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Cell BHK/slam based on small ruminant animal disease virus receptor

A PPR, cell technology, applied in the field of biology, can solve the problems of difficult adaptation of the virus, undetectable virus, low cytotoxicity of the virus, etc., and achieves the effect of convenient and fast spin column, improved cloning efficiency and good stability

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

AI Technical Summary

Problems solved by technology

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 to several generations, and the virus cannot be detected
Seriously affected and limited the isolation, identification, biological characteristics and pathogenic mechanism of PPRV strains collected in the field

Method used

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  • Cell BHK/slam based on small ruminant animal disease virus receptor
  • Cell BHK/slam based on small ruminant animal disease virus receptor
  • Cell BHK/slam based on small ruminant animal disease virus receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Design, synthesize and clone vector pDONRTM221 P5-P2 and expression vector pLenti4 / V5-DEST according to the characteristics of the complete ORF post-translational protein sequence (SEQ ID NO.1) of Peste des petits ruminants virus Slam receptor TM Vector matching primers SEQ ID NO.2 and SEQ ID NO.3, the three sequences are as follows:

[0042] SEQ ID NO.1: MDHKGLLSSNVLLFFSLIIELSCRTGEGLTSSTK

[0043] TIRGQLGSSVLLPLASEEISRSMNKSIHILVTMAESPRDTVKKKIVSLDLRKGDSPRLEDGYEFHLENLSLRIL KSRKEDEGWYFISLEENVSVQHFSLQLKLYEQVSTPQIKVLNSTQEDGNCSLMLACVVEKGDHVTYNWSEEAGA PLLSPTNSSHLLYLTLGPQRANNVYICIASNPISNSSQTFIPWSRCSSRPPESRQWGLYTGLFLGGIVGVIMIL QVVILLLRRRGKTDNYQPTMEAKSLTIYAQVQKSGSVKKRPDPLPAQDPCTTIYVAATEPVPEPIQESGSFTVY ASVTVPES;

[0044] SEQ ID NO. 2: GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGATCACAAAGGGCTCCTCTCCT;

[0045] SEQ ID NO. 3: GGGGACCACTTTGTACAAGAAAGCTGGGTTT

[0046] CAGCTCCTCTGGAACCGTCACA.

[0047] 2. Preparation of total RNA related to the receptor of Peste des petits ruminants viru...

Embodiment 2

[0065] Steps 1-5 are the same as in Example 1.

[0066] 6. Co-transfect 293-FT cells with expression backbone and helper plasmid to obtain replication-defective lentivirus-like particles

[0067] As above, the three auxiliary plasmids pLP1, pLP2, and VSV-G were extracted in large quantities with the endotoxin-free large-scale plasmid extraction kit, and 12ug of the expression backbone, pLP1, pLP2, and VSV-G were added to 750ul opti-MEM, and another 750ulopti-MEM was added. Add 30 ul of p3000 to MEM, and transfect 293-FT cells in sequence according to the instructions of liposome 3000; replace the complete medium 8 hours after transfection, and collect the supernatant of 293-FT cells aseptically for 48 hours. Centrifuge at 5000 rpm at 4°C for 5 minutes, discard the cell debris and precipitate, and the collected supernatant is the replication-defective lentivirus-like particles with the complete ORF of Peste des petits ruminant virus Slam receptors, which are stored in a -70°C r...

Embodiment 3

[0074] 1-7 steps are with embodiment 1.

[0075] 8. BHK / slam cells express the complete ORF of slam and its activity analysis

[0076] Select the BHK and BHK / slam 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 several times with a gun to make the lysate fully contact with the cells. After lysing, centrifuge at 12000rpm for 5min, add 4x sample buffer to the supernatant, boil for 10min, and load the sample for sodium dodecylsulfonate polyacrylamide gel electrophoresis ( 12% SDS-PAGE), the protein glue utilizes BIO-RAD company electroporation instrument (Trans- 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...

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Abstract

The invention provides a cell BHK / slam based on a small ruminant animal disease virus receptor, which is deposited under the accession number of CCTCC NO:C201895, and the small ruminant pestilence virus receptor-intact goat lymphocyte signal activation factor ORF expressed by the cell Is complete and has a function of significantly enhancing the replication of small ruminant animal disease virus,the cell shortens the time of virus collection after cytopathic lesions(from 4-7 days to 3-4 days) through overexpressing of specific cell receptors of the small ruminant animal disease virus, the viral titer of small ruminant animal disease virus (ct value increased from 20.66 to 16.91) is improved, the copy number of the same volume virus (copy number increased from 4.1*10<7> to 5.3*10<8>) wereincreased significantly, and the cost was saved. It provides a good tool for isolation of small ruminant animal disease virus and production of vaccine virus, and also provides a cellular model for the study of pathogenesis of small ruminant animal disease virus. The invention also provides the preparation and application of the cell.

Description

technical field [0001] The 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 based on the PPRV receptor. The cell line is named BQS24, and it was deposited in China Center for Type Culture Collection (Wuhan University, Wuhan, China) on 2018.06.04, with the preservation number: CCTCC NO: C201895. 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 control the epidemic. At the end of November...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867
CPCC07K14/00C12N5/0686C12N15/86C12N2510/00C12N2740/15043
Inventor 吴锦艳田宏尚佑军曹小安惠小婷刘湘涛刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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