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Vero-pAPN (Porcine Aminopeptidase N) cell line and preparation method thereof

A cell line and cell technology, applied in the field of bioengineering, can solve the problems of low efficiency of stable cell lines, difficulties in isolation and culture of PEDV, etc.

Inactive Publication Date: 2018-03-13
杨凌凯瑞生物科技有限公司
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Problems solved by technology

[0005] The purpose of the present invention is mainly aimed at the above-mentioned difficult problems of PEDV isolation and culture involved, to provide a Vero-pAPN cell line, and to provide a method for constructing Vero-pAPN cell lines to solve the problem of low efficiency of constructing stable cell lines

Method used

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  • Vero-pAPN (Porcine Aminopeptidase N) cell line and preparation method thereof
  • Vero-pAPN (Porcine Aminopeptidase N) cell line and preparation method thereof
  • Vero-pAPN (Porcine Aminopeptidase N) cell line and preparation method thereof

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Embodiment Construction

[0014] Construction of Vector CD513B-pAPN

[0015] According to the pAPN sequence in NCBI (sequence number: KU986724), the pAPN gene fragment was synthesized, with restriction sites XbaI and BamHI added at both ends, and a 6×His tag at the C-terminus for subsequent detection. The pAPN fragment was inserted into the CD513B vector through enzyme digestion, ligation, transformation and other genetic engineering techniques, and the resulting new vector was named CD513B-pAPN.

[0016] lentiviral packaging

[0017] HEK293 cells were co-transfected with the constructed vector CD513B-pAPN and three backbone vectors pPACKH1-GAG, pPACKH1-REV, and pVSV-G: 2×10 5 Cells / mL were spread on 6-well plates, and transfected when the cell density reached 80% the next day; 500 μL Opti-MEM was taken, and 0.5 μg pPACKH1-GAG, 0.5 μg pPACKH1-REV, and 0.5 μg pVSV-G were added to it , 1.5 μg CD513B-pAPN, mix evenly, add 6 μL Turbofect, blow and aspirate, mix evenly and let stand for 20 min, drop evenl...

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Abstract

The invention relates to a Vero-pAPN (Porcine Aminopeptidase N) cell line. An adopted construction method is a slow virus mediating method. Compared with a Vero cell line which is constructed by parental Vero cells and an instant transfer mode and is used for expressing pAPN, cells of the cell line disclosed by the invention can stably express APN protein at a higher level and improve the PEDV (porcine epidemic diarrhea virus) breeding titer, and become host cells more suitable for multiplication of PEDV. The cells can be applied to subsequent virus culture, separation and purification, and provide host raw materials for preparation of vaccines.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to constructing a Vero-pAPN cell line stably expressing porcine aminopeptidase N (Porcine Aminopeptidase N, pAPN) and a preparation method thereof. The invention uses a lentivirus-mediated method to construct a stable cell line Vero-pAPN, and the cell can be used for culturing and separating porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDvirus, PEDV), which can infect pigs of various stages and cause acute watery diarrhea, especially in suckling piglets. Severe dehydration can result afterwards, and its fatality rate can be as high as 100%. After the disease was discovered in the UK in the early seventies of last century, Belgium, Holland, Germany, France and other countries have successively reported the occurrence of th...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/57
CPCC12N9/485C12N15/86C12N2740/15043C12Y304/11002
Inventor 张磊戚伟强尹彦龙
Owner 杨凌凯瑞生物科技有限公司
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