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Hybridoma cell strain, getah virus distinguishing monoclonal antibody secreted from hybridoma cell strain, and application

A hybridoma cell line and monoclonal antibody technology, which is applied in the field of bioengineering, can solve the problems of preparation and application of GETV monoclonal antibody not seen in Gaita virus, and achieve the effects of wide application space, strong specificity and high sensitivity

Active Publication Date: 2019-11-22
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no getavirus has been constructed yet, and there are no relevant reports on the preparation and application of GETV monoclonal antibodies

Method used

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  • Hybridoma cell strain, getah virus distinguishing monoclonal antibody secreted from hybridoma cell strain, and application
  • Hybridoma cell strain, getah virus distinguishing monoclonal antibody secreted from hybridoma cell strain, and application
  • Hybridoma cell strain, getah virus distinguishing monoclonal antibody secreted from hybridoma cell strain, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The preparation method of the anti-Gaita virus monoclonal antibody of the present invention comprises the following steps:

[0034] 1. Construction of E2 protein recombinant expression vector

[0035] Gatavirus RNA was extracted and reverse transcribed into cDNA. For specific operation steps, see the RNA extraction manual in the Trizol Invitrogen kit;

[0036] Take 300 μL of the virus solution and extract total virus RNA according to the instructions of TRIZOL RNA Extraction Reagent.

[0037] Reverse transcription reaction system (20 μL): RNA template 13 μL, 5×buffer 4 μL, dNTP (10 mmol / μL) 1 μL, random primer Random (20 pmol / μL) 1 μL, RNase inhibitor (40 U / μL) 0.5 μL, reverse Recorder M-MLV (200U / μL) 0.5 μL; reaction parameters: 42°C for 1h, 95°C for 5min, the cDNA product was used for PCR amplification.

[0038] Referring to the HNJZ-S1 sequence, primers were designed for the E2 protein to amplify the sequence from position 1 to position 304 by PCR (GenBank accessi...

Embodiment 2

[0097] In this example, the specificity and titer of the monoclonal antibody prepared in Example 1 were tested.

[0098] 1. Western blot experiment

[0099] (1) Get the Marc-145 cells infected by Getavirus, and then lyse them with RIPA lysate. After lysis, centrifuge at 12000g / min for 20min at 4°C; collect the supernatant, and detect the protein concentration of the supernatant with a BCA kit

[0100] (2) Add the supernatant of the cell lysate obtained in step (1) into 12% PAGE, electrophoresis at 120V for 1h; then place the PAGE gel on a PVDF membrane, and transfer to the membrane at 40mA for 90min;

[0101] (3) Put the PVDF membrane of step (2) into 5% skimmed milk, block overnight at 4°C, and then use the K3A6B6 monoclonal antibody of the present invention with a volume ratio of 1:2000 of skimmed milk and monoclonal antibody as the primary antibody , 37°C for 1h;

[0102] (4) Take out the PVDF membrane after step (3), wash it three times with PBST buffer, each time for 3...

Embodiment 3

[0119] 1, E2 protein monoclonal antibody blocking ELISA method

[0120] (1) Purification of Mouse Ascites IgG

[0121] Ascites prepared from the K3A6B6 hybridoma cell line with high titer and specific secretion of anti-Gatavirus monoclonal antibody was taken, purified by Protein G, and the protein content was determined after purification;

[0122] (2) Horseradish peroxidase (HRP) labeled IgG

[0123] The purified ascites IgG was coupled to HRP by simple sodium periodate method;

[0124] (3) Blocking ELISA method

[0125] Take the purified His-E2 fusion protein, dilute it to 5.0 μg / mL (the concentration can also be 2.5 μg / mL, 1.25 μg / mL, etc.) with carbonate buffer solution of pH 9.6, and coat the 96-well ELISA plate , and then incubated at 37°C for 2h;

[0126] After incubation, wash with PBST buffer solution with pH=7.4 for 3 times, then add blocking solution 1% BSA (blocking solution can also be 5% skimmed milk, 1% OVA, 2% gelatin, etc.) After blocking for 1 hour, wash...

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Abstract

The invention discloses a hybridoma cell strain, a getah virus distinguishing monoclonal antibody secreted from the hybridoma cell strain, and an application. The hybridoma cell strain is a K3A6B6 hybridoma cell strain secreting the monoclonal antibody for distinguishing getah virus E2 protein. The monoclonal antibody secreted from the hybridoma cell strain can be specially bound with target antigen in high titer, and the monoclonal antibody is prepared into a detection clinical sample for detecting a reagent kit. Specificity is high, sensitivity is high, and stability is good.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. The invention relates to a hybridoma cell line, anti-Gaitavirus monoclonal antibody secreted by the hybridoma cell line and its application. Background technique [0002] Getah virus (GETV) is a member of the genus Alphavirus in the family Togaviridae. The size of the virus particle is about 70nm, spherical structure, with capsule and fiber, the virus genome is a single-stranded positive-strand RNA, and the genome has two open reading frames (ORF), which encode two polyproteins, and are finally cut into 4 There are three nonstructural proteins (NSP1, NSP2, NSP3 and NSP4) and five structural proteins (C, E3, E2, 6K and E1). [0003] In 1955, Scherer et al first isolated the virus from Culex spp.mosquitoes in Malaysia. Serological test results show that GETV is widely distributed in Europe, Asia and Oceania, and GETV antibodies have been detected in various animals such as pigs, horses, c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/569G01N33/577
CPCC07K16/1081G01N33/56983G01N33/577C07K2317/35G01N2333/18
Inventor 王川庆周峰陈陆常洪涛王新卫杨霞刘红英王傲杰王新港
Owner HENAN AGRICULTURAL UNIVERSITY
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