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Yeast cDNA library of hog-cholera-virus-resisting VHH antibody as well as construction method and applications of yeast cDNA library

A technology of swine fever virus and antibody, applied in the biological field, can solve the problems such as the decline of vaccine prevention effect

Inactive Publication Date: 2017-10-31
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, since the 1980s, atypical swine fever (or chronic swine fever) with atypical clinical symptoms and a longer course of disease has become the main form of the disease. The prevention and control of swine fever has encountered new difficulties, and it is necessary to strive for a new research technology or method for the prevention and control of swine fever

Method used

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  • Yeast cDNA library of hog-cholera-virus-resisting VHH antibody as well as construction method and applications of yeast cDNA library
  • Yeast cDNA library of hog-cholera-virus-resisting VHH antibody as well as construction method and applications of yeast cDNA library
  • Yeast cDNA library of hog-cholera-virus-resisting VHH antibody as well as construction method and applications of yeast cDNA library

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The construction of the yeast cDNA library of embodiment 1 anti-swine fever virus VHH antibody

[0068] 1. Materials and methods

[0069] 1.1 Materials

[0070] 1.1.1 Test animals, antigens, adjuvants and swine fever virus antibody detection kits

[0071] 1 male Bactrian camel (1 year old), 1 female Bactrian camel (6 months old); CSFV strain C attenuated vaccine, CSFV antibody ELISA detection kit purchased from IDEXX.

[0072] 1.1.2 Strains and reagents

[0073] 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 mo...

Embodiment 2

[0119] Example 2 Application of the Yeast cDNA Library of Anti-CSFV VHH Antibody in Screening Anti-CSFV VHH Antibody

[0120] 1. Experimental materials and methods

[0121] 1.1 Amplification of classical swine fever VHH gene

[0122] 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, and the upstream primer VF : 5'-CCGGAATTCATGGGTATCAACGCAGAG-3'; VR: 5'-CCCTCGAGCATGGAGACGGTGACC-3', the amplification system is 50ul: 10×PCR Buffer 5ul, dNTP 4ul, primer 1ul, Taq enzyme 1ul, ddH2O 39ul, PCR amplification, 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, and a total of 30 cycles; extension at 72°C for 10 min, and 5 min at 4°C. PCR pro...

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Abstract

The invention discloses a yeast cDNA library of a hog-cholera-virus-resisting VHH antibody as well as a construction method and applications of the yeast cDNA library. According to the yeast cDNA library, the construction method and the applications, the peripheral blood of a camel which is immunized by a hog cholera C strain attenuated vaccine is separated, then lymphocytes are obtained, the total mRNA of the lymphocytes obtained through separation is extracted, cDNA fragments of the hog-cholera-virus-resisting VHH antibody are obtained through an RT-PCR method, the cDNA fragments of the VHH antibody and pGADT7-Rec co-transform yeast competent cells, and thus the yeast cDNA library of the hog-cholera-virus-resisting VHH antibody is obtained. The capacity and polymorphism of the constructed yeast cDNA library are identified, the result shows that the titer of the library is about 5.5x10<6>cfu / ml, the diversity is good, and the recombination rate of the library is 100%. Preliminary functional identification is carried out on the yeast cDNA library of the hog-cholera-virus-resisting VHH antibody by adopting a blood coagulation inhibition test, and the result shows that the hog-cholera-virus-resisting VHH antibody with the neutralizing activity exists in the constructed yeast library. The proposal of the technical scheme provides a platform for screening the hog-cholera-virus-resisting VHH antibody, and a novel technical means is provided for early treatment and diagnosis of hog cholera.

Description

technical field [0001] The invention relates to a Bactrian camel VHH heavy chain antibody library against classical swine fever virus. In addition, the present invention also relates to the construction method, library capacity identification and application of the antibody library. The invention belongs to the field of biotechnology. Background technique [0002] Classical Swine Fever (CSF) is a severe infectious disease of pigs caused by Classical Swine Fever Virus (CSFV). CSFV is an enveloped single-stranded positive-sense RNA virus belonging to the family Flaviridae and the genus pestivirus. Swine fever is one of the most important infectious diseases that harm pigs. The disease exists in more than 40 countries around the world, causing huge economic losses to the pig industry. Immunization prevention is the main technical means adopted by most countries with severe swine fever, and the vaccine immunity rate in my country is as high as 95%. So far, the successful swi...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/81C40B50/06C40B40/02C07K16/10
CPCC07K16/1081C07K2317/569C12N15/1096C12N15/81C40B40/02C40B50/06
Inventor 田宏吴锦艳林彤陈妍尚佑军张克山郑海学王光祥刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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