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Human enterovirus strain and application thereof

A technology of human enteroviruses and viruses, applied in the direction of viruses, antiviral agents, virus antigen components, etc., to achieve good immune protection effect and good immune protection effect

Active Publication Date: 2017-01-11
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, it has been found that gerbils are highly sensitive to EV71, and 21-day-old gerbils are also completely sensitive to EV71, which can be used as a better animal model for virus infection. At present, no one has used gerbils to prepare attenuated strains of EV71 virus.

Method used

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  • Human enterovirus strain and application thereof
  • Human enterovirus strain and application thereof
  • Human enterovirus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Isolation and cultivation of gerbil primary muscle cells

[0023] Select 10 gerbils aged 10-15 days, kill them under humane anesthesia, sterilize them with iodine and 75% alcohol, cut off the hindlimb muscles, break them up, and wash them with MEM culture medium containing 2 times double antibody (purchased from Gibco) for 3 Over time, 0.2% (w / v) collagenase and 0.25% (w / v) trypsin were mixed and digested overnight at 4°C. The digestive solution was repeatedly blown into a suspension with 10 times (w / v) MEM solution, passed through 100-mesh and 200-mesh molecular sieves successively, collected the filtrate, centrifuged at 1000×g for 5-10min, blown the cell pellets evenly, and adjusted the pH to 5- 7. Set at 37°C, 5% CO 2 In the incubator, culture in MEM containing 10% fetal bovine serum for 3 to 4 days, replace the culture medium, the adherent cells are the primary gerbil muscle cells, the micrograph of the isolated primary gerbil muscle cells is shown in fi...

Embodiment 2

[0024] Example 2 Isolation of attenuated strain 157-11-2

[0025] After the primary cells of the gerbil muscle in Example 1 were covered with the culture flask, the culture medium was discarded, and EV71 virus (Genbank ID: KX639820) was inoculated, and the culture bottle was gently shaken to make the virus contact with the primary muscle cells evenly, and then discarded after adsorption at 37°C for 2 hours. Virus liquid, add MEM culture medium containing 3% serum and cultivate for 72-96h, after more than 50% of the cells have pathological changes (expressed as cell shrinkage, rounding and shedding) ( figure 1 In middle B), collect the culture medium, freeze and thaw repeatedly 3 times, and centrifuge at 1000-2000×g for 20 minutes. The supernatant is the EV71 virus solution. The EV71 virus solution is inoculated by a 35°C low-temperature continuous culture method, and repeatedly passaged for 10-20 generations. The attenuated strain 157-11-2 was isolated by plaque method, and th...

Embodiment 3

[0030] Example 3 Determination of the Nucleotide Sequence of the Attenuated Strain 157-11-2 Structural Protein

[0031] The Qiagen RNeasy Mini Kit kit (purchased from QIAGEN Company) extracted the total RNA of the attenuated strain 157-11-2, and the experimental steps were as follows according to the method provided by the kit:

[0032] a) Take 200 μl of virus liquid, add 600 μl of RLT, and vibrate with an oscillator for 1 min.

[0033] b) Add 600 μl of pre-cooled -20°C absolute ethanol and mix well.

[0034] c) Add about 600 μl of the mixed solution to the separation column in two times, centrifuge at 12,000 rpm for 30 s, and discard the filtrate;

[0035] d) Wash the column with 700 μl RW1 washing solution, and centrifuge at 12000 rpm for 30 seconds;

[0036] e) Take 500 μl of RPE wash solution to wash the column, and centrifuge at 12000rpm for 30s;

[0037] f) Take 500 μl RPE and wash again, centrifuge at 12000 rpm for 30 seconds;

[0038] g) The column is vacated at 12...

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Abstract

The invention discloses a human enterovirus strain and application thereof and provides an EV71 (Enterovirus 71) virus attenuated strain. The stable and higher virus titer can be obtained from primary muscle cells of gerbil jird. A virus attenuated strain 157-11-2 has a better immunoprotective effect, and provides a good candidate virus strain for further research and development of vaccines.

Description

[0001] (1) Technical field [0002] The invention relates to an attenuated strain of enterovirus EV71 and its application. [0003] (2) Background technology [0004] In recent years, the epidemic situation of hand, foot and mouth disease (HFMD) in China has shown an upward trend of spread, and the reported cases and deaths of hand, foot and mouth disease rank first among class C infectious diseases. In 2014, the incidence of hand, foot and mouth disease in my country reached about There were more than 2.82 million cases and 508 deaths, nearly double that of 2013. HFMD has become one of the major public health threats in China. Enterovirus 71 (Enterovirus 71, EV71) belongs to the family Picornaviridae Enterovirus genus Enterovirus A, and is one of the main pathogens of hand, foot and mouth disease. EV71 virus infection can cause myocarditis, brainstem encephalitis, aseptic Meningitis, pneumonia, and acute flaccid paralysis similar to polio and other diseases related to the nerv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/125A61P31/14C12R1/93
CPCA61K39/12A61K2039/5254C12N7/00C12N2770/32321C12N2770/32334
Inventor 孙一晟朱函坪夏时畅姚苹苹徐芳杨章女朱智勇卢杭景周小龙
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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