阅读说明：本技术 非洲猪瘟病毒四基因缺失弱毒株及其应用 (African swine fever virus four-gene deletion low virulent strain and application thereof ) 是由 陈鸿军 朱鸿飞 扈荣良 钱莺娟 郭晓宇 李金祥 孙怀昌 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种非洲猪瘟病毒四基因缺失弱毒株,该弱毒株是非洲猪瘟病毒SY18分离株的四基因缺失弱毒株,其缺失以下基因的功能蛋白：CD2v基因编码产物和三个多基因家族基因(MGF360-12L、MGF360-13L、MGF360-14L)编码产物。本发明还公开了上述非洲猪瘟病毒弱毒株在制备预防或治疗非洲猪瘟的疫苗中的应用。本发明的非洲猪瘟病毒弱毒株,能对ASFV亲本毒株的攻击提供完全的免疫保护作用,且安全性高,适于作为预防非洲猪瘟的疫苗候选株。(The invention discloses an African swine fever virus four-gene deletion low virulent strain, which is a four-gene deletion low virulent strain of an SY18 isolate strain of the African swine fever virus, and functional proteins of the following genes are deleted: the CD2v gene encodes a product and three polygene family genes (MGF360-12L, MGF360-13L, MGF360-14L) encode a product. The invention also discloses application of the African swine fever virus low virulent strain in preparation of a vaccine for preventing or treating African swine fever. The African swine fever virus low virulent strain can provide complete immune protection effect on the attack of ASFV parent virulent strain, has high safety, and is suitable for serving as a vaccine candidate strain for preventing African swine fever.)

1. A four-gene-deleted low virulent strain of African swine fever virus is a gene-deleted low virulent strain of a SY18 isolate of the African swine fever virus, and the four-gene-deleted low virulent strain is deleted with the following functional genes:

CD2v gene and three polygene family genes MGF360-12L, MGF360-13L, MGF 360-14L.

2. The African swine fever virus four-gene-deleted attenuated strain of claim 1, wherein the deleted gene sequence comprises: a nucleotide sequence for coding the amino acid sequence of the CD2v protein shown in SEQ ID NO.1, a nucleotide sequence for coding the amino acid sequence of the MGF360-12L protein shown in SEQ ID NO.2, a nucleotide sequence for coding the amino acid sequence of the MGF360-13L protein shown in SEQ ID NO.3 and a nucleotide sequence for coding the amino acid sequence of the MGF360-14L protein shown in SEQ ID NO. 4.

3. The African swine fever virus four-gene-deleted attenuated strain of claim 2, wherein the deleted gene sequence comprises: the nucleotide sequence of the CD2v gene shown in SEQ ID NO.5, the nucleotide sequence of the MGF360-12L gene shown in SEQ ID NO.6, the nucleotide sequence of the MGF360-13L gene shown in SEQ ID NO.7 and the nucleotide sequence of the MGF360-14L gene shown in SEQ ID NO. 8.

4. A recombinant virus is characterized in that the recombinant virus is a recombinant virus with a CD2v gene deleted in a SY18 isolate of African swine fever virus.

5. The recombinant virus of claim 4, wherein the recombinant virus further lacks the following three polygene family genes: MGF360-12L, MGF360-13L, MGF 360-14L.

6. Use of the African swine fever virus four-gene deletion attenuated strain of any one of claims 1 to 3 in the preparation of a vaccine for preventing or treating African swine fever.

7. A vaccine comprising the african swine fever virus four-gene-deleted low virulent strain of any one of claims 1 to 3.

8. the vaccine of claim 7, wherein the vaccine is suitable for oral administration, or intramuscular injection.

9. a test kit for differentiating infection by the african swine fever virus attenuated strain with the four-gene deletion attenuated strain of claim 1, comprising: the primer pair is designed aiming at least one gene of four genes of CD2v and MGF360-12L, MGF360-13L, MGF360-14L of an African swine fever virus SY18 isolate.

10. A test kit for differentiating infection by the african swine fever virus attenuated strain with the four-gene deletion attenuated strain of claim 1, comprising: antibody of protein expressed by at least one gene of CD2v, MGF360-12L, MGF360-13L, MGF360-14L of SY18 isolate of African swine fever virus.

Technical Field

The invention relates to the technical field of bioengineering, in particular to an African swine fever virus four-gene deletion low virulent strain and application thereof.

Background

African Swine Fever (ASF) is an acute severe infectious disease of pigs caused by African Swine Fever Virus (ASFV) infection, which is characterized by Fever and organ bleeding all over the body, and the death rate of domestic pigs can reach 100%. The disease first outbreaks in kenya in 1921, followed by a wide prevalence throughout african domestic and wild pigs, evolving into 24 genotypes. The 20 th century was introduced into europe in the 50 s, and the disease was cured for 40 years throughout europe. However, the disease was again introduced into grurgia from eastern africa in 2007, and then widely disseminated in eastern europe and introduced into elocusk, the far east russia, 2017. In 2018, 8 months, Hurongliang researchers report the epidemic situation of the first African swine fever in China, and the disease spreads to 30 provinces and municipalities in China within a short time of one year, so that the yield is reduced by over 20 percent in the whole country, the loss exceeds billions of yuan, and the prevention and control situation of the disease is extremely severe and complicated at present.

ASFV is a large intracytoplasmic replication virus, and the virion is icosahedral symmetric, 200nm in diameter, and has a concentric structure. The African swine fever virus genome is single-molecule linear double-stranded DNA with covalently closed ends, the total length of the genome is 170-190 kb, and the genome lengths of different strains are different. The whole genome of ASFV contains more than 160 open reading frames, and can code 150-200 proteins. The middle part of the genome is a central conserved region (C region) with the length of about 125kb, two variable regions are respectively arranged at two sides of the C region, the right Variable Region (VR) has the length of 13-22kb and contains 5 multigene families (MGF). This region is involved in mechanisms such as viral antigenic variation, escape from host defense systems, etc.

The vaccine is the most effective and economic means for preventing and controlling infectious diseases, and since the ASF is introduced into Europe, the research and development of the ASF effective vaccine are highly regarded by research institutions in a plurality of countries, especially Europe, and important progress is made, for example, the ASFV inactivated vaccine is found not to induce effective neutralizing antibodies, and the humoral immunity and cellular immunity must be considered at the same time; the subunit vaccine is safer than attenuated live vaccine, and the improvement of the immune efficacy of the ASFV genetic engineering subunit vaccine through the combined expression of different antigen combinations by the virus vector is a feasible idea from the current stage; the attenuated live vaccine by knocking out a virulence gene has the best protection effect and the best development prospect, but has the problems of different effects caused by the deletion of the same gene of different strains, immune side reaction and field virus dispersion caused by insufficient deletion, the loss of the protection effect of attenuated strains caused by excessive weakening caused by multi-gene deletion and the like, and the balance of safety (biological safety risks such as side effect, continuous infection, field virus dispersion and the like) and protectiveness needs to be considered in research.

Due to the complex pathogenic mechanism of ASFV, the viral infection and immune mechanism are not clear, so far, no effective vaccine for preventing and controlling the ASFV is available. Although studies on ASF vaccines have been reported in recent years since the end of the 60's 20 th century, they have not been successfully applied to practice. Hitherto, a plurality of ASFV gene deletion vaccine candidate strains are obtained internationally by adopting a genetic engineering means, and the attenuated vaccine candidate strains are obtained by a method of weakening cells by passage and separating attenuated strains from the nature, wherein some vaccine candidate strains have a 50-100% protection rate against the attack of homologous virus strains in research reports, but have a poor protection effect against the attack of heterologous viruses. In addition, no matter the artificial gene deletion attenuated strain or the natural deletion attenuated strain, the pig has dose-dependent side reaction after immunization, and the immunized pig has the symptoms of mental depression, appetite reduction, fever, viremia, hyperglycoglobinemia, pneumonia, joint swelling, lameness, chronic lesion, cad pig and the like, thereby reducing the actual use value of the vaccine. At present, the safety problem of the African swine fever gene deletion strain and the natural low virulent strain as the vaccine is generally concerned. Due to various reasons, the research and the storage of the African swine fever vaccine are seriously lagged in China, and the research of the African swine fever vaccine is put forward again after the epidemic situation of the African swine fever occurs.

At present, the prevention and control situation of the African Swine Fever (ASF) in China is extremely severe, and the development of safe and efficient ASF vaccines is urgently needed.

Disclosure of Invention

The invention aims to solve the technical problem of lack of safe and effective African swine fever vaccines in China at present, and provides an African swine fever virus gene deletion low virulent strain which can provide complete immune protection effect on the attack of ASFV parent strains, has high safety and is suitable for serving as a vaccine candidate strain for preventing African swine fever.

In order to solve the technical problems, the invention is realized by the following technical scheme:

In one aspect of the invention, an African swine fever virus low virulent strain is provided, the low virulent strain is a gene deletion low virulent strain of a SY18 isolate strain of the African swine fever virus, and functional proteins of the following genes are deleted:

CD2v gene and three polygene family genes MGF360-12L, MGF360-13L, MGF 360-14L.

preferably, the deleted gene sequence comprises: a nucleotide sequence for coding the amino acid sequence of the CD2v protein shown in SEQ ID NO.1, a nucleotide sequence for coding the amino acid sequence of the MGF360-12L protein shown in SEQ ID NO.2, a nucleotide sequence for coding the amino acid sequence of the MGF360-13L protein shown in SEQ ID NO.3 and a nucleotide sequence for coding the amino acid sequence of the MGF360-14L protein shown in SEQ ID NO. 4.

More preferably, the deleted gene sequence comprises: the nucleotide sequence of the CD2v gene shown in SEQ ID NO.5, the nucleotide sequence of the MGF360-12L gene shown in SEQ ID NO.6, the nucleotide sequence of the MGF360-13L gene shown in SEQ ID NO.7 and the nucleotide sequence of the MGF360-14L gene shown in SEQ ID NO. 8.

In another aspect of the invention, a recombinant virus is provided, wherein the CD2v gene of the African swine fever virus SY18 isolate is deleted.

Preferably, the recombinant virus further lacks the following three polygene family genes: MGF360-12L, MGF360-13L, MGF 360-14L.

In another aspect of the invention, the invention also provides application of the African swine fever virus low virulent strain in preparation of a vaccine for preventing or treating African swine fever.

In another aspect of the invention, there is also provided a vaccine comprising an attenuated strain of the african swine fever virus as described above.

The vaccine is preferably suitable for oral, or intramuscular, vaccination.

In another aspect of the present invention, there is provided a detection kit for distinguishing infection of the above african swine fever virus low virulent strain and wild virulent strain, comprising: the primer pair is designed aiming at least one gene of four genes of CD2v and MGF360-12L, MGF360-13L, MGF360-14L of an African swine fever virus SY18 isolate. The African swine fever virus low virulent strain and the wild strain can be distinguished by utilizing the designed and synthesized primer pair through PCR amplification reaction.

In another aspect of the present invention, there is provided a detection kit for distinguishing infection of the above african swine fever virus low virulent strain and wild virulent strain, comprising: antibody of protein expressed by at least one gene of CD2v, MGF360-12L, MGF360-13L, MGF360-14L of SY18 isolate of African swine fever virus. The infection of the African swine fever virus low virulent strain and the wild strain can be distinguished by detecting CD2v, MGF360-12L, MGF360-13L and/or MGF360-14L protein antibodies infecting pig bodies.

The African swine fever virus gene deletion attenuated strain is inoculated to a piglet for immunization for 28 days, and then an ASFV parent virus is used for carrying out a virus challenge test, so that the immunized swine herd is completely protected, the body temperature detected in an observation period is in a normal range, no abnormal clinical expression is seen, the African swine fever virus gene deletion attenuated strain is safe and reliable, and the African swine fever virus gene deletion attenuated strain can be used as a good vaccine candidate strain for preventing African swine fever.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a schematic view of an ASFV recombination deletion gene position and an insertion marker gene expression cassette according to example 1 of the present invention;

FIG. 2 is a view under a fluorescence microscope of the first generation (. about.10) of the ASFV SY 18. delta.C recombinant virus construct of example 1 of the present invention;

FIG. 3 is a view showing the growth of a P2 generation recombinant virus of ASFV SY18 Δ C recombinant virus in example 1 of the present invention (. times.10);

FIG. 4 is a diagram showing the PCR detection results (1 to 23 represent different clones) of the P7 generation of ASFV SY18 Δ C recombinant virus in example 1 of the present invention;

FIG. 5 is a diagram showing the PCR detection results (1 to 23 represent different clones) of the P8 generation of ASFV SY18 Δ C recombinant virus in example 1 of the present invention;

FIG. 6 is a diagram showing the result of PCR detection of the recombinant virus P9-P11 generation ASFV SY18 Δ C in example 1 of the present invention;

FIG. 7 is a diagram showing fluorescence detection results (. times.10) of ASFV SY18 Δ MC4 recombinant virus P2 generation in example 1 of the present invention;

FIG. 8 is a diagram showing the PCR detection results (1 to 23 represent different clones) of the P3 generation of ASFV SY18 Δ MC4 recombinant virus in example 1 of the present invention;

FIG. 9 is a diagram showing the PCR detection results (1 to 23 represent different clones) of the P5 generation of ASFV SY18 Δ MC4 recombinant virus in example 1 of the present invention;

FIG. 10 is a diagram showing the results of PCR detection of ASFV SY18 Δ MC4 recombinant virus P6-P8 in example 1 of the present invention;

FIG. 11 is a diagram showing fluorescence detection results (24hpi) (. 20) of ASFV SY 18. delta. MC4 recombinant virus P8 generation recombinant virus in example 1 of the present invention;

FIG. 12 is a graph of the virus growth curve of example 2 of the present invention;

FIG. 13 is a graph of recombinant virus death in example 3 of the present invention.

Detailed Description

The results of the embodiment of the invention show that: the single deletion of the CD2v gene has limited virulence on Chinese strains, and the previous results also show that after a pig is immunized by a constructed deletion strain of another MGF multigene family 6 gene (containing 6 MGF multigene families including MGF360-12L, 13L and 14L), although the body temperature of the pig can cause a long-term rise phenomenon (not more than 41.3 ℃), the mental state and the feed intake are not influenced.

The invention further performs clone optimization expression on all possible MGF multigene family member genes in the whole genome of the SY-18 strain of the Chinese strain, and screens and determines that the MGF360-12L, MGF360-13L and the MGF360-14L are particularly key to the virus virulence and the immune suppression effect by comparing the suppression activity of the transcription level of the interferon reporter gene. Based on the previous results, the invention further constructs a vaccine candidate strain lacking ASFV CD2v and MGF360 key virulence genes by adopting a genetic engineering method, and after further lacking CD2v genes on the basis of MGF360-12L, MGF360-13L and MGF360-14L gene deletion, the long-time fever caused by virus inoculation is basically eliminated, which shows that although the CD2v single-gene deletion strain has strong virulence, the CD2v gene deletion can further reduce the virulence of the MGF deletion strain. The four-gene deletion strain can be evaluated to have complete protective effect on the attack of ASFV parent strain from the preliminary results of immune efficacy and safety experiments, which shows that the strain can be used as a good candidate strain of African swine fever vaccine.