Construction of gene-deleted attenuated African swine fever virus strain and application of gene-deleted attenuated African swine fever virus strain as vaccine

An African swine fever virus and gene deletion technology, which is applied in the construction and application fields of vaccines, can solve the problems of weakening the virus, and the attenuated strain cannot provide comprehensive protection for animals, and achieves the effect of good safety performance.

Active Publication Date: 2021-05-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Preliminary experiments found that although 20 or 40 single knockouts reduced toxicity, the constructed attenuated strains could not provide comprehensive protection for animals
Moreover, the literature (Afonso CL, Zsak L, Carrillo C C, et al.African swine fevervirus NL gene is not required for virus virulence.Afonso, etc.) discloses that after knocking out the NL gene (i.e. DP71L gene) of the European isolate E70, The virulence of gained ASFV completely disappears; Patent CN110093324A discloses that in Benin 97 / 1 strain, the deletion of DP148R gene makes the virus sufficiently weakened, and the survival rate after immunizing pigs is also 100%; but the literature (Deletion of virulence associated genes from attenuated African swine fevervirus isolate OUR T88 / 3decreases its ability to protect against challengewith virulent virus, Abrams, etc.) disclosed the recombinant virus OUR T88 / 3△DP2 produced by knocking out the two genes of DP71L and DP96R in the natural strain of African swine fever OUR T88 / 3 The immune protection is only 66%. It can be seen that although the above two genes can be knocked out alone to achieve a better immune protection effect, but the combined knockout does not necessarily achieve a better protective effect

Method used

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  • Construction of gene-deleted attenuated African swine fever virus strain and application of gene-deleted attenuated African swine fever virus strain as vaccine
  • Construction of gene-deleted attenuated African swine fever virus strain and application of gene-deleted attenuated African swine fever virus strain as vaccine
  • Construction of gene-deleted attenuated African swine fever virus strain and application of gene-deleted attenuated African swine fever virus strain as vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Construction and purification identification of embodiment 1 attenuated African swine fever virus strain

[0068] 1. Screening expression cassette construction

[0069] To facilitate screening, an expression cassette for the selectable marker gene was constructed.

[0070] Enhanced green fluorescent protein (Enhance Green fluorescent protein, eGFP) gene screening expression cassette construction: refer to literature (O'Donnell V.African Swine Fever Virus Georgia 2007with aDeletion of Virulence-Associated Gene 9GL(B119L), when Administered at LowDoses, Leads to Virus Attenuation in Swine and Induces an EffectiveProtection against Homologous Challenge. JVirol.2015; 89(16):8556-66), amplified the p72 promoter by PCR (from -196nt upstream of the p72 gene to the sequence before +17) , standby; amplification primers are: forward primer 5'-TTATAAAACATATGTTCATAAAAAGGGTCGCCGGAGGAAAAGTC-3' (shown in SEQ ID NO.3) and reverse primer 5'-CTCCTCGCCCTTGTCCACCATATAATGTTATAAAAATAATT-3' ...

Embodiment 2

[0083] The mensuration of embodiment 2 virus titer

[0084] The titer of African swine fever virus adopts half hematosorbate amount (50% haemadsorption, HAD 50 ) means that HAD 50 For the specific operation of the experiment, please refer to the literature (BorcaMV.Development of a highly effective Africanswine fever vir us vaccine by deletion of the I177L gene results in sterile immunity against the current epi demic Eurasia strain.JVirol.2020.pii:JVI.02017-19). It is adjusted appropriately: according to approximately 1x10 in a 96-well plate 5 Cells per well were inoculated with primary PAM cells, and the recombinant virus to be tested was serially diluted 10 times, a total of 7 dilutions, each dilution was 8 wells, and the diluted virus was added to the PAM of a 96-well plate at 100 μL / well, and then added Red blood cells, a total of three replicates. Virus infection can be judged according to the rosette formed by the aggregation of red blood cells around the infected ce...

Embodiment 3

[0085] Virulence Evaluation of Example 3 Gene Deletion Attenuated African Swine Fever Virus Strain

[0086] In order to detect the virulence of the gene deletion attenuated African swine fever virus strain, this experiment used 10 4 HAD 50 The toxicity of the dose was evaluated by intramuscular injection in piglets.

[0087] In this experiment, 17 healthy Landrace piglets negative for African swine fever antigen antibody were divided into 3 groups, including 6 pigs in the gene deletion attenuated African swine fever virus strain Δ20 / 40 group, and 6 pigs in the ASFVCN / GS / 2018 isolate group. Gene deletion attenuated African swine fever virus strain Δ20 / 40 cohabitation group with 5 animals. Measure body temperature changes every day, collect peripheral blood and saliva, refer to literature (KingDP.Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus.J Virol Methods 107:53-61), and use fluorescence quantitative PCR ...

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Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to construction of a gene-deleted attenuated African swine fever virus strain [delta]20/40 and application of the gene-deleted attenuated African swine fever virus strain delta[20/40] as a vaccine. The functions of an MGF-110-9L gene and an MGF-360-9L gene encoding protein are jointly deleted in the African swine fever parent strain ASFVCN/GS/2018 by adopting a genetic engineering means, so that the toxicity of the parent strain is reduced, and the attenuated African swine fever candidate vaccine strain with high safety is obtained; and the attenuated African swine fever candidate vaccine strain is completely attenuated after immunizing a pig, the immunized pig is healthy, 100% immune protection can be provided for attacking (100HAD50 dosage) of ASFV CN/GS/2018 virulent strain, and the attenuated African swine fever candidate vaccine strain can be used as a safe and effective candidate vaccine for preventing and controlling the African swine fever epidemic situation and has great social value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the construction of a gene-deleted attenuated African swine fever virus strain and its application as a vaccine. Background technique [0002] African swine fever (African swine fever, ASF) is caused by the infection of African swine fever virus (ASFV), and is characterized by fever and hemorrhage in pigs. The fatality rate for domestic pigs is as high as 100%. . The disease first broke out in Kenya in 1921 and subsequently became widespread among domestic and wild pigs throughout Africa. It was introduced into Europe in the 1950s, and it took 40 years to eliminate the disease throughout Europe. However, the disease was introduced to Georgia from East Africa again in 2007, and then spread widely in Eastern Europe, and in 2017, it was introduced to Irkutsk in the Russian Far East. In early August 2019, researcher Hu Rongliang took the lead in reporting the fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/90C12N15/34C12N15/85C12N15/65A61K39/12A61P31/20C12R1/93
CPCC12N7/00C12N15/902C12N15/85C12N15/65C07K14/005A61K39/12A61P31/20C12N2710/12021C12N2710/12022C12N2710/12034A61K2039/5252A61K2039/552Y02A50/30
Inventor 郑海学李丹冯涛杨文萍党文茹毅刘华南刘永杰田宏齐晓兰张克山杨帆孙妍刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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