Construction of African swine fever gene deletion low virulent strain and application of African swine fever gene deletion low virulent strain as vaccine
阅读说明：本技术 非洲猪瘟基因缺失弱毒株的构建及其作为疫苗的应用 (Construction of African swine fever gene deletion low virulent strain and application of African swine fever gene deletion low virulent strain as vaccine ) 是由 郑海学 李攀 冯涛 齐晓兰 刘华南 张克山 李丹 吴盼雪 马昭 党文 刘迎琦 于 2020-07-10 设计创作，主要内容包括：本发明属于生物工程技术领域,具体涉及一种可用作疫苗的基因缺失减毒非洲猪瘟病毒株及其构建方法,该病毒株是亲本非洲猪瘟病毒ASFV CN/GS/2018分离株的基因缺失弱毒株。本发明发现了DP71L基因是对猪致病性致弱的新靶标基因,并发现联合丧失MGF360-18R基因、DP71L基因和DP96R基因编码蛋白的功能,降低了亲本毒株的毒性,获得了一株安全性高的减毒非洲猪瘟候选疫苗株；所述减毒非洲猪瘟候选疫苗株高剂量(10<Sup>4</Sup>HAD<Sub>50</Sub>)免疫猪后,对猪完全致弱,免疫猪健康,不发病,与免疫猪同居猪核酸也呈阴性,不水平传播,具有较好安全性；且能够对ASFV CN/GS/2018强毒株的攻毒提供100％免疫保护,可作为安全和有效的防控非洲猪瘟疫情的候选疫苗,具有极大的社会价值。(The invention belongs to the technical field of bioengineering, and particularly relates to a gene deletion attenuated African swine fever virus strain capable of being used as a vaccine and a construction method thereof, wherein the virus strain is a gene deletion weak African swine fever virus ASFV CN/GS/2018 isolateA strain. The DP71L gene is found to be a new target gene for weakening the pathogenicity of the pig, and the combined loss of the functions of MGF360-18R gene, DP71L gene and DP96R gene encoding protein is found, so that the toxicity of parent strains is reduced, and an attenuated African swine fever candidate vaccine strain with high safety is obtained; the attenuated African swine fever candidate vaccine strain has high dose (10) 4 HAD 50 ) After the pigs are immunized, the pigs are completely weakened, the immunized pigs are healthy and do not get ill, the nucleic acid of the pigs living with the immunized pigs is negative and does not spread horizontally, and the safety is better; and can provide 100% immune protection for the challenge of ASFV CN/GS/2018 virulent strain, can be used as a safe and effective candidate vaccine for preventing and controlling African swine fever epidemic situation, and has great social value.)
1. A target gene DP71L for reducing the virulence of African swine fever virus, wherein the nucleotide sequence of the DP71L gene is shown as SEQ ID NO. 3.
2. The application of preparing the African swine fever attenuated strain by jointly losing the functions of MGF360-18R gene, DP71L gene and DP96R gene encoding protein in the African swine fever virus.
3. The use of claim 2, wherein the MGF360-18R gene encodes a protein having the amino acid sequence shown in SEQ ID No.2, the DP71L gene encodes a protein having the amino acid sequence shown in SEQ ID No.4, and the DP96R gene encodes a protein having the amino acid sequence shown in SEQ ID No. 6.
4. The use according to claim 3, wherein the MGF360-18R gene has a nucleotide sequence shown in SEQ ID No.1, the DP71L gene has a nucleotide sequence shown in SEQ ID No.3, and the DP96R gene has a nucleotide sequence shown in SEQ ID No. 5.
5. The use according to claim 2, wherein the method for loss of function of the MGF360-18R gene, DP71L gene and DP96R gene-encoded proteins comprises gene deletion techniques, gene mutation techniques, gene insertion techniques.
6. The application of the African swine fever vaccine prepared by jointly losing the functions of MGF360-18R gene, DP71L gene and DP96R gene encoding protein in the African swine fever virus.
7. The use of claim 6, wherein the MGF360-18R gene encodes a protein having the amino acid sequence shown in SEQ ID No.2, the DP71L gene encodes a protein having the amino acid sequence shown in SEQ ID No.4, and the DP96R gene encodes a protein having the amino acid sequence shown in SEQ ID No. 6.
8. The use of claim 7, wherein the MGF360-18R gene has a nucleotide sequence shown as SEQ ID No.1, the DP71L gene has a nucleotide sequence shown as SEQ ID No.3, and the DP96R gene has a nucleotide sequence shown as SEQ ID No. 5.
9. The use of claim 6, wherein the method for loss of function of MGF360-18R gene, DP71L gene and DP96R gene encoding proteins comprises gene deletion techniques, gene mutation techniques, gene insertion techniques.
10. Use of an attenuated strain of African swine fever virus prepared by the combined deletion of gene fragments in an African swine fever virus, wherein said gene fragments comprise all or part of the nucleotide sequence of the MGF360-18R gene, the DP71L gene and the DP96R gene.
11. The use according to claim 10, wherein the deleted gene fragment has the nucleotide sequence shown in SEQ id No. 7.
12. The use according to any one of claims 2 to 11, wherein the African swine fever virus is genotype II African swine fever virus.
13. The use according to claim 12, wherein the african swine fever virus is an ASFV CN/GS/2018 isolate.
14. A gene-deleted attenuated African swine fever virus is characterized in that the functions of proteins coded by original African swine fever virus MGF360-18R gene, DP71L gene and DP96R gene are lost.
15. The attenuated african swine fever virus with gene deletion according to claim 14, wherein the amino acid sequence of the protein encoded by MGF360-18R gene is shown in SEQ ID No.2, the amino acid sequence of the protein encoded by DP71L gene is shown in SEQ ID No.4, and the amino acid sequence of the protein encoded by DP96R gene is shown in SEQ ID No. 6.
16. The attenuated african swine fever virus with gene deletion according to claim 15, wherein the nucleotide sequence of MGF360-18R gene is shown in SEQ ID No.1, the nucleotide sequence of DP71L gene is shown in SEQ ID No.3, and the nucleotide sequence of DP96R gene is shown in SEQ ID No. 5.
17. The genetically deleted attenuated african swine fever virus of claim 16, wherein the original african swine fever virus is an ASFV CN/GS/2018 isolate.
18. The genetically deleted attenuated african swine fever virus of claim 17, wherein the genetically deleted attenuated african swine fever virus lacks the nucleotide at position 184062-184456 relative to the full-length sequence of the ASFV CN/GS/2018 isolate.
19. An attenuated african swine fever vaccine, comprising the attenuated african swine fever virus having the gene deletion of any one of claims 14-18.
20. A method for preparing attenuated African swine fever virus strain, wherein the function of MGF360-18R gene, DP71L gene and DP96R gene coding protein of original strain is lost by genetic engineering means.
21. The method of claim 20, wherein the method is a homologous recombination technique.
22. The method of claim 21, wherein the method comprises the steps of:
(1) optimizing a pX330 vector; removing nuclear localization signals NLS at two ends of Cas9 enzyme in the pX330 vector by a Clonexpress II one-step cloning method, and naming the NLS as pX330 delta N;
(2) designing target oligonucleotides 181/UK-gRNA-L and 181/UK-gRNA-R, inserting the oligonucleotides into a pX 330-delta N vector in a pairing manner, and preparing positive cloning plasmids pX330 delta N-181/UKL and pX330 delta N-181/UKR; wherein, the nucleotide sequence of 181/UK-gRNA-L is shown in SEQ ID NO.8, and the nucleotide sequence of 181/UK-gRNA-R is shown in SEQ ID NO. 9;
(3) designing upstream and downstream sequences of MGF360-18R gene, DP71L gene and DP96R gene combined fragments as homologous recombination arms, and simultaneously cloning into a pUC19 vector to obtain a recombination transfer vector p 181/UKLR;
(4) inserting eGFP screening expression box gene fragment p72-eGFP-SV40poly in the middle of the gene sequences of the left arm and the right arm of the recombinant transfer vector p181/UKLR to obtain a homologous recombinant transfer vector p 181/UKLR-eGFP;
(5) homologous recombination transfer vectors p181/UKLR-eGFP, pX330 delta N-181/UKL, pX330 delta N-181/UKR and Je
(6) virus strain screening: screening the recombinant virus strain by using a 181/UK-check-F/R primer pair to obtain an attenuated African swine fever virus strain delta 181/UK with a deleted gene fragment, wherein the nucleotide sequence of the 181/UK-check-F/R primer pair is shown as SEQ ID NO. 16-17.
23. The method of any one of claims 20 to 22, wherein the original strain is an African swine fever virus ASFVCN/GS/2018 isolate.
24. The method of claim 23, wherein the attenuated African swine fever virus strain has a deletion of nucleotides 184062-184456 as compared to the full length sequence of the original strain ASFV CN/GS/2018, the deleted nucleotide sequence being shown in SEQ ID No. 7.
25. An attenuated african swine fever virus strain produced by the method of claim 24.
26. An African swine fever vaccine with combined loss of functions of MGF360-18R gene, DP71L gene and DP96R gene coding protein.
27. The african swine fever vaccine of claim 26, further comprising a loss of function of a protein encoded by one or more of a CD2v gene, an MGF360-12L gene, an MGF360-13L gene, an MGF360-14L gene, or an MGF360-505R gene.
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to construction of an African swine fever gene deletion low virulent strain with MGF360-18R gene, DP71L gene and DP96R gene coding protein loss functions and application of the African swine fever gene deletion low virulent strain as a vaccine.
Background
African Swine Fever (ASF) is an acute virulent infectious disease characterized by Fever of pigs and organ bleeding of the whole body of pigs caused by African Swine Fever Virus (ASFV), and the death rate of domestic pigs is as high as 100%. The disease first outbreaks in kenya 1921 and then is widely prevalent in domestic and wild pigs throughout africa. 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. At the beginning of 8 months in 2018, a Hurongrong researcher reports the epidemic situation of the African swine in the first instance of China, and the disease spreads to 30 provinces and municipalities in China within a short time of one year, so that the disease continues to threaten the pig industry, wherein compared with 8 months in 2018, the yield of the domestic pig in 9 months in 2019 is reduced by 40%, the price of pork is doubled since 8 months in 2019, the yield is reduced by more than 40% in China, and the loss is serious. At present, no commercial effective vaccine exists, once the epidemic situation of the African swine fever occurs, the epidemic situation of the African swine fever can only be controlled by a killing means, but the mode not only causes economic loss, but also needs to spend a long time, and can not meet the requirement of large-scale pig raising in China. Therefore, the vaccine is the most effective and economic means for preventing and controlling the virus infectious diseases and is very important for preventing and treating the African swine fever under the mode of pig raising in China. However, the African Swine Fever Virus (ASFV) has a complex structure and a huge genome, most functions are unknown, the infection and pathogenic mechanism are unclear, the theoretical cognition of vaccine creation is limited, the epidemic has been over one hundred years, but no commercial vaccine is developed.
The current preparation of vaccines for african swine fever is mainly done in three ways: firstly, directly inactivating original African swine fever virus to obtain an inactivated vaccine; secondly, screening out virus proteins which effectively induce immune response, and further preparing subunit vaccines and live vector vaccines; thirdly, a gene deletion means is adopted to knock out virulence genes to obtain the recombinant virus vaccine. The first method is the most common and direct method for preparing virus vaccine, but because the African swine fever virus codes proteins such as immunosuppression, immune tolerance and Antibody Dependence Enhancement (ADE), the inactivated vaccine cannot provide effective immune protection in the body of the immunized pig; the difficulty of the second method is that the structure of the African swine fever virus is complex, the protein inducing immune response is not clear at present, and the obtained antigen is not enough to induce strong immune protection. The vaccine which is most expected to break through at present is a gene knockout vaccine, and a vaccine candidate strain which has immunogenicity, can reduce pathogenicity and has an immune protection effect on an immune pig is obtained by knocking out virulence related genes such as immunosuppression, tolerance, ADE and the like. For example, chinese patent CN110551695A provides a four-gene-deleted low virulent strain of african swine fever virus, which is a four-gene-deleted low virulent strain of SY18 isolate of african swine fever virus, and which lacks functional proteins of the following genes: CD2v gene coding products and three polygene family genes (MGF360-12L, MGF360-13L, MGF360-14L) coding products, after the piglets are immunized for 28 days, the piglets are subjected to a challenge test by using ASFV original virus, and as a result, the immunized swinery is completely protected; for example, Chinese patent CN110093324B discloses a gene II type African swine fever virus MGF360-505R deletion and CD2V and MGF360-505R combined deletion gene deletion virus, both strains can provide 100% immune protection for African swine fever Chinese epidemic virulent strains, and the two strains can provide sufficient virulent attack protection 21 days after the immunization.
In summary, the knockout of the virulence gene of classical swine fever virus usually needs to consider immune response and protection to pigs, and also needs to consider pathogenicity and safety. However, the hog cholera virus virulence gene knockout vaccine in the prior art still has the following problems: firstly, different strains have different effects caused by deletion of the same gene, toxicity and pathogenicity residues can be caused by insufficient deletion, the virus titer caused by multi-gene deletion is low, and the risk of reducing immunogenicity or protective action and the like of attenuated strains can be caused; whether the knocked-out virulence genes reduce virus carrying and toxin expelling is an important index of biological safety, and vaccine candidate strains which are eliminated by immunity can be obtained preferably, so that the risk of virus dispersion and toxin expelling is reduced; ③ the complete genome sequencing work of African swine fever is completed, but the number of the regulatory genes and the structural genes which form the African swine fever virus is up to 160, and although the research on the functions of all the genes is a huge project, the comprehensive research on the functions of each regulatory gene and each structural gene is very important for the pathogenic mechanism and the vaccine development.
Therefore, in the preparation process of the attenuated vaccine for African swine fever, the gene function and the influence of the gene function on pathogenicity and immune response are identified, a basis is provided for the selection of a knockout target gene and the construction of gene knockout, the screening of a new target gene is very important, and the safety and the immune effectiveness of a vaccine candidate strain are determined. The DP71L is found to have stronger immunosuppressive action and to be a newly found new target gene; the DP71L, DP96R and MGF360-18R genes are jointly deleted in the original strain of African swine fever, and a high-dose intramuscular injection (each pig is immunized by 10 degrees)4HAD50) The gene deletion strain of the swine which does not cause the disease is negative, and the nucleic acid and the antibody of the swine which live with the immune swine are negative, thereby showing that the strain can not cause the death of the swine caused by intramuscular injection and has no horizontal transmission phenomenon, and showing that the strain is the attenuated African swine fever virus strain with good safety. The African swine fever virus low virulent strain 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.
Disclosure of Invention
Aiming at the technical problems, the invention provides a strategy for constructing an attenuated strain of African swine fever, which is realized by the following technical scheme:
the invention firstly discovers that the DP71L gene has stronger immunosuppressive action and is a newly discovered target gene for weakening the African swine fever virus. Based on the above, the invention aims to provide a target gene DP71L for reducing the virulence of African swine fever virus, wherein the nucleotide sequence of the DP71L gene is shown as SEQ ID NO. 3.
The invention also aims to provide an application of preparing an African swine fever attenuated strain by jointly losing functions of MGF360-18R gene, DP71L gene and DP96R gene encoding proteins in the African swine fever virus; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS and promoter sequence), and the DP96R gene is adjacent to the other end of the DP71L gene and has a connecting sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of the MGF360-18R gene, the DP71L gene and the DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the amino acid sequence of the MGF360-18R gene encoding protein is shown as SEQ ID NO.2, the amino acid sequence of the DP71L gene encoding protein is shown as SEQ ID NO.4, and the amino acid sequence of the DP96R gene encoding protein is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of the MGF360-18R gene is shown as SEQ ID No.1, the nucleotide sequence of the DP71L gene is shown as SEQ ID No.3, and the nucleotide sequence of the DP96R gene is shown as SEQ ID No. 5.
Preferably, the method for losing the functions of the MGF360-18R gene, the DP71L gene and the DP96R gene encoding proteins comprises gene deletion technology, gene mutation technology and gene insertion technology.
The invention also aims to provide an application of preparing the African swine fever vaccine by jointly losing the functions of MGF360-18R gene, DP71L gene and DP96R gene encoding protein in the African swine fever virus; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the amino acid sequence of the MGF360-18R gene encoding protein is shown as SEQ ID NO.2, the amino acid sequence of the DP71L gene encoding protein is shown as SEQ ID NO.4, and the amino acid sequence of the DP96R gene encoding protein is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of the MGF360-18R gene is shown as SEQ ID No.1, the nucleotide sequence of the DP71L gene is shown as SEQ ID No.3, and the nucleotide sequence of the DP96R gene is shown as SEQ ID No. 5.
Preferably, the method for losing the function of the MGF360-18R gene, the DP71L gene and the DP96R gene encoding proteins comprises gene deletion technology, gene mutation technology and gene insertion technology.
It is another object of the present invention to provide a use of an attenuated strain of African swine fever virus by the combined deletion of a gene fragment comprising all or part of the nucleotide sequence of the MGF360-18R gene, the DP71L gene and the DP96R gene in African swine fever virus; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the nucleotide sequence of the deletion gene fragment is shown as SEQ ID NO. 7.
Another object of the present invention is to provide a use of an attenuated african swine fever vaccine prepared by deleting a gene fragment including all or a part of nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene in african swine fever virus; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the nucleotide sequence of the deletion gene fragment is shown as SEQ ID NO. 7.
Preferably, the african swine fever virus is a genotype ii african swine fever virus.
Preferably, the African swine fever virus is an ASFV CN/GS/2018 isolate.
Another objective of the invention is to provide an African swine fever virus with gene deletion, which loses the functions of the proteins coded by the original African swine fever virus MGF360-18R gene, DP71L gene and DP96R gene; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the amino acid sequence of the MGF360-18R gene encoding protein is shown as SEQ ID NO.2, the amino acid sequence of the DP71L gene encoding protein is shown as SEQ ID NO.4, and the amino acid sequence of the DP96R gene encoding protein is shown as SEQ ID NO. 6.
Preferably, the nucleotide sequence of the MGF360-18R gene is shown as SEQ ID No.1, the nucleotide sequence of the DP71L gene is shown as SEQ ID No.3, and the nucleotide sequence of the DP96R gene is shown as SEQ ID No. 5.
Preferably, the original African swine fever virus is an ASFV CN/GS/2018 isolate.
Preferably, the gene deletion attenuated African swine fever virus lacks the nucleotides 184062-184456 relative to the full-length sequence of the ASFV CN/GS/2018 isolate.
Another object of the present invention is to provide an African swine fever attenuated vaccine, which comprises the above-mentioned attenuated African swine fever virus with gene deletion.
Another object of the present invention is to provide a method for preparing attenuated African swine fever virus strains, which comprises the steps of losing the functions of the MGF360-18R gene, DP71L gene and DP96R gene encoding proteins of the original strains by genetic engineering means; since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
Preferably, the method is a homologous recombination technique.
Preferably, the method comprises the steps of:
(1) optimizing a pX330 vector; removing nuclear localization signals NLS at two ends of Cas9 enzyme in the pX330 vector by a Cloneexpress II one-step cloning method, and naming the NLS as pX330 delta N;
(2) designing target oligonucleotides 181/UK-gRNA-L and 181/UK-gRNA-R, inserting the oligonucleotides into a pX 330-delta N vector in a pairing manner, and preparing positive cloning plasmids pX330 delta N-181/UKL and pX330 delta N-181/UKR; wherein, the nucleotide sequence of 181/UK-gRNA-L is shown in SEQ ID NO.8, and the nucleotide sequence of 181/UK-gRNA-R is shown in SEQ ID NO. 9;
(3) designing upstream and downstream sequences of MGF360-18R gene, DP71L gene and DP96R gene combined fragments as homologous recombination arms, and simultaneously cloning into a pUC19 vector to obtain a recombination transfer vector p 181/UKLR;
(4) inserting eGFP screening expression box gene fragment p72-eGFP-SV40poly in the middle of the gene sequences of the left arm and the right arm of the recombinant transfer vector p181/UKLR to obtain a homologous recombinant transfer vector p 181/UKLR-eGFP;
(5) homologous recombination transfer vectors p181/UKLR-eGFP, pX330 delta N-181/UKL, pX330 delta N-181/UKR and
(6) virus strain screening: screening the recombinant virus strain by using a 181/UK-check-F/R primer pair to obtain an attenuated African swine fever virus strain delta 181/UK with a deleted gene fragment, wherein the nucleotide sequence of the 181/UK-check-F/R primer pair is shown as SEQ ID NO. 16-17.
Preferably, the original strain is African swine fever virus ASFV CN/GS/2018 isolate.
Preferably, the attenuated African swine fever virus strain is compared with the original strain ASFV CN/GS/2018 full-length sequence and lacks the nucleotide at 184062-184456, and the deleted nucleotide sequence is shown as SEQ ID NO. 7.
Another object of the present invention is to provide an attenuated African swine fever virus strain prepared according to the above method.
Another objective of the invention is to provide an African swine fever vaccine with combined loss of functions of MGF360-18R gene, DP71L gene and DP96R gene encoding proteins.
Preferably, the functional loss of proteins encoded by one or more genes selected from the group consisting of CD2v gene, MGF360-12L gene, MGF360-13L gene, MGF360-14L gene and MGF360-505R gene is also included.
The invention has the beneficial effects that: the invention obtains an attenuated African swine fever virus strain by jointly losing the functions of proteins comprising MGF360-18R gene, DP71L gene and DP96R gene; the attenuated African swine fever virus strain has obviously weakened virulence, does not cause morbidity and death at high dose, and has 100 percent protection effect on a parent strain ASFV CN/GS/2018 isolate after being immunized by the attenuated African swine fever virus strain. And the pigs living with the immune pigs do not have infection phenomena, and have good safety performance. The strategy provides theoretical basis and practical reference for successfully preparing the African swine fever vaccine in the future, and researchers can finally prepare the safe and effective African swine fever vaccine by simultaneously losing the functions of the MGF360-18R gene, the DP71L gene, the DP96R gene and the protein coded by one or more disclosed genes (such as the CD2v gene, the MGF360-12L gene, the MGF360-13L gene, the MGF360-14L gene, the MGF360-505R gene and the like).
Drawings
FIG. 1 is a schematic diagram of a gene knockout strategy;
FIG. 2 shows the results of microscopic observation of cells of initial passage (F0) and high passage (F6) during the purification of recombinant virus, on a scale of 100 μm;
FIG. 3 shows the results of PCR detection of the cleanliness of a gene-deficient virus strain Δ 181/UK, with p72 as a reference gene and NC as a template with ultrapure water;
FIG. 4 is a graph showing the lethal results of the gene-deleted viral strain Δ 181/UK immunization experiment, wherein the left side represents the immune fraction, the right side represents the challenge fraction, each curve represents one animal, the abscissa represents the days after immunization (dpi) or the days after challenge (dpc), respectively, and the ordinate represents the survival rate; the pathogenicity of the delta 181/UK is evaluated by taking a wild strain ASFV CN/GS/2018as a control in the immunization period, and the immunoprotection effect of the delta 181/UK is evaluated by taking an uninmmunized pig as a control in the challenge period;
FIG. 5 is a graph of the body temperature of animals immunized with a gene-deficient virus strain Δ 181/UK and challenged with a virus portion on the left and a challenged with a virus portion on the right, each curve representing one animal, with the abscissa of the number of days after immunization (dpi) or after challenge (dpc), respectively, and the ordinate of the body temperature; the parent ASFV CN/GS/2018 isolate is used as a control of an immune stage, and an unimmunized pig is used as a control of a challenge stage;
FIG. 6 is a graph showing the results of the virus-carrying in blood of animals immunized with the gene-deficient virus strain Δ 181/UK and challenged with the virus, wherein the left side is the immune portion and the right side is the challenged portion, each curve represents one animal, the abscissa represents the number of days after immunization (dpi) or challenge (dpc), respectively, and the ordinate represents the number of copies of the virus.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.
The experiments described in the following examples obtain biosafety permits and african swine fever laboratory activity permits:
according to the related requirements of biosafety of a Lanzhou veterinary research institute of the Chinese agricultural academy of sciences, a biological safety 3-level laboratory (BSL-3) and related biological safety of African swine fever, the Lanzhou veterinary research institute biological safety committee, the laboratory animal ethics committee, the Chinese agricultural academy of sciences biological safety committee, the Lanzhou veterinary research institute experimental animal ethics committee and the Lanzhou veterinary research institute biological safety committee report step by step, the permission of developing highly pathogenic ASFV pathogens and animal research is obtained by the agricultural department, and the permission is recorded by the agricultural rural department and meets the requirements of national biological safety level.
Experimental cell, viral and plasmid sources described in the following examples:
primary Porcine Alveolar Macrophages (PAM) and primary bone marrow macrophages (BMDM) were taken from healthy SPF Bama minipigs aged 2-4 months, aseptically collected, lysed with red blood cell lysate (purchased from Biosharp), red blood cells were removed, centrifuged at low speed, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium (purchased from Gibco) containing 10% FBS (purchased from PAN), placed at 37 ℃ and 5% CO2Culturing in an incubator. BMDM cell culture was supplemented with additional 10ng/mL final concentration of recombinant porcine GM-CSF (purchased from R) in RPMI 1640 complete medium&D Systems Co.), 5% CO at 37 deg.C2Inducing in an incubator, washing once every 2-3 days, centrifuging the nonadherent cells, adding the nonadherent cells into a new cell dish again, changing the liquid for continuous induction, and freezing and storing after 3-7 days or using. ASFV is amplified by PAM cells, and the virus content is titrated, and BMDM cells are used for plasmid transfection and virus recombination experiments.
The ASFV CN/GS/2018 isolate is from the national African swine fever regional laboratory (Lanzhou), belongs to genotype II, can be obtained by entrustment letter approved by veterinary office of Ministry of agriculture and has a virus titer of 5 × 107TCID50and/mL, which is the 4 th generation virus after PAM cell propagation, and is subpackaged and stored at-80 ℃ for later use.
The pX330 vector, the peGFP-N1 vector and the pUC19 vector were all purchased from Ribo Lai Biotech, Inc., Lanzhou; endotoxin-free plasmid extraction kit, purchased from OMEGA.
The procedures in the experiments are those known in the art unless otherwise specified.
Definition of
The term "loss of function of a protein" refers to the loss of function of a protein encoded by a gene by knocking out, mutating or inserting a part of the gene in a gene segment encoding the protein, so that the protein encoded by the gene is subjected to frame shift mutation. The invention relates to an attenuated African swine fever virus with MGF360-18R gene, DP71L gene and DP96R gene coding protein loss function, which is constructed by knocking out all or part of nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene in the African swine fever virus, so that the function of MGF360-18R gene, DP71L gene and DP96R gene coding protein loss is lost, and the attenuated African swine fever virus is used for producing African swine fever vaccines. Since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
The term "gene deletion" refers to the phenomenon that a segment on a chromosome and a gene carried by the segment are lost together to cause mutation, and the African attenuated swine fever recombinant virus is obtained by deleting part or all of nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, so that the toxicity of parent strains is reduced. Since the MGF360-18R gene is located at one end of the DP71L gene and partially overlaps with the DP71L gene (partial sequence of CDS of MGF360-18R gene and promoter sequence), and the DP96R gene is adjacent to the other end of DP71L gene and contains a linker sequence with partial no gene function in the middle, the loss of the function of the encoded protein can be caused by deletion of all the nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, respectively; in order to simplify the operation steps, partial sequences (partial sequence of CDS and promoter sequence) of MGF360-18R gene, the whole nucleotide sequence of DP71L gene, the partial sequence of DP96R gene and the connection sequence of DP71L gene and DP96R gene without gene function sequence can be deleted by one operation, so that the proteins encoded by MGF360-18R gene, DP71L gene and DP96R gene can not be normally expressed, and the function of the encoded protein is lost; according to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the functions of the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene, such as: frame shift mutations, point mutations, deletions or insertions of nucleotide sequences, etc.
The term "gene mutation" refers to a structural change in the base pair composition or arrangement of genes, i.e., a sudden appearance of a new gene at a site, instead of the original gene, called a mutant gene, which results in the sudden appearance of an unprecedented new trait in the phenotype of the offspring. On the basis of reducing the toxicity of parent strains by deleting the whole nucleotide sequence of the DP71L gene and deleting the partial sequences of the DP96R gene and the MGF360-18R gene, so that the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene cannot be expressed and the functions of the encoded proteins are lost, the attenuated African swine fever recombinant virus is obtained, and the proteins encoded by the MGF360-18R gene, the DP71L gene and the DP96R gene can also be prevented from being expressed and the functions of the encoded proteins are lost by carrying out whole or partial mutation on the MGF360-18R gene, the DP71L gene and the DP96R gene by a person skilled in the art, so that the construction of the attenuated African swine fever recombinant virus is realized.
The gene deletion method generally refers to gene knockout, and is an exogenous DNA introduction technology in which a DNA fragment containing a certain known sequence is subjected to homologous recombination with a gene having the same or similar sequence in a recipient cell genome, is integrated into the recipient cell genome, and is expressed. Methods of gene knockout generally include: homologous recombination technology, random insertion mutation technology and RNA interference technology; wherein, the homologous recombination technique is also called gene targeting, which means that the recombination occurs between the homologous sequence on the exogenous DNA and the chromosome DNA of the receptor cell, and the homologous sequence is integrated to a predetermined position, thereby changing some genetic characteristics, and the recombination aims at knocking out a certain gene; random insertion mutation technology is that certain viruses, bacteria or other gene vectors capable of randomly inserting gene sequences are utilized to carry out random insertion mutation on a cell bank in a target cell genome, and then corresponding gene knockout cells are obtained by screening through corresponding markers; RNA interference technology refers to a reverse genetics technology in which the target mRNA is specifically degraded by double-stranded RNA homologous to the target gene mRNA endogenous to the organism, resulting in silencing of the expression of the target gene.
Although the present invention knocks out only part or all of the nucleotide sequences of the MGF360-18R gene, DP71L gene and DP96R gene by homologous recombination technology, the random insertional mutagenesis technology and RNA interference technology can knock out part or all of the nucleotide sequences of the MGF360-18R gene, DP71L gene and DP96R gene, so that the proteins encoded by the MGF360-18R gene, DP71L gene and DP96R gene cannot be expressed, resulting in loss of functions of the proteins encoded by the MGF360-18R gene, DP71L gene and DP96R gene.
The invention takes ASFV CN/GS/2018 isolate as an example, MGF360-18R gene, DP71L gene and DP96R gene are adjacent genes, and a spacer sequence is also included between the adjacent genes. Wherein the MGF360-18R gene is positioned at the position 183372-184085, the DP71L gene is positioned at the position 184068-184280 and the DP96R gene is positioned at the position 184379-184669 of the whole genome sequence of the ASFV CN/GS/2018 isolate. The spacer sequence does not contain any functional gene sequence; the deleted MGF360-18R gene, DP71L gene and DP96R gene sequences described in the present invention are located at position 184062-184456 (shown as SEQ ID NO. 7) of the whole genome sequence of the ASFV CN/GS/2018 isolate, including a partial spacer sequence.
The term "vaccine" refers to a biological agent capable of providing a protective response in an animal, wherein the vaccine has been delivered and is not capable of causing serious disease. The vaccine of the invention is a genetically engineered gene deletion attenuated virus vaccine, wherein the deleted genes are all or partial nucleotide sequences of MGF360-18R gene, DP71L gene and DP96R gene, and it is understood that the deleted genes can also include all or partial nucleotide sequences of any one or more of genes (such as CD2v gene, MGF360-12L gene, MGF360-13L gene, MGF360-14L gene, MGF360-505R gene, etc.); mutations are understood as changes in the genetic information of the wild-type or unmodified MGF360-18R gene, DP71L gene and DP96R gene in the parent CN/GS/2018ASFV strain. It will be appreciated that recombinant mutants obtained by mutation of MGF360-18R gene, DP71L gene and DP96R gene may also be used as attenuated virus vaccines.
The attenuated african swine fever virus vaccine of the present invention further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immune adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, span, etc.; microbial immune adjuvants such as mycobacteria, lipopolysaccharide, muramyl dipeptide, cytopeptide, lipid soluble waxy D, short corynebacterium; the plant immunologic adjuvant is polysaccharides extracted from plant or large fungi, such as pachyman, carthamus tinctorius polysaccharide, Chinese herbal medicine, etc. And biochemical immune adjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants may be nano-adjuvant biological adjuvants, interleukins, interferons, etc.
The vaccine disclosed by the invention can also be used for preparing a combined vaccine, such as combined with other vaccines of pigs, but the focus is on attenuated live vaccines, particularly the integration of viral genes, such as bivalent vaccine, trivalent vaccine and the like. The combination vaccine may comprise a plurality of attenuated non-swine fever viruses of different genotypes, such that a cross-protective immune response against the plurality of non-swine fever virus genotypes may be induced.
The administration of the vaccines of the present invention may be by any convenient route, for example, intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The attenuated vaccines of the present invention are preferably administered intramuscularly. The vaccine may be administered after a prime-boost regimen. For example, after a first vaccination, the subject may receive a second booster administration after a period of time (e.g., about 7, 14, 21, or 28 days). Typically, the booster is administered at the same or a lower dose than the prime dose. In addition, a third booster immunization may be performed, for example 2-3 months, 6 months or a year after immunization.