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Cell line used for detecting hepatitis A virus titer as well as construction method and application thereof

A hepatitis A virus and construction method technology, applied in the field of qualitative and quantitative detection of hepatitis A virus titer of cell lines and their construction, can solve complicated and tedious operations, long time-consuming detection of hepatitis A virus infectivity titer, etc. question

Active Publication Date: 2018-04-06
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of long time-consuming detection of hepatitis A virus infectivity titer, complex and cumbersome operation, etc., the purpose of the present invention is to provide a fast, simple and accurate cell line for qualitative and quantitative detection of hepatitis A virus titer and Its construction method and application

Method used

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  • Cell line used for detecting hepatitis A virus titer as well as construction method and application thereof
  • Cell line used for detecting hepatitis A virus titer as well as construction method and application thereof
  • Cell line used for detecting hepatitis A virus titer as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Step (1), plasmid construction:

[0077] Using the total RNA in Huh7.0 cells as a template, using P1 and P2 as primers, RT-PCR was used to amplify the MAVS C-terminal gene sequence MAVS (C396-540), after BsrGI / MluI double digestion, insert it into the same LV-EGFP-MAVS(C396–540)-IRES-PURO-WPRE recombinant plasmid was constructed in the enzyme-treated lentiviral expression vector LV-EGFP-IRES-PURO-WPRE vector. After sequencing analysis, the sequence was correct, and it was stored in Standby at -20°C.

[0078] The specific method is as follows:

[0079] PCR amplification system and amplification program

[0080] 1. Synthesize cDNA

[0081] Use takara's PrimeScriptII 1st Strand cDNA synthesis Kit to synthesize cDNA according to the instructions:

[0082] system:

[0083]

Volume (ul)

Huh7.0 RNA

2

Primer P2

0.5

dNTPs

4

RNase free ddH 2 O

6.5ul

Total

13

[0084] 65°C for 5 minutes, followed by a rapid...

Embodiment 2

[0116] Step (1), plasmid construction:

[0117] The total RNA in Huh7.0 cells was used as the template, and P2 and P3 were used as primers to amplify the MAVS C-terminal gene sequence hMAVS (C396-540) by RT-PCR. The LV-mCherry-NLS-hMAVS(C396-540)-IRES-PURO-WPRE recombinant plasmid was constructed from the enzyme-treated lentiviral expression vector LV-mCherry-IRES-PURO-WPRE vector. Store at -20°C for later use. See the structure of the constructed mcherry-NLS-hMAVS(C396-540)-IRES-PURO-WPRE Figure 7 .

[0118] 1. Synthesis of cDNA

[0119] Use takara's PrimeScriptII 1st Strand cDNA synthesis Kit to synthesize cDNA according to the instructions:

[0120] system:

[0121]

Volume (ul)

Huh7.0 RNA

2

Primer P2

0.5

dNTPs

4

RNase free ddH 2 o

6.5ul

Total

13

[0122] 65°C for 5 min, followed by a quick ice bath for 1 min.

[0123] To the above reaction system, add 5*FS buffer: 4ul, DTT: 1ul, RNase inhibitor: 1...

Embodiment 3

[0153] Step (1), plasmid construction:

[0154] Taking the total RNA of tree shrew liver as the template, using P4 and P5 as primers, RT-PCR was used to amplify the MAVS C-terminal gene sequence MAVS (C365-503), after double digestion with BsrGI / MluI, insert into the same enzyme treatment. The LV-EGFP-tMAVS(C365–503)-IRES-PURO-WPRE recombinant plasmid was constructed from the Lentiviral expression vector LV-EGFP-IRES-PURO-WPRE vector, and the sequence was correct after sequencing analysis. ℃ reserve. The structure of the constructed EGFP-tMAVS(C365-503)-IRES-PURO-WPRE is shown in Figure 11 .

[0155] The specific method is as follows:

[0156] 1. Synthesis of cDNA

[0157] Use takara's PrimeScriptII 1st Strand cDNA synthesis Kit to synthesize cDNA according to the instructions:

[0158] system:

[0159]

Volume (ul)

Huh7.0 RNA

2

Primer P2

0.5

dNTPs

4

RNase free ddH 2 o

6.5ul

Total

13

[0160] 65°C for...

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Abstract

The invention relates to a cell line used for detecting hepatitis A virus titer as well as a construction method and an application thereof. According to the invention, by utilizing the characteristicthat 3ABC protease can interact with MAVS, MAVS C end and fluorescent protein are utilized to construct fusion protein which is packaged into lentivirus particles; after cells are infected, the expressed fusion protein is anchored to mitochondrion in cytoplasm to form dotted distribution; under the screening of puromycin, a stable cell line capable of stably expressing fusion protein can be obtained; after such a cell line is infected with HAV, fluorescent protein disperses in the whole cell or is positioned into a cell nucleus; and in the presence of methyl cellulose, fluorescent bacteriophage plaques can be observed, and the infectious titer of HAV can be detected by calculating the quantity of bacteriophage plaques. Compared with conventional methods, according to the method disclosedby the invention, only 1-4 days are needed for detecting HAV titer, and therefore, the virus detection cycle is remarkably shortened, the operation is simple, the sensitivity is high, infection courseof HAV can be intuitively observed, and popularization and application are easy.

Description

technical field [0001] The invention belongs to the technical field of medical biology detection, and in particular relates to a cell line for qualitative and quantitative detection of hepatitis A virus titer, a construction method and application thereof. Background technique [0002] Hepatitis A virus (HAV) infection is considered to be a very important public health problem. In developing or developed countries, HAV is one of the main pathogens that cause acute hepatitis and some acute liver failure. According to statistics, there are approximately 1.4 million cases of hepatitis A virus infection worldwide each year. Due to the successive use of live attenuated hepatitis A vaccine and inactivated vaccine, the spread of hepatitis A virus has been effectively controlled at present. [0003] Hepatitis A virus is a positive-strand RNA virus belonging to the genus Hepatovirus of the Picornaviridae family. HAV infection of cells does not produce cytopathic effects, which bri...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/65C12Q1/70C12R1/91
CPCC07K14/4703C12N15/65C12N15/86C12N2740/15043C12Q1/06
Inventor 寸韡毕研伟龙琼肖红剑李育中
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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