human type-III parainfluenza virus cold-adapted temperature-sensitive strain and application of cold-adapted temperature-sensitive strain

A technology for parainfluenza and cold adaptation, applied in the direction of viruses, antiviral agents, viruses/bacteriophages, etc., to achieve good safety and infection prevention effects

Active Publication Date: 2021-10-01
LANZHOU INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there are no effective drugs and preventive vaccines for the effective treatment of HPIV-3 infection. Therefore, the research and development of cold-adapted temperature-sensitive attenuated vaccines for HPIV-3 is of great significance

Method used

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  • human type-III parainfluenza virus cold-adapted temperature-sensitive strain and application of cold-adapted temperature-sensitive strain
  • human type-III parainfluenza virus cold-adapted temperature-sensitive strain and application of cold-adapted temperature-sensitive strain
  • human type-III parainfluenza virus cold-adapted temperature-sensitive strain and application of cold-adapted temperature-sensitive strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Screening of cold-adapted temperature-sensitive strains of human parainfluenza virus type III

[0031] 1.1 Screening of HPIV-3 positive clinical specimens

[0032] Screening materials: samples of lower respiratory tract secretions from inpatients with lower respiratory tract infection in Maternal and Child Health Hospital of Gansu Province, frozen at -80°C;

[0033] Screening method: Melt the virus screening material at room temperature, then vortex at 12000rpm for 2min; after the concussion, suck the vortex liquid into a 1.5ml centrifuge tube, and centrifuge at 12000rpm for 20min; filter the centrifuged supernatant with a needle filter ( 0.22μm) after sterilizing and filtering, inoculate into 6-well plates grown with WI-38 (human embryonic lung fibroblasts), incubate at 37°C for 2 hours; suck off the supernatant, add 3ml Plasmocin containing 10ug / ml mycoplasma remover (Invivogen, ant-mpt) DMEM medium, placed at 37 ° C, 5% CO 2 Cultivate in the incubator for...

Embodiment 2

[0044] Example 2 Preparation and purification of HPIV3CPW3 virus culture fluid and identification of virus coat protein

[0045] 2.1 Preparation of HPIV3CPW3 virus culture medium

[0046] The HPIV3CPW3 virus strain was inoculated into WI-38 cells at 0.01 MOI, at 25°C, 5% CO 2 After culturing in an incubator for 7 days, the culture supernatant was collected, tested for virus titer by qRT-PCR, and stored at 4°C.

[0047] 2.2 Purification of HPIV3CPW3 virus by ultracentrifugation

[0048] Centrifuge the HPIV3CPW3 virus culture solution prepared in step 2.1 at 8000rpm and 4°C for 30min to precipitate cell debris and collect the supernatant; take 30ml of the supernatant and add it to a 50ml special centrifuge tube, and gently push 15ml50 from the bottom with an 8cm long needle. % sucrose (keep the interface clear during the operation), then put it into an ultracentrifuge (Hitachi cp70mx ultracentrifuge can be selected), and perform ultracentrifugation for 4 hours at 4°C and 35000...

Embodiment 3

[0053] Example 3 Determination of the Whole Gene Sequence of HPIV3CPW3 and Analysis of Genetic Characteristics

[0054] The whole gene sequence of HPIV3CPW3 was determined by next-generation sequencing, and the specific process was completed by Shanghai Bojie Medical Technology Co., Ltd. Sequencing results show that the full gene sequence of HPIV3CPW3 contains 15462 nucleotides. According to nucleotide sequence analysis, the homology between HPIV3CPW3 and HPIV-3 isolate MH678682.1 on GenBank is 99.87%. The sequence conforms to the "6-base principle" of the Paramyxoviridae genome, according to 5'-NP (111-1658)-PP / C(1784-3592)-M(3753-4814)-F(5072-6691)-HN( The sequence of 6806-8524)-L (8571-15347)-3' encodes 6 proteins, and the gene structure is the same as that of the HPIV-3 isolate with known sequence. Description HPIV3CPW3 strain is a type III parainfluenza virus.

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Abstract

The invention provides a human type-III parainfluenza virus cold-adapted temperature-sensitive strain and the application thereof. The human type-III parainfluenza virus cold-adapted temperature-sensitive strain is HPIV3CPW3 and a composition containing the human type-III parainfluenza virus cold-adapted temperature-sensitive strain, the human type-III parainfluenza virus strain HPIV3CPW3 is used as a type-III parainfluenza virus attenuated live vaccine candidate strain. The human parainfluenza III attenuated live vaccine has a dose effect and good safety, has a significant protection effect on infection of a mouse lung HPIV-3 wild strain, and can effectively prevent infection of the parainfluenza III virus with cold adaptation temperature sensitivity. Moreover, the human parainfluenza III virus strain HPIV3CPW3 has the characteristics of cold adaptation and temperature sensitivity and also has the characteristic of temperature sensitivity in the upper respiratory tract and the lower respiratory tract of a mouse, so that the human parainfluenza III virus strain HPIV3CPW3 has important significance in stimulating protective immune response in inoculated mammals without generating serious symptoms; the human type-III parainfluenza virus cold-adapted temperature-sensitive strain can be used for preparing a medicine for causing protective immune response in an object.

Description

technical field [0001] The invention relates to a type III parainfluenza virus, in particular to a cold-adapted temperature sensitive strain of a type III human parainfluenza virus and application thereof. Background technique [0002] Human parainfluenza virus type 3 (HPIV-3) is second only to respiratory syncytial virus and is the main pathogen that easily causes acute lower respiratory diseases such as severe bronchitis and pneumonia in infants and young children. HPIV-3 infection is very common in hospitalized children with acute respiratory tract infection. HPIV-3 outbreaks occur every year, mainly in spring and summer, and epidemiological studies show that 80% of infants under 5 years old have been infected with HPIV -3. In addition, HPIV-3 infection is also an important cause of lower respiratory tract disease and death in elderly patients with chronic diseases and immunocompromised adults. In organ transplantation and immunosuppressed patients, the mortality rate ca...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/155A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2760/18621C12N2760/18634A61K2039/5254
Inventor 白慕群火文关文竹李雄雄王云瑾包红寇桂英韩平王名强
Owner LANZHOU INST OF BIOLOGICAL PROD
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