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ELISPOT detection methods for screening T cell antigen polypeptides and antigen epitopes of African swine fever virus

A technology of antigenic epitopes and dominant antigens, applied in the direction of viral antigenic components, viral peptides, viruses, etc., can solve the problem of no effective vaccine

Active Publication Date: 2020-04-10
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no effective vaccine for ASFV, and there is an urgent need to develop an effective vaccine against the strains circulating in China

Method used

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  • ELISPOT detection methods for screening T cell antigen polypeptides and antigen epitopes of African swine fever virus
  • ELISPOT detection methods for screening T cell antigen polypeptides and antigen epitopes of African swine fever virus
  • ELISPOT detection methods for screening T cell antigen polypeptides and antigen epitopes of African swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Screening Cellular Immunity Dominant Antigen Polypeptides

[0047] 1. Peptide library synthesis

[0048] Referring to the amino acid sequences of the p30, p54, p72 and CD2v genes of ASFV Chinese popular strain ASFV / CN / HLJ / 18 (GenBank entry number: MK333180.1) (Zhao DM, Liu RQ, Zhang XF, Li F, Wang JF, ZhangJW, Liu X, Wang LL, Zhang JE, Wu XZ, Guan YT, Chen WY, Wang XJ, H XJ, BuZG. Replication and virulence in pigs of the first African swine fever virus isolated in China. EMERG MICROBES INFEC8:1,438-447 ), synthesized by GenScript Biotechnology Co., Ltd., each polypeptide contains 15 amino acids, and adjacent polypeptides contain 10 amino acid overlapping sequences. p30 protein synthesizes 37 polypeptides, which are named p30-1 to p30-37; p54 protein synthesizes 35 polypeptides, which are named p54-1 to p54-35; p72 protein synthesizes 127 polypeptides, which are named p72-1 to p72 -127; CD2v protein synthesizes 70 polypeptides, named CD2v-1 to CD2v-70. Simul...

Embodiment 2

[0072] Example 2 ASFV Attenuated Candidate Vaccine Strain rASFVΔCD2V / 360-eGFP-mCherry Immune Challenge Test

[0073] Nine 7-week-old field pigs were randomly divided into two groups, five attenuated ASFV candidate vaccine strain (rASFVΔCD2V / 360-eGFP-mCherry) (patent application number: 201910342578.8) low-dose immunization group, ear numbers were 813, 814, 815, 816, 817; 5 rats in the high-dose immunization group, with ear numbers 819, 821, 824, and 831, respectively. Neck intramuscular injection immunization, 1ml / head, low dose group immunization dose is 10 3 TCID 50 , the immunization dose of the high-dose group was 10 5 TCID 50 . Immunization was boosted in the same way 3 weeks after the first immunization. 2 weeks after the second immunization, 10 2.5 HAD Chinese epidemic strain (ASFV / CN / HLJ / 18) challenge. At the same time, set up the same group as the control group, with one animal in each group. The ear number of the sentinel pigs in the low-dose group was 765, an...

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Abstract

According to the invention, the cellular immune antigen dominant epitopes of four important structural proteins p30, p54, p72 and CD2v of ASFV are screened, and the corresponding enzyme-linked immunospot test methods are established. The screened polypeptides have high amino acid sequence conservation, can effectively induce ASFV specific cellular immune response, and can be used as candidate immunogens for ASFV epitope carrier vaccines. The T cell epitope polypeptides of ASFV structural proteins obtained by the invention are screened, and the corresponding ELISPOT detection methods are established. The invention provides a powerful tool for efficacy evaluation of the ASFV candidate vaccines, and also lays a foundation for the research and development of ASFV polypeptide carrier vaccines.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to the prevention and treatment of animal diseases, and more specifically to the dominant epitope of the immune antigen of African swine fever virus. Background technique [0002] African swine fever (ASF) is an acute, febrile and highly contagious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV) (Gomez-Villamandos JC, Bautista MJ, Sanchez-Cordon PJ, Carrasco L. 2013. Pathology of African swine fever: the role of monocyte-macrophage. Virus Res 173:140-149.). Its clinical symptoms range from acute, subacute to chronic, and its main features are high fever, cyanosis of the skin, extensive hemorrhage of internal organs, respiratory disorder and neurological symptoms, and the morbidity and mortality are almost 100%. Since it was first discovered in Kenya in 1921 (Kleiboeker SB, Scoles GA, Burrage TG, Sur J.1999.African swine fever virus replication i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01A61K39/12A61P31/20G01N33/68G01N33/577C12Q1/02
CPCC07K14/005A61K39/12A61P31/20G01N33/6866G01N33/577G01N33/5038C12N2710/12022C12N2710/12034G01N2333/01G01N2333/57A61K2039/5254A61K2039/552C12N2503/02G01N2500/10
Inventor 步志高刘任强温志远陈伟业山丹王翀柳金雄赵东明何希君
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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