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An expression vector and a Vero cell line expressing pig aminopeptidase N

An expression vector, porcine aminopeptidase technology, applied in the field of bioengineering, can solve the problems of complex culture conditions, slow growth, and biosafety needs to be evaluated, and achieves high efficiency and promotes replication.

Inactive Publication Date: 2017-02-15
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the proliferation ability of this cell line is still different from that of the Vero cell line, and the growth is slow; and its culture conditions are relatively complicated, and growth factors need to be added, which is costly; and epithelial cell lines are not commonly used in vaccine production. system, the practical application is not realistic, and the biological safety has yet to be evaluated

Method used

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  • An expression vector and a Vero cell line expressing pig aminopeptidase N
  • An expression vector and a Vero cell line expressing pig aminopeptidase N
  • An expression vector and a Vero cell line expressing pig aminopeptidase N

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of pAPN eukaryotic overexpression plasmid

[0055] The eukaryotic overexpression plasmid of pAPN uses pcDNA3.1(+)-puro as the vector plasmid. In order to facilitate the detection of the overexpression of pAPN at the protein level in the later stage, a flag-tag tag is added to its N end through a primer. The build process is as follows:

[0056] 1 material

[0057] Porcine small intestine tissue and pcDNA3.1(+) plasmid are kept in this laboratory

[0058] 2 test process

[0059] 1. Construct pcDNA3.1(+)-puro plasmid

[0060] Obtain the puro resistance gene. The sequence of the puro resistance gene is shown in SEQ ID NO: 2. The puro resistance gene fragment is ligated into pcDNA3.1(+) plasmid with restriction enzyme cut sites Sma I and Sal I. pcDNA3.1(+)-puro plasmid.

[0061] 2. Obtain the CDS sequence (SEQ ID NO:1) of pAPN gene

[0062] Since the full length of the mRNA encoding pAPN is 3387bp, the CDS sequence of the complete porcine aminopeptidase N is 289...

Embodiment 2

[0087] Example 2 Screening of Vero cell lines stably expressing pAPN

[0088] 1 material

[0089] The pcDNA3.1(+)-puro-pAPN-flag-N plasmid constructed in Example 1, and the Vero cell line was purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences;

[0090] 2 test process

[0091] 1) Plasmid linearization

[0092] Use pvu I to digest pcDNA3.1(+)-puro-pAPN-flag-N to linearize the plasmid without affecting the expression of pAPN. The linearized plasmid obtained was ethanol-precipitated and then autoclaved with ddH 2 After O is dissolved, measure the concentration for later use. The result of plasmid linearization is as follows figure 2 Shown.

[0093] 2), liposome transfection

[0094] Use the linearized expression plasmid obtained in the previous step 3000 reagent transfection Vero cell line, please refer to Invitrogen for steps TM company's 3000 kit. After transfection, place at 37℃, 5% CO 2 Cultivate for 48h, and screen the clones ...

Embodiment 3

[0108] Example 3: PEDV challenge test to verify the proliferation effect of Vero cell line overexpressing pAPN on PEDV

[0109] 1 material

[0110] The Vero cell line was purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences; Vero-11 was the Vero cell line that was able to stably overexpress pAPN screened in Example 2; the PEDV vaccine virus was a gift from Jilin Zhengye Biochemical Technology Company;

[0111] 2 test process

[0112] The Vero blank control cells and the identified pAPN overexpression positive clonal cell line Vero-11, respectively, according to 1.5×10 6 The density of cells / well is plated in a 6cm plate. After 8 hours, the cell confluence rate is about 70% to 80%. The PEDV virus is inoculated into the corresponding cells at an MOI of 0.1. After 48 hours, the virus cells and RNA in the supernatant and inverted to cDNA. Use TRNzol A directly for RNA in cells + Reagent extraction. Extract RNA from the supernatant, pip...

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Abstract

The invention relates to the technical field of bioengineering, and particularly relates to an expression vector and a Vero cell line expressing pig aminopeptidase N. The cell line is deposited in CGMCC and has an accession number of CGMCC NO.12675. The cell line is liable to be infected by PEDV viruses, and has a function of promoting PEDV replication in host cells. Through PEDV culture utilizing the cell line, viruses having a higher virus copy number and a higher virus titer can be obtained. The cell line can be used for laboratory PEDV virus culture, pathogenesis researching, and the like and can be used for efficient production of PEDV vaccines in the industrialization.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to an expression vector and a Vero cell line expressing pig aminopeptidase N. Background technique [0002] Porcine epidemic diarrhea (PED) is a serious viral infectious disease caused by porcine epidemic diarrhea virus (PEDV). It can cause severe diarrhea, vomiting, and vomiting in lactating piglets. Symptoms such as dehydration, and the fatality rate is extremely high. The disease spreads very widely in Europe and Asia, and it causes serious economic losses to some countries and regions every year, and brings huge harm to the pig industry. Therefore, the prevention and treatment of epidemic diarrhea in pigs is a top priority. [0003] For most viral diarrhea diseases, the prevention and treatment measures generally choose vaccination. Although there are already a variety of vaccine products against porcine epidemic diarrhea virus on the market, the immune effect is not ideal. Th...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N7/00A61K39/12A61P31/14
CPCC12N15/85A61K39/00C12N7/00C12N9/485C12N2770/24351C12N2800/107C12Y304/11
Inventor 唐小春沈立欧阳红生逄大欣任林柱
Owner JILIN UNIV
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