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Yeast cDNA library of anti-SVV (Seneca valley virus) VHH ((variable domain of heavy chain of heavy 2chain) antibody as well as construction method and application of yeast cDNA library

A construction method and virus technology, applied in the biological field, can solve the problems of mixed infection, severe SVV prevention and control, and foot-and-mouth disease prevention and control interference, and achieve good diversity.

Inactive Publication Date: 2018-09-07
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the details of the SVV epidemic are still unknown, there are no commercial vaccines and diagnostic reagents available, SVV is still out of control, and the situation of SVV prevention and control is severe
And because the clinical symptoms are similar to foot-and-mouth disease, and can be mixed infection, it will seriously interfere with the prevention and control of foot-and-mouth disease

Method used

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  • Yeast cDNA library of anti-SVV (Seneca valley virus) VHH ((variable domain of heavy chain of heavy 2chain) antibody as well as construction method and application of yeast cDNA library
  • Yeast cDNA library of anti-SVV (Seneca valley virus) VHH ((variable domain of heavy chain of heavy 2chain) antibody as well as construction method and application of yeast cDNA library
  • Yeast cDNA library of anti-SVV (Seneca valley virus) VHH ((variable domain of heavy chain of heavy 2chain) antibody as well as construction method and application of yeast cDNA library

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Construction of Yeast cDNA Library of Anti-Seneca Valley Virus VHH Antibody

[0063] 1. Materials and methods

[0064] 1.1 Materials

[0065] 1.1.1 Test animals, antigen, adjuvant and Seneca Valley virus antibody detection kit

[0066] 1 male Bactrian camel (1 year old), 1 female Bactrian camel (6 months old); Seneca Valley virus inactivated vaccine, SVV antibody ELISA detection kit purchased from IDEXX.

[0067] 1.1.2 Strains and reagents

[0068] Yeast library-related vectors were purchased from Clontech, restriction endonucleases were purchased from NEB, camel peripheral blood lymphocyte isolation kit (product number: LTS1076) were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., Trizol, nickel affinity Chromatographic resin, DNA fragment recovery kit and plasmid extraction kit were OMEGA products, SMART cDNA library construction kit (product number: 634901) and PCR-related reagents were purchased from Treasure Biotech, and molecular biolog...

Embodiment 2

[0115] Use of the Yeast cDNA Library of Anti-Seneca Valley Virus VHH Antibody in Screening Anti-Seneca Valley Virus VHH Antibody

[0116] 1. Experimental materials and methods

[0117] 1.1 Amplification of the Seneca Valley VHH gene

[0118] use The Plasmid Midi Kit OMEGA Yeast Plasmid Extraction Kit extracted the yeast library plasmid in Example 1, and designed amplification primers according to the structural characteristics of VHH, and introduced EcoRI and XholI restriction sites at the 5′ ends of the upstream and downstream primers, respectively. The primer sequences are:

[0119] VF: 5'-CCGGAATTCATGGGTATCAACGCAGAG-3';

[0120] VR: 5’-CCCTCGAGCATGGAGACGGTGACC-3’, the amplification system is 50μl: 10×PCR Bμffer 5μl, dNTP 4μl, primer 1μl, Taq enzyme 1μl, ddH 2 O 39 μl for PCR amplification, the reaction conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 50 s, annealing at 57°C for 40 s, extension at 72°C for 1 min, a total of 30 cycles; extensi...

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Abstract

The invention discloses a yeast cDNA library of an anti-SVV (Seneca valley virus) VHH ((variable domain of heavy chain of heavy 2chain) antibody as well as a construction method and an application ofthe yeast cDNA library. Lymphocyte is obtained through separation in SVV inactivated vaccine immunized camel peripheral blood, a cDNA fragment of the anti-SVV VHH antibody is obtained with an RT-PCR (reverse transcription-polymerase chain reaction) method, the cDNA fragment and Pgadt7-Rec are used for co-transformation of yeast competent cells, and the cDNA library of the anti-SVV VHH antibody isobtained. Results show that the tilter of the library is about 7.2*10<6> cfu / ml, and the recombination rate of the library is 100%. Further preliminary functional identification on the library shows that the constructed yeast library has the anti-SVV VHH antibody. A platform is provided for screening of the anti-SVV VHH antibody, and a novel method is provided for early treatment and diagnosis ofSeneca valley.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a Bactrian camel VHH heavy chain antibody library against Seneca Valley virus. In addition, the present invention also relates to the construction method, library capacity identification and application of the antibody library. Background technique [0002] Seneca valley virus (SVV), also known as Seneca virus A (SVA), belongs to the genus Senecavirus of the Picornaviridae family (a different genus from the same family as foot-and-mouth disease virus), and is also a member of the genus sole member. The genome is single-stranded positive-sense RNA. Like other members of the picornaviridae family, SVV has a non-enveloped capsid approximately 25-30 nm in diameter with icosahedral symmetry. The SVV genome is approximately 7.2 kb in length and contains a unique open reading frame (ORF) flanked by a 5' untranslated region (MTR) (approximately 668 nucleotides) and a 3' untranslated region (...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/81C12N1/19C40B50/06C40B40/02C07K16/10
CPCC07K16/1009C07K2317/22C07K2317/569C12N15/1096C12N15/81C40B40/02C40B50/06C12Q2531/113C12Q2521/107
Inventor 郑海学田宏杨帆石正旺吴锦艳朱紫祥李丹张克山曹伟军刘永杰郭建宏何继军马旭升茹毅李林林刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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