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Separated nucleic acid and application thereof

A technology of nucleic acid and carrier, applied in isolated nucleic acid and its application field, can solve the problem that there is no preventive vaccine and therapeutic drug for Zika virus, and achieve good stability

Active Publication Date: 2019-01-04
BRAVOVAX +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are currently no commercial Zika virus preventive vaccines and therapeutic drugs

Method used

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  • Separated nucleic acid and application thereof
  • Separated nucleic acid and application thereof
  • Separated nucleic acid and application thereof

Examples

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preparation example Construction

[0069] The method for preparing Env protein provided by the embodiment of the present invention comprises culturing the above-mentioned recombinant cells or recombinant bacteria. Specifically, the method includes culturing the above-mentioned recombinant cells or bacteria, and then isolating and purifying the Env protein from the culture, which is the Zika virus antigenic protein.

[0070] The Zika virus vaccine provided by the embodiment of the present invention includes the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned recombinant adenovirus or the Env protein prepared by the above-mentioned method for preparing Env protein.

no. 1 example

[0073] The pAd5-CMV-M-Env-PolyA plasmid was prepared.

[0074] 1. Construction of transfer plasmid (pDONR221-M-Env-PolyA)

[0075] 1.1. Gene synthesis and primer design

[0076] According to the sequence shown in SEQ ID NO.3, let Gene Synthesis Company synthesize the M-Env sequence.

[0077] Using the M-Env sequence (sequence shown in SEQ ID NO.3) as a template, upstream and downstream primers (primers were synthesized by Invitrogen) were designed for PCR amplification.

[0078] Wherein, the sequence of the upstream primer (M-Env-F) is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATGGCTGTGACGCTCCCCTCCCATTC-3' (as shown in SEQ ID NO.8);

[0079] The sequence of the downstream primer (SV40-R) is as follows: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGATGATAAGATACATTGATGAGT-3' (as shown in SEQ ID NO.9).

[0080] 1.2. PCR amplification

[0081] Amplification system: 2 μL of upstream and downstream primers, 1 μL of gene synthesis plasmid template, 25 μL of Primer STAR mi...

no. 2 example

[0092] Preparation of linearized viral vector rAd5-M-Env-PolyA.

[0093] 3. Amplification of Adenoviral Vectors

[0094] 3.1. The pAd5-CMV-M-Env-PolyA plasmid sequenced correctly in step 2.3 was transformed into Escherichia coli TOP10 competent cells, and spread on a solid LB plate containing Amp antibiotics. Negative control for comparison: Transform Escherichia coli ccdBsuvival competent cells (purchased from Invitrogen, USA) with pAd5-CMV / V5-Dest plasmid (purchased from Invitrogen, USA), and spread on solid LB plates containing Amp antibiotics.

[0095] 3.2. The next day, pick out the single clones on the LB plate respectively, and inoculate them in the liquid medium containing 200mL Amp. pAd-CMV / V5-Dest plasmid.

[0096] 4. Adenoviral vector linearization treatment The pAd5-CMV-M-Env-PolyA plasmid and pAd-CMV / V5-Dest plasmid obtained in step 3.2 were respectively treated with PacI restriction endonuclease (purchased from NEB, USA), 37 Digest for 3 hours at ℃, and the di...

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Abstract

The invention discloses separated nucleic acid and an application thereof, and relates to the technical field of biological molecules. The base sequence of the nucleic acid is as shown in any of (1)-(3), the base sequence is as shown in SEQ ID NO. 3, the base sequence from first to 225 is as shown in SEQ ID NO. 1, the base sequence from 226 to 1737 encodes Env protein, the amino acid sequence of the Env protein is as shown in SEQ ID NO. 4, the base sequence from first to 225 encodes M protein, the base sequence from 226 to 1737 is as shown in SEQ ID NO. 2, and the aminoacid sequence of the M protein is as shown in SEQ ID NO. 5. The nucleic acid can express immunizing antigen of Zika viruses, and research of Zika virus vaccines or Zika virus therapy drugs is facilitated.

Description

technical field [0001] The invention relates to the technical field of biomolecules, in particular to an isolated nucleic acid and its application. Background technique [0002] Zika virus (Zika virus) was first discovered in 1947 in the Zika Forest of Uganda, Africa. It is parasitic in rhesus monkeys and is mainly transmitted through mosquito bites. At present, about 80% of Zika virus-infected individuals do not show obvious clinical symptoms, and a small number of infected individuals with clinical symptoms show fever, rash, arthralgia, muscle pain, headache, vomiting and conjunctivitis. It usually lasts for more than 1 week and is relatively mild. [0003] Zika virus vectors are Aedes aegypti and Aedes albopictus, which are active year-round in many parts of the world, including most of the southern coastal states of the United States and many resort areas in Latin America and the Pacific Ocean. Apart from early prevention, there is no effective treatment for Zika virus...

Claims

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Application Information

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IPC IPC(8): C12N15/40C07K14/18C12N15/861C12N7/01A61K39/12A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/53A61P31/14C07K14/005C12N7/00C12N15/86C12N2710/10043C12N2770/24122C12N2770/24134Y02A50/30
Inventor 谌章舟慕婷赵萍董威
Owner BRAVOVAX
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