μl MgCl2, 1μl dNTP, 0.5μl upstream primer, 0.5μl downstream primer, 1μl single colony suspension, 13.3μl ddH2O

Amplification program: 95°C for 5min; 95°C for 30s, 58°C for 30s, 72°C for 60s; repeat steps 2-4 for 40 cycles, 72°C for 10min, 12°C for 5min.

The amplified product was subjected to 0.8% agarose gel electrophoresis, and the results are shown in Figure 1 and Figure 2, Figure 1 is the identified MS2 positive clone, and Figure 2 is the identified NS5 positive clone. The positive clones that amplified the target band were successfully constructed, and the single colony was inoculated in 10 ml of Cm and Kan resistant LB liquid medium, cultured at 37°C and 220rpm. The bacterium is preserved, and the bacterium is named pET-30a-MS2-ACYCD-NS5, which is a recombinant bacterium containing a double vector.

2. Induced expression and packaging to obtain Zika virus pseudovirion particles

Streak the recombinant bacteria pET-30a-MS2-ACYCD-NS5 prepared above and containing double vectors on the plate to make a single colony grow, pick a single colony and inoculate it in the LB liquid medium with Cm plus Kan resistance, 37 Cultivate overnight in a 220rpm constant temperature shaking incubator. Inoculate the overnight cultured bacterial solution in the new Cm plus Kan-resistant LB liquid medium at a ratio of 1:100, in a 37°C, 220rpm constant temperature shaking incubator to OD600=0.4-0.6, and add 0.5M IPTG to the final The concentration is 1mM, cultured at 22°C, 200rpm for 6h, and the bacteria are collected;

Use lysate (100mM Tris-HCl (pH8.0), 10% glycerol, 1% Triton X-10) to resuspend the bacterial body, place in the ice-water mixture, make the ice-water mixture cover the liquid surface of the bacterial suspension, use Ultrasonic cell disruptor crushes the bacterial suspension; ultrasonic crushing parameters are: ultrasonic 3s, dwell 4s, crushing time 15min, crushing power 60% of the total power; centrifuge the crushed solution at 12000rpm for 5min, collect supernatant to obtain Zika virus pseudovirion particles.

3. Verification of fake virus particles

The Zika virus pseudovirion particles prepared in the above two steps were subjected to nucleic acid extraction (extracted according to the Tiangen virus RNA extraction kit) to obtain RNA.

Using RNA as a template, RT-PCR and PCR verification (7500 real time PCR) were performed with the following primers.

The primers are: ZK-NS5-F: AGGCATGGGGGAGGATTAGT

ZK-NS5-R: CCCATCCAATGGTCCTCGTT

RT-PCR system: 20 μl system amplification, 10*Buffer 2 μl, AMV-RT 0.5 μl, Taq DNA polymerase 0.5 μl, MgCl2 1.2 μl, dNTP 1 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, template 2 μl, EvaGreen 0.6 μl, ROX 0.2μl, ddH2O 11μl

PCR amplification program: 50°C for 30 min, 95°C for 5 min; 95°C for 15 s, 58°C for 30 s, 68°C for 20 s; repeat steps 3-5 for 32 cycles, 68°C for 10 min, 12°C for 5 min.

PCR system: 20μl system amplification, 2μl 10*Buffer, 0.5μl Taq DNA polymerase, 1.2μl MgCl, 1μl dNTP, 0.5μl upstream primer, 0.5μl downstream primer, 2μl template, 0.6μl EvaGreen, 0.2μl ROX, 11.5μl ddH2O

RT-PCR amplification program: 95°C for 5min; 95°C for 15s, 58°C for 30s, 68°C for 20s; repeat steps 3-5 for 32 cycles, 68°C for 10min, 12°C for 5min.

The amplification results are shown in Figure 3. It can be seen that the Zika virus NS5 fragment is obtained, which proves that the obtained pseudovirion particles are Zika virus pseudovirion particles.

Claims (10)

1. A Zika virus pseudovirus particle, which is a pseudovirus particle formed by encapsulating the Zika virus NS5 gene; The coat protein is composed of phage MS2 maturation enzyme and phage MS2 capsid protein; The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2. 2. a method for preparing Zika virus pseudovirus particles described in claim 1 is to adopt MS2 phage packaging system to package Zika virus NS5 gene, obtain Zika virus pseudovirus particles; The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2. 3. The method according to claim 2, characterized in that: said adopting the MS2 phage packaging system to package the Zika virus NS5 gene is to express the phage MS2 maturation enzyme encoding gene, the phage MS2 capsid protein encoding gene and the bacteriophage in the host bacterium. The Zika virus NS5 gene is packaged to obtain Zika virus pseudovirion particles containing the Zika virus NS5 gene; The nucleotide sequence of the Zika virus NS5 gene is the 19th-2727th position of sequence 2. 4. The method according to claim 2 or 3, characterized in that: The method comprises the steps of: 1) Introduce the fragment containing the Zika virus NS5 gene, the phage MS2 maturation enzyme coding gene and the phage MS2 capsid protein coding gene into the host bacteria to obtain recombinant bacteria; The fragment containing the Zika virus NS5 gene is composed of an MS2 phage packaging site recognition fragment and the Zika virus NS5 gene; 2) Inducing and culturing the recombinant bacteria to obtain Zika virus pseudovirion particles. 5. The method according to claim 4, characterized in that: The fragment containing the Zika virus NS5 gene is introduced into the host bacterium through the recombinant vector A, The recombinant vector A is a vector obtained by inserting a fragment containing the Zika virus NS5 gene into an expression vector; The fragment containing the Zika virus NS5 gene is a DNA molecule shown in sequence 2 in the sequence listing; The phage MS2 maturation enzyme coding gene and the phage MS2 capsid protein coding gene are introduced into the host bacterium through the recombinant vector B, The recombinant vector B is a vector obtained by inserting a fragment containing a phage MS2 maturation enzyme coding gene and a phage MS2 capsid protein coding gene into an expression vector; The fragment containing the gene encoding phage MS2 maturation enzyme and the gene encoding phage MS2 capsid protein is the DNA molecule shown in sequence 1 in the sequence listing. 6. The method according to claim 5, characterized in that: The expression vector in the recombinant vector A is the same as or different from the expression vector in the recombinant vector B. 7. The method according to claim 5 or 6, characterized in that: The expression vector in the recombinant vector B is pET-30a+; The expression vector in the recombinant vector A is pACYCDuet-1; The host bacteria is Escherichia coli. 8. The method according to claim 5 or 6, characterized in that: In the step 2), after the induction culture, the following steps are further included: crushing the cells after the induction culture, collecting the supernatant of the crushed product, and obtaining Zika virus pseudovirion particles. 9. the Zika virus pseudovirus particle described in claim 1 or the Zika virus pseudovirus particle prepared by the method described in any one of claim 2-8 is used as the contrast reference substance in the Zika virus nucleic acid detection reagent applications in Or the Zika virus pseudovirus particle described in claim 1 or the Zika virus pseudovirus particle prepared by the method described in any one of claims 2-8 is used for the application in the preparation of Zika virus nucleic acid detection reagent. 10. the Zika virus pseudovirion obtained by the arbitrary described method of claim 2-8.