Dengue virus (DENV)-like particle as well as preparation method and application thereof

A dengue virus, virus-like technology, applied in the field of genetic engineering, can solve the problems of secreting and expressing four types of dengue virus virus-like particles that have not yet been found, and achieve the effect suitable for large-scale production

Inactive Publication Date: 2012-02-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the secretion and expression of four types of dengue virus virus-like particles in the Pichia pastoris eukaryotic system

Method used

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  • Dengue virus (DENV)-like particle as well as preparation method and application thereof
  • Dengue virus (DENV)-like particle as well as preparation method and application thereof
  • Dengue virus (DENV)-like particle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Determination of the optimal construction strategy for DENV1-4VLPs gene expression elements

[0034] 1. DENV1~4 virus culture and virus RNA extraction

[0035] DENV-1 GZ01 / 95 strain, DENV-2 ZS01 / 01 strain, DENV-3 H87 strain and DENV-4 H241 strain were respectively inoculated on C6 / 36 cell monolayer, and cultured in MEM containing 2% inactivated calf serum cultured at 37°C until the appearance of cytopathic changes reaching "+++~++++", and the supernatant was collected for RNA preparation. Total RNA was extracted from the culture supernatant using Trizol reagent. Trizol ? LS reagent is a product of Invitrogen, USA.

[0036] 2. Preparation of DENV1-4 prME gene elements

[0037] Using ThermoScript TM RT-PCR System (Invitrogen, USA) reverse-transcribed DENV1-4 RNA to synthesize the first strand of cDNA.

[0038] Reaction system: template RNA (5-10 μg) 8 μl, primer R 2 μl, dNTPs (100mM) 2 μl, DEPC water 3 μl. The total volume was 15 μl. The a...

Embodiment 2

[0042] Example 2 Construction and Identification of Recombinant Vectors Capable of Secreting and Expressing DENV1-4 VLPs

[0043] 1. Construction of recombinant vector

[0044] The prME gene prepared in Example 1 was double digested with Bsp119 I and Xba I, and the target fragment was recovered, and directional ligated with the pGAPZαA vector (product of Invitrogen Company) through the same double digestion, and the ligated product was transformed into E. coli DH5α (product of Stratagene, USA) competent cells, single clones were picked and inoculated in LB medium containing antibiotics and cultured overnight at 37°C with shaking. The correctly identified plasmids were selected and named: pGAPZ-α-PrMED1, pGAPZ-sPrME472-D2, pGAPZ-sPrME-D3 and pGAPZ-sPrME-D4, and the correctly identified recombinant vectors were transformed into P. pastoris yeast cells to obtain stable The expressed engineering bacteria were respectively preserved in the Chinese Type Culture Collection Cente...

Embodiment 3

[0055] Example 3 Preparation and Identification of DENV1~4VLPs

[0056] 1. Separation of virus-like particles by sucrose density gradient centrifugation

[0057] Harvest the cultured yeast cells, use 10-50% sucrose density gradient ultracentrifugation to purify the DENV1-4 VLPs in the supernatant of the cell lysate, absorb 30-40% of the flocculent virus-like particle layer for SDS-PAGE and Western Blotting detection. Figure 7 It is the Western blot identification chart of purified VLPs.

[0058]2. Detection of virus-like particles by electron microscopy

[0059] The solution layer containing virus-like particles obtained by the above purification method was observed by negative staining electron microscope, as shown in Figure 8 As shown, both 30 nm and 55 nm diameter VLPs were detected. Use the anti-DENV-2 serum of certain dilution concentration to carry out immunoelectron microscope observation, such as Figure 9 , VLPs with diameters of 30 nm and 55 nm were also...

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Abstract

The invention discloses a dengue virus (DENV)-like particle as well as a preparation method and the application thereof. P. pastoris yeast strains transformed by recombinant carriers of virus-like particles (VLPs) of DENV1-4, which are respectively pGAPZ-Alpha-PrME-D1-X33, pGAPZ-sPrME472-D2-X33, pGAPZ-sPrME-D3-X33 and pGAPZ-sPrME-D4-X33, are respectively preserved at China center for type culture collection (CCTCC) at Wuhan University on March 14th, 2011 and have the serial numbers of CCTCCM2011063, CCTCCM2011064, CCTCCM2011065 and CCTCCM2011066. The VLPs of the DENV1-4 are obtained by transfecting the recombinant carriers stated in claim 1 with P. pastoris yeast cells X33 and enabling the recombinant carriers to secrete the VLPs. According to the preparation method, a P. pastoris yeast system not relied on carbinol is adopted to prepare the DENV-like particle, which is not reported at home and abroad yet, so the innovativeness is achieved. The DENV-like particle prepared from the system can be used for preparing vaccines, and various defects in the research of dengue vaccines such as attenuated live vaccines, subunit vaccines, carrier vaccines and the like are overcome; and the DENV-like particle has the advantages of safety and high efficiency, and is suitable for large scale production.

Description

technical field [0001] The invention relates to the field of genetic engineering, and the specific content is a method for preparing dengue virus-like particles using Pichia pastoris and its application. Background technique [0002] Dengue virus (DENV) is transmitted by Aedes mosquitoes and can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in humans. ). DHF / DSS is a serious condition with a high mortality rate, and its pathogenesis has not yet been elucidated. Due to the lack of effective dengue virus vaccines and the practical difficulties in controlling mosquito vectors, the geographical and epidemic intensity of dengue virus infection is constantly expanding. At present, dengue fever is making a comeback in many parts of the world, and has become an arbovirus disease with the widest distribution, the most incidence and great harm in the world. Therefore, it has become a very urgent problem to strengthen the research on the pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N7/04C12N15/81A61K39/12A61P31/14C12R1/84
CPCY02A50/30
Inventor 江丽芳黎孟枫刘岩周俊梅
Owner SUN YAT SEN UNIV
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