Construction of attenuated African swine fever virus strain without twelve genes and application of attenuated African swine fever virus strain as vaccine

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an attenuated African swine fever virus strain without twelve genes and application of the attenuated African swine fever virus strain as a vaccine. According to the attenuated African swine fever vaccine strain, G-ACD-00190, MGF 110-9L, G-ACD-00210, MGF 360 (9L, 10L, 11L, 12L, 13L and 14L) and MGF 505 (1R, 2R and 3R) genes are deleted in an ASFV CN / GS 2018 virus in a combined manner, so that the toxicity of a parent strain is reduced, and the attenuated African swine fever vaccine strain is obtained; after the attenuated African swine fever vaccine strain is used for immunizing pigs, the pigs are completely attenuated, the immunized pigs are healthy, viruses cannot be detected in the blood of the pigs after immunization, 100% immune protection can be provided for attack of ASFVCN / GS / 2018 virulent strains, and the attenuated African swine fever 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 an attenuated African swine fever strain Δ20 / M9 lacking twelve genes 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 report...

AI Technical Summary

Problems solved by technology

However, in the prior art, the virulence gene knockout vaccine of classical swine fever virus also considers the following factors: (1) whether the knockout virulence gene reduces virus transmission, detoxification, and whether it causes tissue damage and affects production performance is a factor of biological safety. An important indicator, which is also an important indicator of the continuous improvement of the safety of gene deletion vaccines; (2) Different strains have different effects of deleting the same gene, and insufficient deletion has residual virulence and pathogenicity; and viruses caused by multiple gene deletions Low titer will also reduce the risk of immunogenicity or protective effect of the attenuated strain; (3) Although the whole genome sequencing of African swine fever has been completed, there are more than 160 regulatory genes and structural genes that make up the African swine fever virus One, although the project is huge, the function of regulatory genes and structural genes is crucial to its pathogenesis and vaccine development; (4) The selection of joint knockout virulence genes is very important, and different selections will produce different or even opposite immune protection

[0005] Therefore, although the knockout of virulence genes is a strategy for constructing attenuated strains of African swine fever, knocking out the same gene in different viruses will also lead to significant differences in effect, and the virulence genes in the process of joint knockout Selection is also crucial, and not all virulence genes combined knockout will achieve the effect of attenuation protection

For example, 1. the shoulder protection effect is different after knocking out the NL gene (i.e. DP71L gene) in different types of African swine fever virus, wherein, the virulence of the African swine fever virus strain obtained by knocking out the E70NL gene disappears completely; The NL gene was knocked out in Pr4, and the strain Pr4-ΔN-SL was obtained at 10 2 TCID 50 Under the dose, although the death and onset time of pigs were delayed, the lethality rate was close to 86%, and the virus strain Mal-ΔNL obtained by knocking out the NL gene in Mal still maintained a very high virulence, and the virus strain Mal-ΔNL still maintained a very high virulence at 10 2 TCID 50 The lethal rate under the dosage is 100%; the Benin 97 / 1 strain with DP148R gene deletion can also make the virus fully attenuated, and the survival rate after immunizing pigs is also 100%, but the survival rate after the DP148R gene deletion of the Chinese epidemic strain immune strain is only 0%; ②Although it is disclosed that the joint knockout of MGF 505 / 530 family genes and MGF360 family genes can produce certain attenuation protection, but the joint knockout of MGF360, MG F 505 / 530 and B119L does not have attenuation protection However, joint deletion of several members of MGF360 / 505 in E70△NL (E70 strain with NL gene knockout) will lead to enhanced toxicity of the attenuated strain (E70△NL); Combined knockout of CD2v and MGF-360-505 family genes, such as combined knockout of CD2v and MGF-360-12L, MGF-360-13L, MGF-360-14L, deletion of all MGF-360-505R genes, and CD2v and The combined deletion of all genes of MGF-360-505R; however, although the obtained attenuated strain will provide a certain immune protection effect, after the pig is immunized, the virus can still be detected in the pig serum, and the safety still has problems; Moreover, the inventor found that the joint deletion of MGF505-7R, MGF110-9L, DP71L and DP148R genes, the recombinant virus constructed had poor immune protection effect, and the protection rate was only 50%.

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