Method for cultivating hepatitis e virus

Method for cultivating hepatitis e virus

  • CN 105,062,979 A
  • Filed: 08/21/2015
  • Published: 11/18/2015
  • Est. Priority Date: 08/21/2015
  • Status: Active Application
First Claim
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1. cultivate a method for hepatitis E virus, it is characterized in that carrying out as follows:

  • (1) by HEV viral solution after 0.22 μ

    m of membrane filtration is degerming, add the dual anti-solution of viral solution volume 1 ~ 2%, 4 DEG C process 1h ,-80 DEG C save backup, and measuring its viral copy number through Real-timeqPCR is 2 ×

    10 5~ 2 ×

    10 6copy number/mL, wherein dual anti-solution is the solution containing 400U/mL penicillin and 1000U/mL Streptomycin sulphate;

    (2) A549 cell is pressed 2 ×

    10 5/ hole is seeded in 6 orifice plates, with the DMEM substratum containing mass percent 10% foetal calf serum at 37 DEG C, 5%CO 2static gas wave refrigerator cell in incubator, until cell grows up to individual layer;

    (3) be seeded in monolayer cell by the HEV viral suspension of step (2) by every hole 100 μ

    L, put 37 DEG C and hatch 1 hour, every 15min slightly shakes up once, and virus is fully adsorbed onto on cell;

    Abandon supernatant, 1 ×

    PBS washed cell 3 times, add the DMEM substratum containing mass percent 2 ~ 5% serum in pregnant late period, add final concentration is 0.01mMMgCl simultaneously 2the protection integrity of virion and the adsorption of enhanced virus and cell, be placed in 37 DEG C, 5%CO 2continue in incubator to cultivate;

    (4) inoculate HEV virus after 6 days A549 cell there is pathology phenomenon, multigelation, collect sick cell and nutrient solution ,-80 DEG C of preservations are stand-by.

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