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A kind of cell line for detecting hepatitis A virus titer and its construction method and application

A technology of hepatitis A virus and its construction method, which is applied in the field of cell lines and its construction for qualitative and quantitative detection of hepatitis A virus titer, which can solve complex and cumbersome operations, long time-consuming detection of hepatitis A virus infectivity titer, etc. Problems, to achieve good repeatability, shorten the time of marketing immunization, and ensure the quality of the effect

Active Publication Date: 2021-06-29
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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  • A kind of cell line for detecting hepatitis A virus titer and its construction method and application
  • A kind of cell line for detecting hepatitis A virus titer and its construction method and application
  • A kind of cell line for detecting hepatitis A virus titer and its construction method and application

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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), and after double digestion with BsrGI / MluI, it was inserted into the The 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 dNTP 4 RNase free ddH 2 o

6.5ul Total 13

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

Embodiment 2

[0116] Step (1), plasmid construction:

[0117] Using the total RNA in Huh7.0 cells as a template, using P2 and P3 as primers, RT-PCR was used to amplify the MAVS C-terminal gene sequence hMAVS (C396-540), and after double digestion with BsrGI / MluI, it was inserted into the The LV-mCherry-NLS-hMAVS(C396-540)-IRES-PURO-WPRE recombinant plasmid was constructed in the enzyme-treated lentiviral expression vector LV-mCherry-IRES-PURO-WPRE vector. After sequencing analysis, the sequence was correct and the Store at -20°C for later use. The structure of the constructed mcherry-NLS-hMAVS(C396-540)-IRES-PURO-WPRE is shown in Figure 7 .

[0118] 1. Synthesize 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 dNTP 4 RNase free ddH 2 o

6.5ul Total 13

[0122] 65°C for 5 minutes, followed by a rapid ...

Embodiment 3

[0153] Step (1), plasmid construction:

[0154] The total RNA of the tree shrew liver was used as a template, and P4 and P5 were used as primers to amplify the MAVS C-terminal gene sequence MAVS (C365-503) by RT-PCR. After double digestion with BsrGI / MluI, insert it into the LV-EGFP-tMAVS(C365–503)-IRES-PURO-WPRE recombinant plasmid was constructed in the lentiviral expression vector LV-EGFP-IRES-PURO-WPRE vector. After sequencing analysis, the sequence was correct and it was stored at -20 ℃ for later use. 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. Synthesize 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 dNTP 4 RNase free ddH 2 o

6.5ul Total 13

[0160] 65°C for 5 minutes, follo...

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Abstract

The invention relates to a cell line for detecting the titer of hepatitis A virus, its construction method and application. The invention utilizes the property that 3ABC protease can interact with MAVS, constructs a fusion protein between the C-terminal of MAVS and fluorescent protein, and packages it into lentiviral particles. After infecting cells, the expressed fusion protein will be anchored on the mitochondria in the cytoplasm to form Dot-like distribution, under the selection of puromycin, a stable cell line that stably expresses the fusion protein can be obtained. When the cell line is infected by HAV, the fluorescent protein will diffuse throughout the cell or localize to the nucleus. In the presence of methylcellulose , fluorescent plaques can be observed, and the infectious titer of HAV can be detected by counting the number of plaques. Compared with the traditional method, the method of the present invention only needs 1-4 days to detect the HAV titer, significantly shortens the virus detection cycle, and is simple to operate and has high sensitivity, can directly observe the infection process of HAV, and is easy to popularize and apply.

Description

technical field [0001] The invention belongs to the technical field of medical biological detection, and in particular relates to a cell line for qualitative and quantitative detection of hepatitis A virus titer, its construction method and application. 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 causing acute hepatitis and some acute liver failure. According to statistics, there are approximately 1.4 million hepatitis A virus infections worldwide each year. Due to the successive use of live attenuated hepatitis A vaccine and inactivated vaccine, the transmission of hepatitis A virus has been effectively controlled at present. [0003] Hepatitis A virus is a positive-sense RNA virus belonging to the family Picornaviridae and the genus Hepavirus. HAV will not produce cytopathic effect after infecting cells, which brings cert...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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