Recombinant replication-defective adenoviral vector H5N1 subtype influenza genetic engineering vaccine

A genetically engineered vaccine, replication-deficient technology, applied in the field of recombinant genetically engineered vaccines, can solve problems such as high production costs, single administration method, and dependence on transportation and storage

Inactive Publication Date: 2012-06-06
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

H5N1 influenza virus vaccines are still widely produced in chicken embryos. Although these vaccines have a good immune effect, they are expensive to produce, and must rely on low-temperature "cold chain" for transportation and storage. During the pandemic, it was limited by factors such as restrictions on the supply of chicken embryos

Method used

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  • Recombinant replication-defective adenoviral vector H5N1 subtype influenza genetic engineering vaccine
  • Recombinant replication-defective adenoviral vector H5N1 subtype influenza genetic engineering vaccine
  • Recombinant replication-defective adenoviral vector H5N1 subtype influenza genetic engineering vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant replication-deficient adenovirus expressing influenza virus A / Swine / Fujian / 1 / 2001 (H5N1) HA gene of the present invention

[0037] A. Obtaining the immunogen

[0038] A / Swine / Fujian / 1 / 2001 (H5N1) (purchased from the Animal Influenza Laboratory of the Ministry of Agriculture, Harbin Veterinary Research Institute) was proliferated in SPF chicken embryos (purchased from the Experimental Animal Center of Harbin Veterinary Research Institute) for 48 hours, and the allantoic fluid was harvested , adding Trizol reagent (Gibco BRL) to extract RNA, and using universal primer uni12 (sequence: 5'-AGCAAAAGCAGG-3') to reverse transcribe to obtain influenza virus HA gene cDNA.

[0039] B. Preparation, collection and verification of recombinant adenovirus rAd-H5HA-EGFP Using the cDNA obtained in the above A as a template to amplify the HA gene by polymerase chain reaction (PCR), the primers used: upstream primer HAF: 5'CCC GTC GAC ATG GAGAAA ATA...

Embodiment 2

[0043]Example 2 Proliferation and purification of rAd-H5HA-EGFP

[0044] When the HEK293 cells grew to 80%-90% saturated, the recombinant virus rAd-H5HA-EGFP was inoculated at a multiplicity of infection m.o.i=5-10. After 3-4 days, when the cell lesion reaches 70-90%, collect the cell solution in a centrifuge tube, centrifuge at 3500g for 15 minutes to harvest the cells, transfer the supernatant to a sterile beaker for storage, and use 10ml of the supernatant to resuspend the cell pellet Break the cell pellet 3 times in a freeze-thaw water bath at -80°C / 25°C (the maximum water temperature of the water bath should not exceed 25°C), centrifuge again at 3500g for 15 minutes to remove cell debris, separate the supernatant and mix it with the retained supernatant, reconstitute The purification process of the virus was carried out according to the AdenoPACK adenovirus purification kit of Sartorius AG TM 100RT instructions, the obtained recombinant virus was aliquoted and frozen at ...

Embodiment 3

[0045] Example 3 Determination of rAd-H5HA-EGFP infectious titer (TCID 50 )

[0046] Take one T75 cell flask of HEK293 cells pre-cultured with culture medium (Dulbecco's Modified Eagle Media, DMEM for short) + 10% FBS (fetal bovine serum), wait until the cells grow to 80%, collect the cells and digest the cells with trypsin And count. Prepare cell suspension in DMEM with 5% FBS, each plate needs 11mL with a concentration of 1×10 5 / mL of cell suspension. Press 100μL per well (ie about 1×10 4 cells) into a 96-well plate (add another 96-well plate if a set of replicate experiments is to be performed). Dilute the adenovirus sample by aseptic operation: take 10 μL of high-concentration recombinant adenovirus solution for serial 10-fold dilution, and use DMEM with 5% FBS as the diluent. Do another set of repeated experiments, and then carry out the second set of dilutions according to the above steps. The detection process adopts the rapid adenovirus infectious titer (TCID) p...

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Abstract

The invention provides a recombinant replication-defective adenoviral vector H5N1 subtype influenza genetic engineering vaccine, which uses a replication-defective human adenovirus 5 with the combination deletion of E1 and E3 as a vector, uses HEK293 cells of the integrated adenovirus E1 gene as a packaging cell line. The E1 region is provided with an H5N1 subtype influenza virus envelope glycoprotein encoding gene HA and a green fluorescent protein tracer gene EGFP. The H5N1 subtype influenza virus envelope glycoprotein encoding gene HA is from the influenza strain A / Swine / Fujian / 1 / 2001(H5N1).

Description

technical field [0001] The present invention relates to a recombinant genetic engineering vaccine, specifically, the present invention relates to a recombinant genetic engineering vaccine for preventing animal H5N1 subtype influenza, more specifically, the vaccine involved in the present invention is based on the H5N1 subtype influenza strain glycoprotein HA As the antigen gene, the replication-deficient human adenovirus with combined deletion of E1 and E3 is used as the carrier. The invention also relates to HEK293 cell virus cultures of the vaccine. Background technique [0002] Highly pathogenic H5N1 subtype avian influenza is an important disease that endangers the development of the poultry industry in the world. It can cause 100% death of infected poultry flocks. It is listed as a Class I severe infectious disease by the World Organization for Animal Health. The widespread spread and prevalence of avian influenza has caused serious losses to the world's poultry indust...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/145A61P31/16
Inventor 乔传玲吴运谱杨焕良陈艳辛晓光陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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