Cell BHK/slam/v based on a small ruminant pestilence virus receptor

A technology of Peste des petits ruminants and cells, which is applied in the field of biology, can solve the problems of limited virus strains, low toxicity of virus cells, and impact, and achieve the effect of improving cloning efficiency, good stability, and high purity

Pending Publication Date: 2019-01-25
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the cells in the known field of biology are either difficult to adapt to the virus, or the virus cell has a low toxicity. In some cells, the virus has not been adapted for several generations, an

Method used

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  • Cell BHK/slam/v based on a small ruminant pestilence virus receptor
  • Cell BHK/slam/v based on a small ruminant pestilence virus receptor
  • Cell BHK/slam/v based on a small ruminant pestilence virus receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Design, synthesize and clone the vector pDONRTM221P5-P2 and the expression vector pLenti4 / V5-DEST according to the characteristics of the PPRV receptor itself and its coding functional region V gene after translation (SEQ ID NO.1) TM Vector matching primers SEQ ID NO.2 and SEQ ID NO.3, the three sequences are as follows:

[0046] SEQ ID NO. 1: LDLRKGDSPRLEDGYEFHLENLSLRILKSRKED EGWYFISLEENVSVQHFSLQLKLYEQVSTPQIKVLNSTQEDGNCSLMLACVVEKGDHVTYNWSEEAGAPLLSPT NSSHLLYLTLGPQHA;

[0047] SEQ ID NO.2: GGGGACAACTTTGTATACAAAAGTTGTAATG

[0048] CTAGATCTGCGGAAAGGTGACT;

[0049] SEQ ID NO. 3: GGGGACCACTTTGTACAAGAAAGCTGGGTT

[0050] GGCATGCTGAGGGCCAAGAGTGAG.

[0051] 2. Preparation of total RNA related to the receptor of Peste des petits ruminants virus

[0052] Select a healthy goat, collect 50ml of whole blood from the jugular vein, add it to the glass container with anticoagulant before, shake well, add diluent in equal amount, shake well, add goat lymphocyte separation medium...

Embodiment 2

[0068] Steps 1-5 are the same as in Example 1.

[0069] 6. Co-transfect 293-FT cells with the expression backbone and helper plasmids to obtain replication-defective lentivirus-like particles. As above, use the endotoxin-free large-scale plasmid extraction kit to extract a large number of three helper plasmids, pLP1, pLP2 and VSV-G, 750ul opti -Add 12ug of the expression backbone, pLP1, pLP2 and VSV-G to the MEM, add 30ul of p3000 to the other 750ul opti-MEM, and transfect 293-FT cells in sequence according to the instructions of liposome 3000; 8h after transfection The complete medium was replaced, and the 293-FT cell supernatant was aseptically collected for 48 hours. Centrifuge at 5000rpm at 4°C for 5min, discard the cell debris and precipitate, and the collected supernatant is the replication-defective lentivirus-like particles with the receptor of Peste des petits ruminants virus, which are stored in a -70°C refrigerator until the target cells are infected.

[0070] 7. P...

Embodiment 3

[0075] 1-7 steps are with embodiment 1.

[0076] 8. BHK / slam / V cell expression slam / V and its activity analysis

[0077] Select BHK and BHK / slam / V cells in good growth state, digest the normal cells with trypsin, discard the trypsin, collect the cells in the maintenance solution, freeze and thaw repeatedly in the refrigerator, add 500ul RIPA cell lysate (Solabu ), pipet with a gun several times to make the lysate fully contact with the cells, centrifuge at 12000 rpm for 5 minutes after lysis, add 4x sample buffer to the supernatant, boil for 10 minutes, and load the sample on sodium dodecyl sulfate polyacrylamide gel Electrophoresis (12% SDS-PAGE), the protein gel was electroporated using BIO-RAD company ( Turbo) was transferred to a PVDF (polyvinylidene double oxide membrane) transfer membrane, transferred for 7 minutes, washed with PBS 5 times, shaken for 5 minutes each time, and TBST containing 5% skimmed milk powder was added dropwise to block overnight. Wash the membr...

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Abstract

The invention provides a cell BHK/slam/v based on a small ruminant pestilence virus receptor, the accession number of which is CCTCC NO:C201739, and a small ruminant pestilence virus receptor-goat lymphocyte signaling activation factor V (GLSAF-V) expressed by the cell can enhance the small ruminant pestilence virus replication function, overexpressing of specific cell receptors of the small ruminant pestilence virus by the cell shortens the time of virus collection (from 4-7 days to 3-4 days), the viral titer of small ruminant pestilence virus was increased (ct value increased from 20.19 to 17.01), the copy number of the same volume virus was also increased (copy number increased from 5.6*10<6> to 4.9*10<8>), and the cost was saved. It provides a good tool for isolation of small ruminantpestilence virus and production of vaccine virus, and also provides a cellular model for the study of pathogenesis of small ruminant pestilence virus. The invention also provides the preparation and application of the cell.

Description

technical field [0001] The present invention relates to the establishment of cell lines in the field of biology, in particular to the construction and application of a receptor cell line of Peste des Petits Ruminants virus, specifically a cell BHK / slam / v based on the Peste des Petits Ruminants virus receptor. The cell line is named BHK / Slam / V, and was deposited in the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on July 6, 2017, with the preservation number: CCTCC NO: C201739. Background technique [0002] Peste des petits ruminants (PPR) is caused by Peste despetits ruminants virus (PPRV), an acute, hot and highly contact disease characterized by fever, stomatitis, diarrhea, and pneumonia in sheep, goats, and especially lambs. infectious disease. In 2007, Peste des petits ruminants was first introduced into the Ngari area of ​​Tibet, my country. The Ministry of Agriculture, together with the local government, took effective measures to quickly ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867
CPCC07K14/70503C12N15/86C12N2510/00C12N2740/15043
Inventor 吴锦艳尚佑军田宏曹小安王耀杰张吉利刘湘涛刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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